H i s t o c h e m i c a l J o u r n a l 24, 461 - 634 (1992)

461

Abstracts of the 9th International Congress of Histochemistry and Cytochemistry MAASTRICHT,

THE

NETHERLANDS,

30 AUGUST

-- 5 SEPTEMBER,

1992

PLENARY LECTURES LI

In Situ Hybridization Coming of Age. M. van der Ploeg, T. Raap, R. Dirks and J. Wiegant Department of Cytoehemistty and Cytometry, Leiden University, The Netherlands. Through the combined efforts of several groups, in situ hybridization (ISH) methodology has reached a high detection sensitivity (defined as the smallest DNA target detectable); a high DNA resolution (defined as the smallest distance in kilobasepairs between two DNA targets which can be resolved microscopically) and a high multiplicity (defined as the number of different DNA targets that can be identified simultaneously). It is also possible to detect mRNA's by ISH. Single copy DNA sequences as small as 1 kb can be localised in metaphase or interphase chromosome preparations. Using chromatin of varying states of decondensation, resolutions > t 0 kb can be reached. Multiplicity - - currently about 12 targets simultaneously - - is likely to increase, particularly by use of image cytometry. Finally, the combination of ISH and immunocytochemistry allows to visualize not only the presence, absence or amplification of genes and gene transcripts, but also that of the cognate proteins or peptides. The speed, the accuracy, the relative ease of these nonradioactive procedures, and the fact that they permit to extract detailed molecular data from morphologically intact cells and chromosomes, has increased their importance in many biomedical fields.

P D G F and cytokines are currently used in these studies. Labelling with fluorescent dyes is preferred over colour staining because of its greater sensitivity. In situ hybridization for the localization, and Northern and slot blots for quantitation of the mRNAs of the different proteins complete this program. Technical aspects of these new methods will be discussed and new data from our own research will be presented. It is hoped that these methodological advances will provide a better understanding of biological processes and of the pathogenesis of human diseases.

L3

Exploring Cell Physiology with Machine-Vision Light Microscopy. D. L. Taylor Pittsburg, USA.

L4

Nerve-Immune System Interactions. D. Felten Rochester, USA.

L5

Histochemistry: From Tissues to Molecules. J. Roth Zfirich, Switzerland.

L2

Histochemistry in Cardiovascular Biology: New Frontiers. J. Schaper Max-Planck-Institute, Bad Nauheim, Germany. Histochemical methods in the conventional sense of localization of enzymes are still useful tools in histology, but in research they have been widely replaced by immunocytochemistry using monoclonal antibodies. Our own experience using these methods in cardiovascular research is concerned with: 1. studies of human myocardium afflicted by either coronary heart disease or dilated cardiomyopathy and 2. studies of angiogenesis in the chronically ischemic dog or pig heart. In addition, cells isolated and cultivated are investigated for comparison with tissue sections. Antibodies against the various proteins of the contractile machinery and o f the cytoskeleton of the cardiomyocyte, against extracellular matrix components and their receptors, antibodies for blood cell typing and against growth factors such as a F G F and

L6 Peroxisomes: Elucidation of Their Structure and Function by Cytochemistry. H. D. Fahimi Institute for Anatomy and Cell Biology (If), University of Heidelberg, D-6900 Heidelberg, FRG. The general scientific interest in peroxisomes has grown significantly in the past few years because of their involvement in several important metabolic processes and the recent discovery of more than a dozen inherited diseases characterized by the morphologic absence or biochemical dysfunction of this organelle. In my presentation the current methods for light and electron microscopic cytochemical and immunocytochemical investigation of peroxisomes are briefly reviewed and their contribution to the elucidation of the biology and pathobiology of this organelle discussed. The most common method for the detection of peroxisomes is based on the visualization of the peroxidatic activity of

462 catalase with the 3,3'-diaminobenzidine (Fahimi, J. Cell Biol. 43, 275, 1969). The application of the cerium technique recently has revealed the localization of different oxidases in distinct specific domains within the peroxisomal matrix: urate oxidase in crystalline cores, D-amino acid oxidase in central matrix and a-hydroxy acid oxidase in peripheral matrix. Moreover, the marginal plates of bovine renal peroxisomes were isolated and found to consist exclusively of a-hydroxy acid oxidase B (Zaar, V61kl, Fahimi, J. Cell Biol. 113, 113, 1991). The immunocytochemical labelling with antibodies to peroxisomal membrane proteins (Baumgart et al, J. Cell Biol. 108, 2221, 1989) and the application of computer assisted three dimensional reconstruction of proliferating peroxisomes (Yamamoto and Fahimi, J. Cell Biol. 105, 703, 1987) have elucidated the morphological basis of biogenesis of peroxisomes. Those observations together with recent biochemical studies by Lfiers and V61kl (in preparation) suggest that peroxisomes grow by forming small buds which gradually grow to regular-sized mature peroxisomes. Supported by SFB 352 of DGF, FRG. L7

Contributions of Histoehemistry in Raising Questions and Answering Some. S. S. Spicer Medical University of South Carolina, Charleston, South Carolina U.S.A. Radioautography with 3~SO4 = and cationic stains have revealed in many epithelial cells, previously unknown sulfated glycoconjugates. Labelled lectins have localized neutral glycoproteins thought to be cell specific and not priorly detected. Questions then arise about the biologic significance of these constituents. By localizing proteins of known activity, immunocytochemistry has contributed knowledge of cell function and classification. Thus, immunostaining for specific hormones settled questions about functional cell types in pituitary and pancreas. Immunolocalization of carbonic anhydrase (CA) clarified mechanisms of acidification in stomach and alkalinization in proximal duodenum and pancreatic ducts. Two types of CA-rich intercalated cells can be distinguished in distal nephron: one immunopositive also for basal band III protein and an acidifier of urine, the other

Plenary lectures devoid of band III protein and an alkalinizer. Demonstration of CA in distinctive fibrocytes under urothelium and in inner ear identified a subclass of fibrocytes active in ion transport. Immunocytochemistry of isozymes CA I, II and III revealed variable distribution of these enzyme subtypes and pointed to possible linkage between an isozyme and a compatible ion transport mechanism. A mouse line rendered genetically deficient in CA II lacked immunoreactivity for the enzyme and evidenced arteriolar calcification, establishing this line as an experimental disease model.

L8 New Trends in Immunohistochemistry. P. K. Nakane Department of Anatomy HI, Nagasaki University School of Medicine, Nagasaki 852, Japan. Unlike chemical and physical matters, living organisms are with a fixed genetic program. When the program is comprehended, one can deduce what were the PAST activities, what are the PRESENT activities and what will be the FUTURE activities of the cells and tissues. Immunohistochemical methods have contributed greatly for the comprehension of the program during the past three decades. By localizing proteinaceous antigens immunohistochemically, one recognizes what genes had been transcribed; and by localizing mRNA by in situ hybridization, one identifies what genes are being transcribed. A new trend is to expand application of immunohistochemistry to prognosticate what genes would have been transcribed in cells if the cells were not killed. Several approaches have been conceptualized to peak the FUTURE. One is to differentiate active and inactive chromatins in nuclei and identify genes in each of them. The differentiation of the chromatins may be accomplished either by selective extraction or capitalizing on the accessibility of exogenous proteins such as nuclease, polymerase, antibodies to the active chromatin but not to the inactive chromatins. The identification of genes in each chromatin is done then by utilizing methods such as in situ polymerase chain reaction, in situ hybridization, in situ nick translation, histochemistry and immunohistochemistry in combination. Those genes in the active chromatin but are not being transcribed are those genes which will be or can be transcribed in the FUTURE.

463

Invited symposia INVITED SYMPOSIA

S1. I N S I T U HYBRIDIZATION FROM BENCH TO BEDSIDE

S1.1

Genomics and molecular cytogenetics. Jr. W. Gray

Abstract not received

S1.2

PRINS (Primed In Situ Labelling) of Nucleic Acids. J. Hindkjaer, J. Koch, S. Kolvraa, H. Fischer, J. Mogensen, S. Pedersen and L. Bolund Institute of Human Genetics, Aarhus University, Denmark.

PRINS is a method for sequence specific labelling of nucleic acids in situ, which may be faster, milder and more sensitive than traditional in situ hybridization. Short unlabelled DNA probes are hybridized to the complementary nucleic acid sequences in situ. Labelled nucleotides are then incorporated by a suitable polymerase with the probe as primer and the target nucleic acid as template, thus labelling the site of hybridization. Since the probes are unlabelled, one can use very high probe concentrations giving high hybridization efficiency without background problems. The labelling is independent of probe length since the sensitivity is determined by the primer extension. This allows the detection of minor sequence variations, especially since the match at the 3' end of t h e probe is crucial for priming. Synthetic oligonucleotides can be used as primers, obviating the need for cloned probes. PRINS allows the visualization of unique DNA sequences in combination with high quality Q-banding for cytogenetic mapping. Preliminary results indicate that relaxation of torsional strain in the DNA template during chain elongation greatly improves the degree of labelling. The PRINS method can also be used to visualize mRNA variants, thus permitting functional cytogenetic analyses and, possibly, the detection of somatic mutations in expressed genes at the single cell level. Finally, PRINS is so mild, that it opens up for flow fluorimetric analysis and sorting of cells and chromosomes with respect to their contents of specific nucleic acid sequences.

$1.3

Quantitative Reverse Cytogenetics: A Powerful New Method for Molecular Cytogenetic Analysis of Solid Tumors. A. Kallioniemi, O. Kallioniemi, D. Sudar, D. Rutovitz, J.

W. Gray, F, Waldman and D. Pinkel Div. Molecular Cytometry, Dept. Lab. Med., U.C. San Francisco, CA 94143 - 0808, USA.

Cytogenetic studies in solid tumors are difficult because of the low yield of mitoses. Molecular studies of tumor DNA, in turn, can only probe one defined genomic region at a time. We have developed a powerful new method for surveying the entire genome for DNA sequence copy number variation in a single hybridization. The method is based on the competition

between biotinylated total tumor DNA and digoxigeninlabeled normal genomic reference DNA during hybridization to normal metaphase chromosomes. The relative amount of bound tumor and normal DNA at a given locus in the target chromosomes is dependent on the relative abundance of those sequences in the two DNA samples. After immunofluorescent staining with avidin-FITC (green) and anti-digoxigenin Rhodamine (red), variation of DNA sequence copy numbers in the tumor are detected as variations in the ratios of green and red fluorescence along each chromosome. Using five fibroblast cell lines with X chromosome aneusomies ( 1 - 5 copies) we verified that there was a linear correlation between the green/red ratio of the X chromosome and the degree of aneusomy. Using the method, several genetic changes such as aneusomies, deletions and amplifications, some in previously unknown loci, have been found in cancer cell lines and primary tumors.

S1.4 Visualizing the Multistep Process of Human Tumorigenesis by In Situ Hybridization and Immunohistochemistry. W. N. Hittelman, D. H. Shin, N. Voravud, J. Y. Ro, J. S. Lee and W. K. Hong Departments of Medical Oncology and Pathology, Univ. of Texas M. D. Anderson Cancer Center, Houston, Texas, 77030, USA.

Human aerodigestive tract tumor development has been proposed to be a multistep process driven by carcinogeninduced genetic alterations. To determine the relationship between genetic changes and their phenotypic consequences at the tissue level during tumorigenesis, paraffin-embedded head and neck squamous cell carcinoma specimens containing adjacent premalignant lesions have been examined for chromosome polysomy (by in situ hybridization), dysregulation of proliferation (PCNA immunohistochemistry), and epidermal growth factor receptor expression. Normal appearing epithelium adjacent to the tumor showed increased frequencies of cells with polysomies for chromosomes 7 and 17 compared to normal control epithelium. The fraction of cells exhibiting polysomy increased as tissue passed through hyperplasia, dysplasia, to tumor. Similarly, proliferative dysregulation was increasingly observed during histological progression. Interestingly, increased PCNA expression was first observed in the parabasal region of the epithelium, suggesting a defect in the downregulation of proliferation. Increased PCNA labelling in the basal layer occurred later in histological progression and was, in some cases, associated with an increased EGFR expression. These results suggest that a field cancerization process is associated with tumorigenesis in the aerodigestive tract, and markers of genetic change and proliferative dysregulation might serve as intermediate markers for chemopreventive trials. Moreover, this in situ approach should allow one to determine the tissue consequences of specific genetic alterations during tumor development.

Invited s y m p o s i a

464

$2. I N S T R U M E N T A T I O N

IN CYTOMETRY

AND

MORPHOMETRY

$2.1 Image Information

$2.3 Detection and Quantitation of Fluorescent Signals

G. Brugal Laboratoire TIM3, Universit~Joseph Fourier, Grenoble, France.

H. Tanke, N. P. Verwoerd, E. J. Hennink, A. K. Raap, W. C. R. Sloos and V. Vrolyk Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.

$2.2 Morphometry in a Working Laboratory Environment

$2.4 Three-dimensional Reconstruction and Quantitation

G. Slavin St. Bartholemew's Hospital Medical College, London, UK.

M. Robert-Nicoud Grenoble, France.

$3. DEVELOPMENTS

IN DIAGNOSTIC

MOLECULAR

$3.1

Histochemical Techniques in Molecular Pathology. R. DeLellis Boston, USA.

$3.2

Polymerase Chain Reaction In Situ, Technical Aspects and Potential Application. H. Wolf Boston, USA.

$3.3 Interphase Karyotyping in Tumor Pathology. A. H. N. Hopman, M. Vallinga, P. Poddighe and F. C. S. Ramaekers Dept of Molecular Cell Biology and Genetics, University of Limburg, 6200 MD Maastriet, The Netherlands.

$3.4 Mutation of the p53 Gene and Accumulation of the p53 Protein. Common Steps in Human Cancer. D. P. Lane, C. Midgley, S. Picksley and C. Stephen CRC Laboratories, University of Dundee, Dundee, DDI 4HN. The human gene for p53 is located on chromosome 17p and

$4. I N T R A V I T A L

PATHOLOGY

encodes a 393 amino acid nuclear phosphoprotein. Somatic mutation of this gene is found at high frequency in most human cancers. Germ line mutations in the p53 gene are responsible for the Li-Fraumeni cancer family syndrome and may also be involved in other high risk cancer families. The normal protein is a sequence specific DNA binding protein that has many of the properties of a transcription factor. We have expressed p53 in bacterial systems and used it to make new monoclonal and polyctonal antibodies. These new antibodies allow the detection of p53 in routine clinical samples including formalin fixed paraffin embedded sections, cytology samples and by ELISA in tumour extracts. The results of these assays for p53 protein have been compared with the sequence of the p53 gene in matched samples. Many point mutations in p53 are associated with accumulation of the protein and alteration of its conformation. The nature of this conformational change has been defined using phage epitope mapping libraries. Mutation in the p53 gene may not be the sole cause of protein accumulation and in a variety of systems we have established that other genetic events are required. A range of genotoxic agents stabilise the normal p53 protein and this may explain the growth arrest induced by these agents. The acute sensitivity of tumour ceils to these drugs may then result from the absence of functional normal p53 in tumours. Since in these cells there is no growth arrest and the agents are thus more toxic. This model may also explain the high frequency of chromosomal aberation in tumours with mutant p53.

MICROSCOPY

$4.1 Imaging Organeile and Cytoskeleton Dynamics. D. G. Weiss Inst. Zool., Techn. University Munich, D-W8046-Garching, FRG.

The developments in microelectronics and computer design that enable one to digitize and process microscope images in "real time", i.e. at video rates, led to a renaissance of light microscopy in the form of video microscopy. Since living cells could now be studied in great detail that previously was only

accessible by electron microscopy of dehydrated samples, cell motility is one field benefitting considerably from these developments. The study of the motion of identified intracellular organelles and microtubules (25 nm) which are far smaller than the limit of resolution of light microscopy has become possible by video-enhanced contrast (VEC) microscopy. Such studies have revealed that in animal cells microtubules are motile and highly dynamic and serve as tracks for organelle movement. VEC microscopy allowed for the first time to detect and assay the activity of microtubule-

Invited symposia dependent motor enzymes (cytoplasmic dynein, kinesin) so that they could be purified and characterized. Since in plant cells organelle movements occur along actin filaments we tested their possible involvement in animal cells using preparations of cytoplasm extruded from squid giant nerve fibers. We found a new type of organelle motility that did not follow microtubules but used tracks that were invisible by VEC microscopy. The presence of a network of actin filaments (typically 6 nm diameter) was demonstrated in these regions by video-intensified fluorescence microscopy (VIM) of unfixed preparations stained with rhodamine-phalloidin. The new type of motile activity along "invisible" filaments was shown to be dependent on the presence of the actin filament network but moving organelles were also able to switch from actin filaments to microtubules (Kuznetsov, Langford, Weiss: Nature 356, April 23, 1992). We can say that video microscopy allowed us to go beyond the classical limits of light microscopy: it enabled us to see smaller objects than before, to work at extremely low fluorescence intensities, and to generate contrast where none could be generated by conventional techniques. Our findings indicate that in the cytoplasm of neurons tubulin-based and actomyosin-based motilities coexist and may collaborate to generate organelle movements. (Supported by DFG)

$4.2

Quantitative Studies of Microcirculation in Transparent Invertebrates using Video Imaging and Fluorescent Probes, R. Paul Institute of Zoology, University of Diisseldorf, FRG. The physiology of systemic interactions in animals is a complex field. Mostly only one variable is measured by the applied techniques, and the frequent invasiveness of them tend to negatively influence normal function. The new video imaging techniques together with fluorescent probes are valuable tools for the physiologist to make systemic functions visible and to overcome problems related to former techniques. Essential for their application is the existence of transparent organisms which are abundant among waterliving invertebrates, but there are also transparent fish and transparent developmental stages in higher vertebrates. During the process of developing and applying basic 'optophysiological' techniques, we focused first on the relationships between blood gas transport and cell function. A former study (Paul et al., 1991: Naturwissenschaften 78, 134-135) in the tarantula Eurypelma californicum has surprisingly shown functional adaptations of haemolymph distribution in the open vascular system (revealed by immunofluorescence microscopy using a monoclonal antibody specific for the oxygen carrier haemocyanin) on the oxidative capacity of muscle cells (studied by succinic dehydrogenase activity). Microcirculation in the muscle tissues

465 of the transparent spider Pholcus phalangioides has now been studied using the strong autofluorescence of haemolymph cells and fluorescent microspheres: depending on muscle type, flow patterns and velocities vary. Haemolymph flow in an open circulatory system is probably directed by the varying adhesion of tissue sheaths as revealed by contrast enhancement techniques.

$4.3

Vital Microscopy of Chromatin for the Study of Chemically Induced Disturbances of Mitosis In Vitro. D. Schiffmann, H. Stopper and U. De Boni* Institute of Toxicology, W-8700 Wu'rzburg, Germany; ~Dept. of Physiology, University of Toronto, MSS IA8 Toronto, Canada. Some carcinogens (e.g asbestos, synthetic estrogens) have been reported as non-genotoxic. Evidence has accumulated that such compounds cause mitotic disturbances resulting in displacement, nondisjunction and subsequent loss of chromatin. Nonrandom loss of specific genomic elements has already been observed in a number of transformed cell lines and tumors. Most likely, these phenomena are associated with the loss of tumor suppressor genes and therefore responsible for the development of malignancy without involvement of gene mutations. - - We have developed a new method to monitor the arrangement and distribution of chromatin elements in living cells throughout the course of mitosis: The cells are labelled with a DNA-specific stain (e.g. Bisbenzimide) and kept at 37~ on an inverted fluorescence microscope. The cells are monitored using a 100 x oil immersion phase contrast objective. UV flux is reduced by use of neutral density filters to a level permitting analysis of spatial position of chromatin elements, but avoiding UV-damage to cells. A high resolution Silicon Intensifier Target Camera (SIT) is required for visualization. This camera is connected to a video image processor system which enhances video signals for increased contrast. This method allows a detailed analysis of the origin of mitotic disturbances and their relation to the various phases of mitosis. - - Our results indicate that different functional elements (kinetochores, tubulin, chromatin structure etc.) can be affected in different mitotic stages depending on the mode of action of the inducing agent. As an example, the synthetic estrogen diethylstilbestrol causes dislocation of chromatin elements as early as prophase which persist throughout mitosis and form micronuclei during cytokinesis. In contrast, tubulin-related metaphase disturbances are repaired, presumably at a "mitosis checkpoint" resulting in normal exit from mitosis.

$4.4

Relating IntraceUular Motility with Locomotion. G. Dunn London, UK.

466

$5. D E V E L O P I N G

Invited symposia CONCEPTS

IN CELL DIFFERENTIATION

S5,1 Epithelial Differentiation Markers W. Franke Heidelberg, Germany.

$5.2 The Proliferation Marker K i - 67: Diagnostic & Prognostic Value and Molecular Characterization. J. Gerdes, C. Schlfiter, M. Duchrow, C. Wohlenberg, M. H. G. Becker, G. Key and H.-D. Flad Div. Molecular Immunology, Forsehungsinstitut Borstel, D-2061 Borstet.

It iS now almost ten years since a unique monoclonal antibody was described which reacted selectively with the nuclei of proliferating cells a. A recent data base search revealed that 560 publications referring to this antibody and its applications have appeared since that time. These papers confirmed the value of antibody K i - 67 for the assessment of cell growth in clinical samples, and in particular for assessing the growth fraction of human neoplasms (for review sees). Despite these well-documented diagnostic and prognostic applications of K i - 6 7 , the antigen that is defined by this antibody was not known. Recently, we could demonstrate that K i - 67 detects a double band (345kD & 395kD) in Western blots of lysates obtained from proliferating cells, which was absent in lysates obtained from quiescent cells 3. Using immunocloning approaches, we have isolated and sequenced a 1095 bp eDNA fragment encoding for parts of the Ki - 67 antigen 3. Using this K i - 67 DNA as probe in plaque hybridization techniques and chromosomal walking and anchor priming with PCR, we have now cloned, sequenced, and aligned in an open reading frame the entire K i - 6 7 eDNA of 9700 bp. The structure of this eDNA is thus far unique: The centre is formed by a 6845bp exon, in which a highly conserved motif of 62 bp is repeated for 16 times. Using synthetic peptides, we could clearly demonstrate that this 62 bp element encodes for the epitope recognized by antibody K i - 67. We have now produced a number of new monoclonal antibodies by immunizing mice with a recombinant K i - 6 7 gene product (see abstract of Michael H. G. Becker et al.). One of these antibodies can be applied on paraffin sections (see abstract of Giorgio Cattoretti et al.). 'Gerdes, J., Schwab, U., Lemke, H., Stein, H., Int., J Cancer 1983; 31:13-20. 2Gerdes, J., Seminars in Cancer Biology, 1:199-206. 3Gerdes, J., Li, L., Schlfiter, C., et al. Am. J. Pathol. 1991; 138:867-873.

S5.3

Transforming Growth Factor fls in Early Muriue Development. C. M u m m e r y , H. Slager, K. Lawson and J. van den Eijnden-Van Raaij Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht.

Cell-to-cell interactions are important in the development of all complex organisms, but molecules that may mediate these regulatory interactions have only recently been identified. In the early murine embryo, peptide growth factors with their respective receptors are prime candidates as mediators of such

AND PROLIFERATION

interactions on the basis of their differential regulation in time and space. Using embryonal carcinoma (EC) and embryonic stem (ES) cells as models for early differentiation, we have described changes in RNA expression and protein secretion of the three isoforms of transforming growth factor/3 (TGF//~_3) as endodermal and mesenchymal derivatives form. TGF/~'s are multifunctional regulators of growth and differentiation that can alter the expression of a spectrum of genes depending on the nature of the target ceil. They have been implicated in mesoderm formation in explants of Xenopus embryos; we present preliminary evidence for a similar effect on the promotion of mesoderm formation in EC and ES cells, particularly for TGFfl2. Using a specific antibody, we have also shown that TGF//2 is expressed in the early embryo in a manner predicted by the results in EC and ES cells and further that microinjection of neutralizing antibodies may alter development. The implications of these findings will be discussed.

$5.4 Characterisation of Human Mitotic Cyclins in the Cell Cycle. J. AT. Pines The Wellcome/CRC Institute, Tennis Court Rd., Cambridge, UK.

The mitotic cyclins are proteins implicated in the induction of mitosis, that are marked by their accumulation in interphase followed by dramatic destruction at mitosis in each cell cycle. We have been characterising A- and B- type human cyclins during the cell cycle. We have shown that the levels of cyclins A and B 1 are controlled at both the level of transcription and post-translationally. Cyclin A synthesis begins at the start of S phase, whereas cyclin B1 synthesis starts in the middle of S phase. Both cyclins are stable throughout interphase but are rapidly and specifically destroyed in mitosis. The human A and B1 type cyclins both associate with and activate protein kinases of the cdc2 family; cyclin A associates with p33 cdkaand cyclin B binds to p34cdc2 itself. In collaboration with Dr. J. Nevins (Duke University), we have demonstrated that the cyclin A-p33 Cd~zcomplex is associated with the transcription factor E2F and that this complex also contains the p107 retinoblastoma-related protein. The associated protein kinase activities of cyclin A and B1 appear to be under different forms of regulation. The cyclin B-p34r complex accumulates in an inactive form throughout G2 phase, and is only activated at the beginning of mitosis when p34 ~c2 is dephosphorylated on residues T14 and Y15. By contrast the cyclin A-p33 cd~2 complex is active as soon as it is formed in S phase. Human cyclins A and B1 differ in their sub-cellular location. Cyclin A is a nuclear protein from the time of its synthesis onwards. In mitosis cyclin A associates with chromosomes in prophase but not in metaphase, and it is degraded before the cells enter anaphase. On the other hand, cyclin B1 accumulates in the cytoplasm throughout late S and G2 phases. Most of the cyclin B1 rapidly moves at the start of mitosis, before nuclear lamina breakdown, tn the nucleus, cyclin B1 binds to condensed chromosomes and to the mitotic spindle in prophase and metaphase. Cyclin B1 is abruptly destroyed at the metaphase-anaphase transition, and thus the inactivation of the cyclin Bl-p34 c~c2 kinase activity is implicated as one of the major control steps in the exit from mitosis.

Invited symposia

467

$6. TRENDS IN A U T O R A D I O G R A P H Y

$6.1 Electron Microscopic Radioautography Using Cryo-Techniques. T. Nagata Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 390, Japan.

The techniques of demonstrating diffusible compounds in electron microscopic radioautography consist of two methods, i.e. precipitation fixation and freezing fixation. We have developed cryo-techniques in combination with dry-mounting radioautography at the electron microscopic level during these 30 years. Materials used were cultured cells or tissues obtained from various organs of mouse and rat, such as the liver, pancreas, eye and others which were labelled with 3H-thymidine, 3H-uridine, 3H-leucine, 3H-glucose, 3H-glycerol, 3H-labelled drugs, and various inorganic radionuclides. The cells and tissues were quickly frozen either in iso-pentane cooled with liquid nitrogen at - 1 6 0 ~ or in contact with copper blocks cooled with liquid nitrogen at -196~ After the cryofixation, the following procedures can be divided into 3 categories, i.e., dry cryosectioning with a cryoultramicrotome followed by freeze-drying, freeze-drying and embedding, and freeze-substitution and embedding. The embedded tissues were dry-sectioned without using any water and they were drymounted with Konica NR-H2 emulsions by a wire-loop method using dioctyl sodium sulfosuccinate to prevent bursting while they are air-dried. The dry films were exposed, developed and observed in intermediate high voltage electron microscopes at accelerating voltages of 2 0 0 - 4 0 0 kV. As the results, various kinds of diffusible compounds were demonstrated to localize diffusely in the cytoplasm and nuclei or specifically located over some cell organelles.

S6.2

Cell Kinetic Studies of Various Cell Populations (Normal and Tumor Cells) using 3H-Thymidine Radioautography. B. Maurer-SchuRze and 1. D. Bassukas inst. f. Med. Strahlenkunde, University of Wffrzburg, FRG.

3H-thymidine (3H-TdR) autoradiography is still the main method applied in studies of cell proliferation and mode of growth in normal and tumor cell populations. Out of the various cell kinetic methods using 3H-TdR the determination of the labelling index (LI = number of labelled cells to all cells of a cell population) is most frequently applied. It provides a rough estimate of the proliferative activity of a cell population and is a prognostic indicator in the case of various malignancies. More detailed cell kinetic parameters can be obtained by the percent-labelled-mitoses method, the grain count halving method and the double labelling method with 3H- and ~4C-TdR. The duration of the cell cycle and its phases as well as the mode of growth under normal and perturbed conditions have been studied for a great variety of normal and tumor cell types using these methods. Combined with some mathematical modelling regulation of proliferation and differentiation in

various tissues as well as kinetic compartments throughout cell lineages can be explored. Also the extent of the growth fraction (GF) and the exchange of cells between GF and nonGF can be studied quantitatively by these methods. The mechanism of action of cytotoxic drugs, irradiation and other therapeutic regimens have been studied in a great number of ascites and solid animal tumors. Recently cell kinetic methods have been applied to study cell proliferation of human tumors after transplantation into nude mice. It has been shown that the cell kinetic parameters change after xenotransplantation, particularly the GF of human tumor cell populations increases considerably. This has implications on the conclusions which generally are drawn from the nude mouse model to the conditions in human beings.

$6.3

Receptor Autoradiography Combined with Immunocytochemistry. M. Sar Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill, NC 27599, USA.

A combined technique of autoradiography and immunocytochemistry has been developed in our laboratory to study interaction between steroid hormones and neuropeptides, protein hormones, neurohormones or neurotransmitters in the brain. In this procedure autoradiograms prepared after radiolabelled compound are processed for immunostaining with antibodies to neuropeptides, neurotransmitter synthesizing enzymes (TH, DBH) or receptor proteins. For colocalization of steroid hormone and antibodies to neuropeptide autoradiograms are prepared from the animals treated with colchicine prior to the administration of radiolabelled steroid horrnone. With the application of this technique, dopaminergic neurons of the hypothalamus, nor adrenergic neurons of the lower brain stem, gabanergic neurons of the preoptic area have shown to contain [3HI estradiol in their nuclei. [3HI progestin has also been localized in dopaminergic neurons of the hypothalamus and DBH containing neurons of the nucleus tractus solitarius. Colocalization of [3HI estradol with GHRH, SRIF, B-endorphin, neuropeptide-Y and ANF cells has been observed but not with LHRH neurons. Demonstration of steroid hormone association with its receptor protein in brain and uterus was possible utilizing autoradiogram of [3HI estradiol or [3H] progestin and antibodies to estrogen or progesterone receptor proteins. Furthermore, immunocharacteristic of steroid hormone target cells in rat anterior pituitary has been identified. (Supported by NIH Grant NS-17479).

$6.4 High Resolution Autoradiography in Electron Microscopy. M. A. Williams Dept of Biomedical Science, University of Sheffield, UK.

Invited symposia

468

$7. RECENT TRENDS IN EM CYTOCHEMISTRY

$7.1

Recent Developments and Applications of PostEmbedding Quantitative Immunocytochemistry. M. Bendayan and L. Ghitescu Department of Anatomy, Universit( de Montr(al, Montr(al, Quebec, Canada.

In recent years two alternative immunogold probes have been introduced. The protein G-gold and the protein A/G-gold complexes. These probes are by various aspects superior to the protein A-gold or the IgG-gold complexes because of their broader spectrum of reactivity with immunoglobulins of various mammalian species and particularly with the mouse monoclonal antibodies. They have also been found to be excellent for immunoblot analysis and therefore represent highly versatile and convenient probes providing labellings of high resolution. They were thus applied for quantitative immunocytochemistry in the study of the dynamics of cellular phenomenon such as vascular permeability and endocytotic activities. For this, hapten-tagged serum proteins were injected into the circulation and traced at different time points by post-embedding immunogold techniques. Various serum proteins including albumin, immunoglobulins and transferrin were tagged with dinitrophenol, fluorescein or digoxigenin prior to their introduction into the circulation. They were revealed by the protein A / G gold complex in conjunction with anti-hapten antibodies in various tissues and cells allowing for the study of the dynamics of the transendothelial transport through vascular walls, the glomerular filtration and the renal epithelial endocytotic activity. These studies have demonstrated that the hapten-tagged proteins combined to quantitative high resolution post-embedding immunocytochemistry represent a powerful approach for the morphological study of cellular dynamics under very favourable conditions in terms of resolution and physiological significance. Supported by the Medical Research Council of Canada.

$7.2

Application of Cryofixation, Freeze-Drying and Low Temperature Embedding for Immunocolloidal Gold (IEM) Localization of Sarcoplasmic Reticulum (SR) Proteins in Muscle. A. O. Jorgensen*, A. C.-Y. Shen*, S. A. LittlC and L. J.

McGuffeC *Dept. of Anatomy & Cell Biol., U. of Toronto, Canada and ~Dept. of Pharm., U. of New Mexico, N.M, U.S.A.

To optimize the use of monoclonal and site specific antibodies as probes for immunolocalization of intracellular antigens it is essential to also optimize the maintenance and accessibility of epitopes of the respective antigen. To this end we have previously reported on the use of cryofixation, freeze-drying and low temperature embedding in Lowicryl K4M (Jorgensen & McGuffee, J. Histochem. and Cytochem. 35, 723 (1987)) for IEM localization of SR proteins (Ca~+-ATPase and calsequestrin) and T-tubular proteins [ei-DHPR(Jorgensen et al, J. Cell Biol. 109, 135 (1989) and TS28 (Jorgensen et al., J. Cell Biol. 110, 1173 (1990)] in muscle tissue. To improve the

simultaneous visualization of colloidal gold particles and muscle membranes (SR, T-tubules and sarcolemma) muscle tissues were low temperature embedded in LR White instead of in Lowicryl K4M. Applications of this specimen preparation to the localization of SR proteins including Ca z+release channels (ryanodine receptor and IP3 receptor) will be presented. Supported by MRC of Canada (AOJ), HSFC Ontario (AOJ) and NIH HL 37015 (L.J.M.).

$7.3

Organelle Plasticity in Phagocytic Leukocytes. J. M. Robinson Ohio State University, Columbus, OH, 43210, U.S.A.

Certain organelles within phagocytic leukocytes display remarkable structural plasticity upon activation with appropriate stimuli. Human neutrophils contain a novel compartment which is characterized by the presence of the enzyme alkaline phosphatase (AlkPase). In resting cells this enzyme is present in small membrane-bound compartments within the cytoplasm and to a lesser extent on the cell surface. Upon stimulation with a chemotactic peptide or an active phorbol ester the AlkPase activity is rapidly upregulated to the cell surface. This process is unusual in that the small AlkPasepositive compartments fuse and then form tubular arrays which become continuous with the cell surface. The formation of these tubular structures is sensitive to the microtubuledepolymerizing drug nocodazole. Murine macrophages undergo a number of remarkable structural alterations upon activation with stimuli such as phorbol esters. The lysosomal compartment in these cells display elaborate alterations in structure following phorbol stimulation. These structures rapidly elongate and form elaborate tubular/reticular structures which have a close spatial association with cytoplasmic microtubules. Rapid alterations in macrophage microtubules, which may be important in the regulation of the formation of the tubular/reticular structures, also occurs during this time period.

$7.4

Cytochemistry, Autoradiography, In Situ Nucleic Acid Hybridization: Complementary Tools for Studying the Functional Organization of Cell Nucleus. F. Puvion-Dutilleul and E. Puvion Laboratoire de Biologic et Ultrastrueture du Noyau (UPR272, CNRS) BP n ~ 94801 Villejuif Cedex, France.

A number of new preparative methods for electron microscopy have been developed during the last twenty years. Used in parallel, they provide complementary information and make possible the study of the composition of defined cellular structures at in situ level. Cells infected with adenovirus type 5 (Ad5) are very suitable to study the function-structure relationships since the development of Ad5 induces reproducible nuclear alterations. In order to elucidate the precise location of the progeny AdS genomes in HeLa

Invited symposia nuclei and to determine their sites of transcription and replication, we present the techniques which have already brought conclusive data in the study of adenovirus infection or which could provide new informations in the future, and we examine their advantages and limits. Several methods concomitantly detect both cellular and viral nucleic acids in a section. The osmium ammine complex of Cogliati and Gautier clearly reveals different degrees of compaction of DNA in the nuclear compartments, but does not permit the identification of the various types of Ad5 DNA. The method of high resolution autoradiography provides functional data. A short pulse labelling reveals the

469 sites of nucleic acid synthesis whereas additional chase periods reveal the dynamics of the incorporated radioactive molecules. In situ hybridization which reveals defined nucleic acid sequences is a powerful tool for localizing Ad5 and their specific transcripts in biological material. We use biotinlabelled viral DNA probes and ultrathin sections of Lowicrylembedded infected cells. With appropriate pro-treatments of sections, in situ hybridization provides specific identification of the different types of viral DNA: total viral DNA, or only its single-stranded (ss) portions, or only its double-stranded (ds) portions. Viral RNA molecules can also be specifically detected.

$8. CYTOCHEMISTRY OF N E U R O N A L MESSENGERS

$8.t

Cytochemistry of Neuronal Messengers. T. Hokfelt Department of Histology and Neurobiology, Karolinska Institute, S-104 O1 Stockholm, Sweden.

The complexity of the nervous system including the diversity of neurons and glial cells with an array of processes extending over shorter or longer distances requires detailed morphological analysis at the light and electron microscopic levels. During the last decades a number of powerful histochemical methods have been developed suitable for analysis of the nervous system providing complementary data to earlier light and electron microscopic structural analysis. These methodologies include histochemical methods of various types, for example formaldehyde induced fluorescence, immunohistochemistry and in situ hybridization. This can be combined with retrograde and antrograde tracing methods which allow definition of transmitter specific projections. In the present symposium examples of some of these techniques will be presented encompassing both the most recent developments in tracing techniques and in in situ hybridization allowing the use of both radioactive and none radioactive probes and double labelling experiments. Moreover, it has been recognized that so called immediate early genes can be visualized both with immunohistochemistry and in situ hybridization providing a new tool to map functional circuits in the nervous system.

$8.2 New Cytochemical Approaches to Tracing of Neuronal Pathways. F. G. Woutertood Department of Anatomy, Vrije Universiteit, A msterdam, The Netherlands. Traditionally, neuroanatomy has been dealing with the cytoarchitectonic delineation of nuclear areas in the brain and with the tracing of fiber pathways that connect these areas. The Golgi silver impregnation method was used to study the shape and dendritic geometry of individual neurons. Knowledge of function was based on electrophysiological, and mare recently, on histochemical data. The application of immunohistochemistry has fundamentally changed our approach to brain research as well

as our view of the brain. Areas in the brain are nowadays studied using primarily immunohistochemical markers, whereas the traditional cytoarchitectonic stains are applied only for the comparison of the newly described areas with brain maps determined in the classical way. New tracing methods based on immunocytochemistry have been developed, as well as combinations of tracing methods and immunocytochemistry to determine the neurochemical substances involved in the pathways and the target neurons. Golgi impregnation has been supplanted by intracellular injection in slices of fixed brain. Most powerful are combinations of these methods. Knowledge of the neuron populations, the neuroactive substances that the cells contain, and their connectivity has provided new openings towards the functional interpretation of brain areas and the interconnecting pathways,

$8.3 Functional Neurochemical Mapping Using c-Fos, c-Jun and Other Immediate-Early Gene Markers. S. P. Hunt Molecular Neurobiology Unit, MRC Centre, Addenbrookes Hospital Site, Hills Road, Cambridge CB2 2QH, UK. Immediate-early genes (IEGs) were originally identified as a class of genes which were rapidly and transiently expressed in cells following stimulation by growth factors but which were not themselves dependant upon new protein synthesis for their induction. Many of these IEG products have subsequently been shown to be transcription factors, binding to DNA. The subsequent demonstration of IEG expression within the central nervous system (CNS) led to the suggestion that genes such as c-fos and c-jun could be involved in triggering the molecular changes underlying long term change. At the same time, evidence was accumulating to suggest that, at least in neuronal cell lines, IEG expression could be increased by depolarization of the cell membrane and that the involvement of growth factors or second messengers was by no means essential. This led to the hypothesis that the appearance of, for example, Fos-like proteins could be used as activity markers at the single cell level within the nervous system. However it is now clear that IEG expression within the CNS is not simply a result of neuronal activation but is related to the long term consequences of stimulation. The appearance of IEG mRNA

470

Invited symposia

or protein is dependant upon numerous criteria which vary according to the brain area under study. In the spinal cord, for example, activation of high threshold nociceptive sensory afferents, but not low threshold 'touch' afferents is necessary to activate c-fos and other IEGs in neurons of the dorsal horn. Moreover, this pattern can change and evolve with time and may be causally linked to changes in peptide gene expression which can follow injury. In contrast to these rapid changes in gene expression, recent evidence now suggests a role for c-jun in the regenerative response of neurons, appearing only at around 24 h following axon damage. The analysis of IEG

$9. C Y T O C H E M I S T R Y

expression is therefore emerging as a powerful new tool in our understanding of events following stimulation of the CNS.

$8.4 Molecular Anatomy of Neuronal Interactions by Combined In Situ Hybridization and Immunocytochemistry. B. Bloch Bordeaux, France.

OF PLANTS

$9.1

Cytoskeletal Dynamics in Living Plant Cells. P. Hepler, A. Cleary, U. Meindl, P. Wadsworth, G. Wasteneys and D. Zang Dept. of Biology University of Massachusetts, Amherst, MA, USA.

$9.2

Imaging Calcium Dynamics in Living Plant Cells. N. D. Read Molecular Signalling Group, Institute of Cell and Molecular Biology, Edinburgh University, Edinburgh EH9 3JH, UK.

Living systems have fashioned a complex network of signalling capabilities around Ca 2+. The direct demonstration of signal response coupling via Ca 2+ requires the measurement and localization of changes in cytosolic free Ca 2+ ([Ca2+]~) in living cells. In the last few years we have had considerable experience in imaging [Ca2% dynamics in a wide range of plant and fungal cells. We are presently employing three different technologies towards this end: ratio imaging of cells loaded with fluorescent dyes (Indo-1 and Fura-2); confocal microscopy of cells loaded with fluorescent dyes (Fluo-3 and Calcium Green); and imaging, using a photon counting camera, of luminescence from plants genetically transformed with the aequorin gene. This paper will cover three aspects of our recent work: (1)problems with loading fungal hyphae with fluorescent dyes; (2) confocal imaging of [Ca2% during red light-mediated photomorphogenesis, and the use of caged Ca -'+ and caged InsP3 to dissect apart and define components of the signal transduction pathway; and, (3) imaging [Ca2+]~in whole seedlings subjected to cold shock, touch and wounding. $9.3

Cell Wall Subunit Affinodetection and Topochemistry of the Assembled Composite. B. Vian Laboratoire des Biomembranes et Surfaces Cellulaires Vdgdtales, ENS, 46 rue d'Ulm, 75231 PARIS Cedex 05, France.

Higher plant cell walls are mutifunctional constructions built up from cellulose and an heteromolecular matrix. In the recent years we have focused on the fact that many cell walls have an ordered architecture revealing a helicoidal pattern (1). The paper presents recent data concerning: a) the interactions of wall polymers in heteromolecular fractions from controlled dissociated walls; b) the emerging sites of the polymers in the cell and the spatial distribution of cellulose and of some

matrix polymers in helicoidal walls from both elongating and non elongating cells. Wall subunits were targeted mainly by means of affinity probes bound to fluorescent or gold markers (2). The probes were the enzyme cellobiohydrolase, CBH1 for cellulose, monoclonal antibodies JIM 5 or JIM 7 for homogalacturonan sequences according to their degree of esteriflcation (3), and a polyclonal antibody for targeting hydroxyproline-rich structural glycoproteins (HRGPs). Affinity labelling was complemented by subtractive cytochemistry and by the labelling of the available anionic groups by means of cationic gold particles. On the heteromolecular fractions the available residual charges appeared present along the microfibrils organizing an anionic surfacting coat.Data confirm that co-distribution and a strong association exist between cellulose and some major matrix components, thus strengthening the morphogenetic role of the latter. (1) Roland, J. C., Reis, D., Vian, B. & Roy (1989). Biol. Cell 67, 209 - 220 (2) Vian, B. & Roland, J. C. (1991) . Biol. Cell 71, 4 3 - 5 5 (3) Knox, J. P., Lindstead, P. J., King, J., Cooper, C. & Roberts, K. (t990). Planta 181, 512-521.

$9.4 Morphological Dynamics of the Endoplasmic Reticulum Imaged in Living Plant Cells. H. Quader Zellenlehre, Fakultdt ffir Biologie, University of Heidelberg, Im Neuenheimer Feld 230, D-6900 Heidelberg 1, Germany.

Isolated vesicles of the endoplasmic reticulum (ER) as obtained by cell fractionation procedures, hardly represent the morphological situation in vivo of this continuous, highly anastomosing membrane meshwork. Three morphologically distinct modifications of the ER can be distinguished in so different plant cells as epidermal cells, root hairs, protonema cells of mosses, by fluorescence microscopy employing the vital fluorochrome DiOC6 or video-enhanced differentialinterference-contrast microscopy: two tubular forms, a peripheral network closely associated with the plasma membrane and long tubular strands deeper in the cytoplasm, and flat sheet-like lamellae fitted into or closely associated with the peripheral network. These modifications reversibly change into one another very rapidly depending on the environmental conditions. At the extreme, the ER may occur entirely in tubular form, e.g. at low temperature (<7~ or after acidification of the cytosol, or mainly as large fiat sheets after raising the temperature (>35~ interfering with the cellular calcium distribution, or elevating the cytosolic pH.

Invited s y m p o s i a

471

The tubular ER moves along actin filaments with myosin most likely functioning as linking and motor protein. Protons and calcium seem to play a major regulatory role in ER shaping. Both cations affect the ER morphology either directly by causing the fusion of parts of the tubular ER to flat sheets or

S10, ENZYME

HISTOCHEMISTRY:

IN SITU

the decay of the latter to tubular elements, or indirectly by destabilizing the link between the actin filaments and the ER. The movement of the ER and its 3D-arrangement has been studied in different cell types by eonfocal laser scanning microscopy.

STUDY

$10, I

Enzyme Histochemistry: I n Situ Study of Cell Metabolism. C. J, F. Van Noorden Laboratory of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, The Netherlands. The efforts made in the past two decades to validate quantitative enzyme histochemical methods enable the analysis of kinetic parameters of enzymes in their cellular environment. These parameters can be essentially different from biochemically determined parameters in dilute solutions with protein concentrations of 0.1% or less. Protein concentrations in mammalian cells are usually 15-25~ A particular enzyme of interest may be diluted in the cytoplasm but a large variety of other macromolecules take up a substantial fraction of the total cytoplasmic volume. Enzymes perform their biological task in the crowded cytoplasm in a different way than in a dilute solution. Moreover, many enzymes can be found either in a soluble form or associated with membranes or the cytoskeleton as part of the regulation of their activity. Quantitative histochemical methods for the demonstration of catalytic activities of enzymes in unfixed tissue sections or cells allow the study of these posttranslational regulation mechanisms. For glucose-6phosphatase it was found that both KM and Vma~values were significantly different in periportal and perieentral zones of rat liver. In fact, KM values were found to increase linearly with V .... values, irrespective of sex and feeding condition, showing that the enzyme exists in a high affinity configuration at low enzyme concentrations. At high enzyme concentrations the enzyme transforms to a low affinity configuration by a hysteretic mechanism (Jonges et al., J. Biol. Chem. 267, 1991: 4878-4881). A similar kinetic behaviour was also described for Ca2+-ATPase in rat muscle (Blanco and Sieck, Histochem. J. 24, 1992: in press). These findings clearly indicate that the in situ analysis of cellular metabolism reveals a different kinetic behaviour of enzymes than in dilute solutions and may be a more reliable method to study the metabolism as it occurs in vivo.

St0.2 In Situ Measurements of Enzyme Activities in the Brain. P. Kug/er Dept. of Anatomy, University of Wr~rzburg, Wurzburg, FRG. Catalytic enzyme histochemistry applied to brain sections allows both localization and quantification of enzyme activities (Kugler, P., Int. J. Biochem. 23: 6 5 7 - 6 6 1 , 1991). This study refers to the histochemistry of dehydrogenases in

OF CELL

METABOLISM

the rat hippocampus which is a well defined brain area. We have studied three mitochondrial enzymes involved in glutamate and GABA metabolism, i.e. NAD-linked isocitrate dehydrogenase (ICDH), glutamate dehydrogenase (GDH) and GABA transaminase (GABAT). It was shown that 1) the use of appropriate tetrazolium salts in combination with tissue stabilizers, exogenous electron carriers and inhibitors of the respiratory chain offered satisfactory conditions for the qualitative and quantitative evaluation of dehydrogenase activities; 2 ) G D H activity was restricted to astrocytes, whereas ICDH and GABAT seemed to be localized in both neurons and astrocytes; 3 ) i n spite of different levels of activities and cellular localizations ICDH, GDH and GABAT were significantly correlated in their hippocampal distribution. It is concluded that the enzymes demonstrated might be involved in a layer-specific cooperation to produce or catabolize glutamate and GABA, and thus to control aminoacidergic transmission. - - This study was supported by the Deutsche Forschungsgemeinschaft. S10.3

Measurements of Local Substrate Concentrations in Tissue Sections using a Bioluminescence Technique. S. Wa[enta and W. Mueller-Klieser Institute of Physiology and Pathophysiology, University of Mainz, Duesbergweg 6, D-6500 Mainz, FRG. The metabolic status of malignant tumors are characterized by a pronounced spatial variation in tissue oxygenation, in pH and in the fraction of proliferating cells (Vaupel et al., Cancer Res. 49: 6449, 1989). The resulting heterogeneous metabolic micromilieu represents a crucial problem in non-surgical cancer therapy. To further describe the metabolic status of such tissues, a bioluminescence technique was developed allowing for the detection of local ATP, glucose, and lactate concentrations in consecutive cryosections. Cryosections were made from rapidly frozen tumors, picked up on cover glasses and were heat inactivated for t0 minutes at 100~ For measurement, a solution containing all necessary enzymes and cofaetors for substrata specific luminescence reaction is frozen in a mould and covered with the tissue section. After controlled thawing the enzyme reaction starts, and the luminescence is registered with high spatial resolution by an imaging photon counting system (Argus 100, Hamamatsu Europe, Herrsching, FRG) connected to a microscope (Axiophot, Zeiss, Oberkochen, FRG). The resulting intensity distributions are transferred into concentration values using appropriate tissue standards with known substrata contents. The evaluation of local concentrations in viable and necrotic tissue areas is performed by comparision with parallel sections stained with H.E. using a specially designed computer software. (Supported by the BMFT 01 ZO 8801)

472

Invited symposia

s10.4 Immunocytochemical Detection of Cathepsins and Cystatins at LM and EM Level to Elucidate their Physiological Functions. N. Katunuma Tokushima, Japan.

S l l . H I S T O C H E M I S T R Y OF R E C E P T O R S Sll.1

Histochemistry of Receptors: An Up-Date on Ligand Binding Autoradiography. J. M. Palacios Research & Development Department, Laboratorios Almirall, Cardener, 68-74, 08024 Barcelona, Spain.

Receptor autoradiography (RA) visualizes, at the regional or light microscopic level, radiolabelled receptor binding sites. Like its parent technique, high affinity binding analysis, receptor autoradiography is well established as an essential widely used research methodology. When used in combination with image analysis, RA provides spatial mapping of receptor localization at moderately high resolutions, in combination with full quantitation of ligand affinity and/or receptor densities. RA suffers from a number of limitations. A major problem is discrimination of sites of origin for receptors. Demonstration that a given receptor originates in a specific cell population could only be made indirectly. Another limitation of RA is not inherent to the technique itself, but arises from the lack of selectivity of many ligands. Ligands previously considered as selective for a receptor population do, in fact, bind to several subtypes of the same receptor class. Advances in the understanding of the molecular biology of receptors, such as the cloning of the genes coding for a large and ever-increasing number of receptor molecules have produced developments in receptor analysis. Knowledge of the sequence of the mRNA coding for receptor proteins has made possible the development of nucleic acid probes. These can be used in combination with the technique of in situ hybridization to visualize perikarya that express the mRNA for a particular receptor with unprecedented selectivity, allowing direct demonstration of sites of active synthesis of receptor proteins. Combination of in situ hybridization and receptor autoradiography with highly selective liagands provides precision in characterizing receptor populations. Sll.2

Molecular Neuroanatomy of Receptors and Enzymes - - Targets for Psychoactive Drugs. G. Richards Pharma Division, Preclinical Research, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland.

In order to provide a more rational basis for developing psychoactive drugs, an in-depth knowledge of the structure, cellular expression and regulation of their molecular targets is essential, particularly considering the anatomical complexity of the CNS. In neuroscience research the application of quantitative receptor/enzyme radioautography with image analysis and in situ hybridization histochemistry - - high

resolution protein and transcript mapping techniques, respectively - - has been instrumental in this regard. To exemplify this research, the characteristics of two drug targets in the CNS - - GABAA receptors and monoamine oxidases - will be described. They are the recognition sites of future generation drugs for the treatment of anxiety, epilepsies and sleep disturbances on the one band, and depression, Parkinson's disease and Alzheimer's disease on the other. GABAA receptors in the CNS - - the targets for the therapeutic effects of benzodiazepines - - are transmembrane heterooligomeric (pentameric?) glycoproteins which probably exist in structurally and functionally diverse forms. The 5 polypeptide subunits (a,/3,y,6,p) and a total of 15 variants, which have been cloned to date, have a distinct pattern of mRNA expression in the CNS, creating new opportunities for drug development - - namely their targeting to specific subpopulations of GABAA receptors. Monoamine oxidases (MAO-A and MAO-B) - - isoenzymes identified by substrate selectivity, inhibitor sensitivity and primary structure - - oxidatively deaminate neurotransmitterand xenobiotic amines in the CNS and periphery. In human brain, serotonin and noradrenaline are preferentially metabolized by MAO-A, the trace amines phenethylamine and methylhistamine by MAO-B but dopamine, tyramine and octopamine by both enzymes. Paradoxically, MAO-A is expressed in noradrenergic neurons but MAO-B in serotoninergic and histaminergic neurons as well as in glia.The physiological role of MAOs - - in transmitter inactivation - - as well as their pathophysiological roles - - in neurodegenerative diseases - - provide drug targets for selective, reversible inhibitors with unique and even unexpected therapeutic profiles.

Sll.3 Mapping of Receptors for Catecholamines and Peptides. R. Elde, A. P. Nicholas, V. A. Pieribone and T. H6kfelt Department of Histology & Neurobiology, Karolinska Institutet, S-104 O1 Stockholm, Sweden. Several receptors have been recently cloned, including adrenergic and peptide receptors, the latter providing further evidence for a functional role of peptides in the nervous system. We have synthesized oligonucleotide probes against a l b , a2b (brain and kidney) /31 and /32 adrenoreceptor mRNAs, as well as to the neurotensin and substance P receptors. Both central nervous system tissue and peripheral organs have been analysed. In the brain, differential distribution patterns were observed. For example, a l b mRNA was seen in cerebral cortex, thalamus, dorsal and medullary raphe, as well as in liver, heart and kidney. Brain a2b mRNA was found in the hippocampus, spinal grey matter, striatum, cerebral and cerebellar cortices, as well as in

Invited s y m p o s i a

473

s3nnpathetic and dorsal root ganglia and in the adrenal medulla. fll mRNA was seen in cerebral cortex and heart, while/32 mRNA was in hippocampus, trachea and lung. The kidney a2b probe labelled kidney, but not brain. Neurotensin mRNA was observed in several brain regions with particularly high levels in the doparnine cells of the substantia nigra and in the cholinergic forebrain cells. Substance P receptor mRNA had a wide distribution in brain and spinal cord, for example in the caudate nucleus and in the medulla oblongata. These results provide

S12. NEUROHISTOCHEMISTRY

complementary information to earlier binding studies and open up possibilities to study regulation of receptor synthesis.

Sll.4 Sorting of Glucose Transferase in Mammalian Cells. Jo W. Slot Dept of Cell Biology, ]~4edical School, University of Utrect, The Netherlands.

OF DEVELOPMENT

5t2.1

Spatio-Tempora| Patterns of Hormone and Hormone Transcription Factor Gene Expression During the Development and Mature Function of the Neuroendocdne System. L. W. Swanson Department of Biological Sciences, Hedco Neuroscience Building, me 2520, University of Southern California, Los Angeles, California, 90089-2520, USA.

The core of the neuroendocrine system consists of the hypothalamus and pituitary, and the mechanisms that interrelate these two organs. We have used this system as a model for studying the spatio-temporal patterns of gone expression that may be involved in establishing a functional system during the development of the rat embryo, as well as in the modulation of gone expression during different functional states of the adult organism. In the embryo, we have examined the spatio-temporal pattern of expression for the various genes that are involved in the synthesis of the classical anterior pituitary hormones and it was found that there is an interesting compartmental pattern of expression, which is also characterized by distinct temporal patterns of expression. In addition, we have correlated these patterns with the pattern of expression of two putative transcription factors that are thought to be involved in regulating the expression of growth hormone, thyrotropin releasing hormone, and prolactin. In the adult rat, it is now becoming clear that individual hypothalamic neurosecretory neurons can not only synthesize multiple neuropeptides, but that the genes regulating the synthesis of these neuropeptides may be differentially regulated by different physiological and behavioral factors that fall under the broad category of stress. 512.2

Molecular Neuroanatomical Indications for Gene Repair in Post-Mitotic Vasopressin Neurons of Brattleboro Rats. F. W. Van Leeuwen Netherlands Institute for Brain Research, Amsterdam, The Netherlands. The homozygous Brattleboro rat (di/di) is a mutant strain of the Long Evans rat which exhibits a recessively inherited hypothalamic diabetes insipidus. The frame-shift mutation is due to a single base deletion in exon B of the vasopressin (VP) gone and results in a different C-terminus of the VPprecursor. This altered VP precursor cannot be translocated at the endoplasmic reticulum since it remains anchored in the

AND AGING

membranes of this organetle (Schmale etal., Eur. J. Biochem. 182, 625, 1989). This precursor therefore never reaches the Golgi apparatus and does not undergo axonaI transport from the magnocetlular hypothalamic nuclei to the terminals in the neural lobe. An intriguing finding was that a small but linearly increasing number of solitary hypothalamic neurons (up to 3%) of the di/di rat undergoes a switch to a heterozygous phenotype. In addition to the mutant VP precursor they express the wild-type VP precursor, which is axonally transported (Van Leeuwen et al., PNAS 86, 6417, 1989). The molecular event generating this somatic reversion occurring in neurons after termination of their proliferation (foetal day 15) is presently being worked out. Preliminary data (Evans et al., Soc. Neurosci. 17, 380, 1991) indicate that the revertant phenotype is due to single base insertions in the region of the deletion in exon B. It has been reported in the ostrich that at the very place of the G deletion even an amino acid insertion occurs indicating that this area can represent a potential "hotspot recombinational" area similar to the rosy locus of drosophila rnelanogaster and the gene responsible for Duchenne muscular dystrophy. In conclusion, the di/di rat model has shown that alterations at the level of DNA of postmitotic neurons are possible.

512.3

Axonai RNA in the Hypothalamo-neurohypophyseal System: Structures and Functional Implications. G. 1=:.Jirikowski Dept. of Neuropharmacology, Scripps Res. Inst., La Jolla, USA. mRNAs coding for oxytocin (OT) or vasopressin (VP) have been detected in axonat secretory vesicles in the rat hypothalamoneurohypophysial system. Magnocellular hypothalamic neurons of Brattleboro rats are capable of accumulating, transporting and translating exogenous VP RNA, thus leading to a temporary correction of diabetes insipidus. Poly A ( - ) RNA proved to be much more effective than poly A( + ) RNA. In further studies we used primary cultures of dissociated fetal rat hypothalamus !n order to investigate the fate of radiolabelled VP RNA added to the culture medium. Autoradiograms revealed that a portion of neurons in vitro accumulates RNA in processes and perikarya. This group of cells includes the VP immunoreactive perikarya but is not limited to this ceil type. We could further demonstrate in such experiments that depolarization of cells with 50mM KC1 results in increased amounts of radioactivity in the culture medium, indicating a stimulus dependent release of RNA. This effect was in part Ca + + dependent. Synthetic Arg-VP

474

Invited symposia

also stimulated release of incorporated exogenous VP RNA. Our data indicate that hypothalamic neurons can accumulate, transport and translate exogenous RNA and that RNA can also be secreted in a stimulus-dependent manner. It is possible that exchange of RNA occurs in the hypothalamus under physiological conditions thus representing a novel way of interneuronal signalling.

S12.4 Histochemistry of the Human Hypothalamus in Development and Aging. D. F. Swaab Netherlands Institute for Brain Research, Amsterdam, The Netherlands.

S13. I M M U N O H I S T O C H E M I S T R Y : N E W S O L U T I O N S FOR O L D P R O B L E M S S13.1

Modern Microscopical Imaging Methods. J. M. Polak and A. E. Bishop Department of Histochemistry, Royal Postgraduate Medical School, DuCane Road, London W12 ONN, UK.

The huge advances made in the understanding of neuroendocrine and endothelial cell biology by the advent of immunocytochemistry are being built upon with the development of new investigative morphological methods. Immunocytochemical techniques have been refined and are now applied, at the light and electron microscopical levels, in the study of not only bioactive products but also their precursor or abnormal molecular forms. Gene expression can be examined in histological preparations by in situ hybridization. The results of this technique, viewed together with data from immunocytochemistry of stored products, can reveal not only sites of expression of specific nucleic acid sequences but also the rates of product synthesis. In vitro autoradiography can be used to localize, characterize and quantify binding sites on the surface of neuroendocrine cells, and if the nucleotide sequence has been disclosed the mRNA can also be localised at the cellular level. In combination, these methods provide the investigator with the means not only to identify neuroendocrine and endothelial cells but also to derive morphofunctional information on their biology.

S13.2 Immunocytochemistry in Combination with other Techniques. G. V. Childs Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, TX 77555-0843, USA.

For several decades immunolabelling has been a valuable tool for identifying sites of synthesis, storage or binding of antigens. As multiple labelling techniques were improved, one could determine sites of multiple antigens in the same tissue sections or cell population. However, with immunolabelling alone, one could not distinguish functionally different sites,

S14. C Y T O C H E M I C A L

MARKERS

such as sites of binding from sites of storage. Thus, target cells might be mislabelled as source cells. During the past decade, the sensitive technology developed by immunocytochemists has been adapted to allow the detection of probes other than antibodies. These probes include biologically active ligands that detect target cells, or complimentary DNA, RNA, or oligonucleotide probes to detect mRNA and sites of synthesis. With basic dual detection systems the researcher can now detect expression of multiple functions in a given cell or tissue section. The first part of the presentation illustrates how affinity cytochemistry combined with immunocytochemistry is used to study physiological changes in pituitary target cells for releasing hormones. The second part of the presentation will show how in situ hybridization can be combined with immunolabelling to identify the source of a regulatory factor in the pituitary that inhibits the release of follicle stimulating hormone (follistatin). Dual in situ hybridization and immunolabelling labelling studies can also be used to detect and characterize multipotential pituitary cells. Studies of plasticity in the pituitary cell population during the estrous cycle show that some of these augment the gonadotropes just before the midcycle surge. Supported by NIH R01 HD 15472, NIH R01 DK 39553; NSF IBN-9117897. S13.3

Quantitative Immunocytochemistry. T. Cowen and T. Andrews Department of Anatomy and Developmental Biology, Royal Free Hospital School of Medicine, London UK.

s13.4 Recombinant Monoclonal Antibodies in an In Vitro Immune System. H. Hoogenboom Cambridge Centre for Protein Engineering, Hills Road, Cambridge CB2 2QH, UK.

AS DIAGNOSTIC

S14.1 Problems and Possible Solutions using Cytochemieal Markers in Toxicology. G. R. Bullock K125.10.16, Ciba-Geigy A G, CH-4OO2-Basel, Switzerland.

The question often arising in toxicology is how to establish

TOOLS

IN TOXICOLOGY

early predictors for potential toxicological changes. Previously much reliance was placed on gross a)morphological changes such as alterations in structure, deposition of abnormal material e.g. amyloid and b) histochemical changes such as up/down-regulation of enzymes. In addition, cell proliferation leading to benign/malign tumours was and is of paramount importance or alternatively cell death as in

Invited symposia

475

irreversible neuronal toxicity. All of this, however, is classical toxicology as we know it. Now there exists many opportunities to incorporate the newer methodology deriving from molecular biology and allied techniques. Whilst more knowledge has been gained re abnormal cell constituents such as the appearance of Glial Fibrillary Acidic Protein (GFAP) in the brain, greater emphasis should be placed on more subtle changes such as induction of c-fos protein following treatment with neurotoxic compounds or cardiotoxicity following DNA intercalation by adriamycin. Predictors for cell proliferation (cyclins), cell differentiation, breaks in nucleic acids and mitotic aberrations (by 'in situ' hybridization) and sensitive cellular changes such as upregulation of growth factors are all now within the remit of the toxicologist. These developments will be discussed in particular with regard to cardiovascular and brain toxicology and the potential for reducing the drug test period.

S15. A D V A N C E D

MICROSCOPICAL

Imaging of Cells and Tissues by Confocal Microscopy. B. A m o s .MRC Laboratory o f Molecular Biology, Hills Rd, Cambridge, CB2 2QH, UK.

S15.2

Time Resolved Microscopy. H. Tanke, N. P. Verwoerd, J. Bonnet, C. R. G. van de Geest and E. J. Hennink Department of Cytochemistry and Cytometry, Leiden University, The Netherlands.

2-Photon Imaging in Biological Microscopy. W. Webb Ythaea, USA.

P. 1t. Bach Faculty of Science Polytechnic of East London, Romford Rd, London E15 LZ4, UK.

S14.3 The Use of Isolated Cells in Toxicity Testing. A. J. Guillouzo Hdpital de Pontchaillou, 35033 Rennes, France.

S14.4 The Use of Bone Proteins as Cytochemical Markers of Bone Metabolism. P. Clezardin Lyon, France.

TECHNIQUES

$15.1

$I5o3

s14.2 Application of Fluorescent Probes and Confocal Scanning Laser Microscopy Data for Toxicology.

S15.4 Atomic Force Microscopy for High Resolution Imaging of Biological Surfaces. J. H. Hob and P. K. Hansma Department of Physics, University of California Santa Barbara, Santa Barbara, California 93106, USA. The Scanning Tunneling Microscope started a revolution in microscopy that has led to the emergence of a family of Scanning Probe Microscopes. These instruments have in common the feature of passing a probe in close proximity to a surface, and collecting spatially resolved information about properties such as tunneling current, physical topography, ion conductance or temperature. One of the most promising members of this family of instruments for biological applications is the Atomic Force Microscope (AFM, also known as the Scanning Force Microscope, SFM). The AFM has now been used to image samples ranging from whole cells to single ion channels. On hard flat samples this microscope can often resolve atoms, though on soft biological materials the lateral resolution is currently in the range 1-50 nm. Its ability to image in fluids, including physiological buffers, and to gather dynamic data point to a bright future in biology. In addition to imaging, the AFM also can be used for experimental manipulation of membranes and molecules, and can measure a variety of local physical and biophysical surface properties. Future technological developments, particularly in the area of tip structure and composition, will improve resolution and extend the types of local properties that can be examined.

476

Minisymposia

MINISYMPOSIA

M16. APOPTOSIS M16.1 Ultrastructural Localisation of BCL-2 and the Epstein-Barr Virus Protein BHRF-1 to the Outer Mitochondrial Membrane. P. Monaghan, D. Robertson and T. F. Hickish Institute of Cancer Research, Haddow Laboratories, 15, Cotswold Road, Sutton, Surrey SM2 5NG, England. The BCL-2 gene was first recognised in follicular B-cell lymphomas with the t(14; 18) translocation. This translocation brings the BCL-2 gene to the immunoglobulin heavy chain locus, leading to de-regulation and overexpression of the mRNA and protein. The gene codes for a 26KDa protein of unknown function. Light microscope immunolocalisation of BCL-2 has demonstrated the protein in a variety of normal tissues cells which are characterised by apoptic cell death suggesting that the BCL-2 protein protects cells from apoptosis. Because of the uncertain function of the BCL-2 protein it is important to determine its subcellular location. Previous cell fractionation studies have proposed an inner mitochondrial membrane location for BCL-2. We have determined the locatisation of the BCL-2 protein in lymphoma and breast tumour cell lines and biopsies by cryo-sectioning, low temperature embedding and freeze substitution. In all cases BCL-2 was predominantly associated with the outer mitochondrial membrane (approx. 50% of label). A closely related protein BHRF1 with a 40% homology to the BCL-2 gene, but unknown function also localises to the outer mitochondrial membrane.

M16.2 A New Method to Detect Apoptosis in Paraffin Sections: In $itu End-Labelling of Fragmented DNA. J. Wijsman, R. Jonker, C. J. Cornelisse and J. H. van. Dierendonck Departments of Surgery and Pathology, University of Leiden and TNO Radiobiological Institute (RJ), Rijswijk, The Netherlands. A new staining technique to facilitate the detection of apoptosis in formalin-fixed paraffin embedded tissue sections is described. As extensive DNA fragmentation is one of the characteristics of apoptosis, these breaks can be visualized by an in situ endlabelling technique (ISEL). After protease treatment to permeate the tissue sections, biotinylated nucleotides are in situ incorporated in DNA breaks by polymerase and subsequently visualized by DAB staining via peroxidase-conjugated avidin. Staining of cells with the morphological characteristics of apoptosis was demonstrated in prostate and uterus after castration, tumors, lymph node follicles and embryos. Apoptotic cells could be discriminated from areas of labelled necrotic cells, in which DNA degradation occurs as well. We used H&E stained sections of involuting prostates of castrated rats to validate the ISEL method for the quantification of apoptotic cells. A high correlation was found between the fractions of ISEL labelled cells and the fractions of apoptotic cells morphologically determined in adjacent H&E stained sections.

M16.3 Endonuclease Activation in Apoptotic Cells. V. G. Evans and I. D. Bowen School of Pure and Applied Biology, University of Wales College of Cardiff, UK. The classic diagnostic feature of cells undergoing apoptosis or programmed cell death is the appearance of DNA fragments, cut to multiples of 200bp by a Ca z+ and Mg 2+ dependent endonuclease. A cytochemical method was used to check results which showed that this endonuclease had been activated independently of apoptosis-inducing treatment in murine thymocytes. Samples, air-dried on microscope slides, were fixed in Bouin's fixative and then put through the Feulgen reaction. Apoptotic cells show dark purple stained condensed chromatin and are easily distinguishable from normal or necrotic cells. Results of the cell counts showed the expected levels of apoptosis, depending on treatment. Endonuclease activation, visualized as DNA laddering on agarose gels, occurred in the identical samples regardless of treatment. Rapid endonuclease activation has been reported in the literature, as has the induction of apoptosis by various stress factors. Here it is proposed that endonuclease activation can occur independently from the genetically governed programmed cell death as a response of cells which are susceptible to the induction of apoptosis and which are undergoing fast-acting, irreversible damage. Therefore, endonuclease activation should be regarded as indicative of apoptosis only in conjunction with other morphological or biochemical features of apoptotic cells.

M16.4 Cell Death and Growth Arrest: Integral Components of Tissue Homeostasis. P. A. Hall, P. J. Coates, B. Ansari, J. Kearsey, J. Whitaker and N. Sawhney Dept of Histopathology, UMDS, St Thomas's Campus, London, UK. That the regulation of populations of cells in normal tissues involves control of cell proliferation and differentiation has long been realised. In addition, it has been well documented that pathological processes may be associated with abnormalities of these processes. There is now considerable information concerning the mechanisms of cell proliferation and differentiation, but these are only one facet of the mechanisms that regulate tissue homeostasis. We argue that considerable attention should be placed on two other primary controls of cell number and function in tissues: namely programmed cell death and growth arrest. Recently molecular biological methods have led to the identification of genes involved in these processes and the generation of antibodies that recognise the products of these genes. For example, recent data points to a role for genes such as bcl-2, myc and p53 in programmed cell death. In addition two novel cDNAs (RP-2 & RP-8) have been described that specifically expressed by thymocytes undergoing programmed cell death and a number

Minisymposia

477

of genes are expressed in growth arrest (eg. TI-I, prohibitin, gas & gadd). We have been defining the distribution of expression of such genes in normal and pathological tissues, as well as in vitro model systems, using molecular methods including in situ hybridisation. In addition, we have been generating novel antibodies raised against genetically engineered recombinant proteins. Of considerable interest is the observation that the in vivo or in vitro treatment of cells with chemotherapy, radiation or heat shock can induce programmed cell death and the expression of growth arrest genes.

radiation-induced apoptosis. Radiation induced apoptosis could almost completely be blocked in IL-3 dependent cell lines after addition of IL-3. Double labelling of a cell surface marker and the DNA strand breaks allowed for further analysis of subpopulations of cells. This method proved valuable in the fields of AIDS and of hyperthermia research.

Ml6.5 Detection of Apoptosis with In Situ Nick Translation.

M. G. Ormerod, X.-M. Sun, R. T. Snowden and G. M. Cohen MRC Toxicology Unit, Woodmansterne Road, Carshalton, Surrey SM5 4EF, England.

R. R. Jonker, J. G. J, Bauman, J. H. Wijsman* and J. W. M. Visser Institute of Applied Radiobiology and Immunology, TNO Rijswijk, and *Dept. of Surgery, Leiden University, The Netherlands. A technique was developed for the detection and quantification of apoptosis in individual cells using In Situ Nick Translation (ISNT). The technique is based on the detection of DNA degradation, caused by activation of endogenous endonucleases. This degradation is an early and specific characteristic of apoptosis. DNA breaks could readily be detected by enzymatic incorporation of labelled nucleotides, starting at DNA strand breaks. Biotin- or digoxigenin labelled nucleotides were stained and flow cytometry was used for quantification. The fluorescence of cells in apoptosis was up to 100 fold stronger than in control cells. ISNT also allowed for easy and objective detection of apoptosis in paraffin-embedded material by microscopy. The method was used to detect apoptosis in haemopoietic cells and cell lines after irradiation. The effect of radiation on the induction of apoptosis could be detected down to 0.25 Gy ),-irradiation in subpopulations of bone marrow cells and thymocytes. We showed that growth factors can inhibit

M16.6 Characterisation and Quantification of Apoptosis by F l o w Cytometry,

Conventional methods for detecting apoptotic cells (light and electron microscopy, DNA gel electrophoresis) do not tend themselves to quantification. Flow cytometric methods have the advantage that measurements are made on large numbers of individual ceils yielding good quantitative data. Cells can also be sorted for further morphological or biochemical analysis. Several authors have reported that, in a DNA histogram, apoptotic cells give a ' s u b - G l ' peak. This method is limited in its use because it uses fixed cells. In an alternative method, unfixed cells are incubated with the bis-benzimidazole, Hoechst 33342; the apoptotic cells take up this dye more rapidly. Non-viable ceils can be enumerated at the same time by addition of propidium iodide. We have used this method to examine the effects of various inhibitors on the induction of apoptosis in an IL-3 dependent murine cell line after withdrawal of IL-3 and in rat thymocytes treated with glucocorticoids or DNA topoisomerase poisons. The basis of the assay lies in a change in the properties of the membranes of apoptotic cells. These changes have been further characterised in rat thymocytes using a series of derivatives of fluorescein diacetate.

M17. E N Z Y M E H I S T O C H E M I S T R Y M17.t

Microfluorometric Kinetic Analysis of Cathepsin B Reaction in Single Human Thyrocytes Using an Image Analysis System. L. Kayser and P. E. Hoyer Institute of Medical Anatomy, Dept A, The Panum Institute, University of Copenhagen, Denmark. Dept of Medicine E, Frederiksberg Hospital, Frederiksberg, Denmark. The activity of the cysteine proteinase Cathepsin B has been assessed in unfixed single human thyroid epithelial cells using an image analysis system (Image-l), The reaction was continuously monitored at room temperature by measuring the increase in fiuorescense intensity due to formation of a Schiff-base product formed by using N-CBZ-ala-arg-arg-4methoxy-2-naphthylamide as substrate and 5-nitrosalicylaldehyde as coupling agent (Van Noorden et al., Histochem J 1987; 19: 483). Contribution from non-specific fluorescent signals was eliminated by adjusting the video signal to zero using leupeptin - - a specific inhibitor of cathepsins B, H and L. A plot of fluorescense intensity versus time showed a lag

period of 5 ( 4 - 8 ) rain, followed by a linear increase for 5 ( 3 - 8) rain. Then at the same time as crystallization began to occur in the cytoplasm, a marked decrease in reaction rate was seen for 3 (1 - 4 ) rain followed by a new linear increase for 11 ( 8 - 14) rain. The second linear part of the curve was not as steep as the first one. In conclusion, microfluorometry can be used for kinetic analysis of Cathepsin B activity in single cells. The reaction demonstrates a biphasic pattern which may be due to a change in activity after crystallization of the final fluorescent reaction product began. M17.2

Analysis of Cathepsin B Activity in Human Pancreatic Cancer Cells with Confocal Scanning Laser Microscopy. C. J. F. van Noorden ~, I. M. C. Vogels ~, G. N. Jonges ~ and J. van Marle 2 ~Laboratory of Cell Biology and Histology, and 2Department of Electron Microscopy, Academic Medical Centre, University of Amsterdam, The Netherlands.

478 Many different proteolytic enzymes may play a role in various aspects of tumour growth and metastasis. Cathepsin B, a proteinase of the cysteine class, has been implicated in the ability of tumour cells to degrade the laminin component of the basement membrane and extracellular matrix compounds, which facilitates tumour cell invasion. Cathepsin B activity has been detected in the lysosomes of normal cells. In cancer cells, the enzyme is probably processed in a different way and its presence has not only been shown in the lysosomes, but also on the plasma membrane and extracellularly. In the present study, cathepsin B activity has been investigated in xenografts of a poorly differentiated human pancreatic tumour cell line grown in nude mice. The catalytic activity of the enzyme was analyzed with a histochemical fluorescence method instead of determining the mere presence of enzyme molecules by immunohistochemical means because large quantities of the proteinase may occur as inactive proenzymes or as enzymeinhibitor complexes. The fluorescence enzyme assay has been performed on unfixed cryostat sections using 5-nitrosalicylaldehyde as coupling agent. The formation of fluorescent final reaction product was monitored continuously during incubation using confocal scanning laser microscopy. Cathepsin B activity was found to be localized in lysosomes and at the plasma membrane of the pancreatic tumour cells. A high activity was not only determined in the border area between viable tumour cells and necrotic areas within the tumour but also in the connective tissue surrounding the tumour tissue. The activity could be monitored during incubation in each of the subcellular compartments of the tumour cells. It appeared that confocal scanning laser microscopy enables kinetic analysis of enzyme reactions in situ with a high 3D resolution, which is impossible to obtain in actually cut thin sections (0.5 - 1/am) due to the limited amounts of enzyme present in such thin sections.

M17.3

Probes for Detecting Enzymatic Activity in Single Cells. R. P. Haugland, R. Haugland, Z. Huang, K. Larison, M. N. Malekzadeh, P. Millard, J. Naleway, N. Olsen, V. Paragas, D. Robinhold, V. Singer, S. Wells, W. You and Y. Z. Zhang Molecular Probes Inc., Eugene, OR 97405, USA. We have developed several approaches for the fluorescent histochemical detection of enzymatic activity in single cells using fluorogenic substrates. In some cases we have accomplished this in living cells. The challenge has been to improve retention of the fluorescent product in the cell as well as to facilitate loading of the substrate into the cell. At least four approaches have been used: l)precipitation of an insoluble fluorescent product, 2)formation of a lipophilic product that accumulates in the cell membranes, 3) use of a fluorogenic substrate that is also glutathi0ne-reactive to yield a product with good retention in the cytoplasm of living cells and 4) formation of a fluorescent product that accumulates in intracellular organelles or binds to other cellular components. Among the enzymes that have been successfully detected by at least one of these approaches are /~-galactosidase and /3-glucuronidase (both intrinsic and as the result of gene fusions), a-fucsoidase, fl-glucosidase, hexoasaminidase, neuramidase, aryl sulfatase, alkaline and acid phosphatase,

Minisymposia various peptidases, and peroxidases. We have also demonstrated the potential of the substrates that yield fluorescent precipitates for significantly improving the detection levels in assays such as in situ hybridization. This research has been supported in part by N.I.H grant GM 38987 to R.P.H.

M17.4 Advances with Secretory Proteinases in Rat Submandibular Glands. J. R. Garrett, D. K. Shori, G. B. Proctor and K. M. Chan Oral Pathology, K.C.M.D.S., The Rayne Institute, London SE5 9NU, England.

Numerous kaUikrein-like proteinases occur in the granular tubules of rat submandibular glands. Differences in histochemical staining of the tubules using D-Val-Leu-Arg MNA (extensive and uniform) and Z-Arg MNA (irregular and patchy) suggested that the proteinases may be identifiable with different peptide substrates. The constituent proteinases in glandular homogenates or saliva can now be identified after IEF by the development of fluorescent bands in overlay membranes impregnated with fluorogenic substrates. The underlying proteinases can then be excised and eluted for further biochemical testing. We have developed methods whereby tissue kallikrein can be identified catalytically by the use of D-Val-LeuArg-AFC in the presence of SBTI and tonin by means of Z-ValLys-Lys-Arg AFC in the presence of aprotinin. Ratios of the different proteinases in sympathetic saliva have been found to be the same as in the glands but to differ from those in parasympathetic saliva, which contains a greater percentage of more glyeosylated tissue kallikrein, suggesting that it is synthesised and secreted constitutively during parasympathetic stimulation. Assessment after degranulation indicates that tissue kallikrein is resynthesised more rapidly than the other proteinases. Sexual dimorphism between secretory proteinases has been identified histochemically and biochemically, which supports the belief that hormones influence the individual synthetic pathways differently. Streptozotocin-induced diabetes reduced synthesis of all proteinases; insulin, however, evoked a more rapid recovery of tissue kallikrein than of the other proteinases, indicating that insulin exerts a more direct influence on kallikrein synthesis. Supported by a Wellcome Project Grant and K.M.R.T.

M17.5

A Novel Fluorescent Method for Detection of Subpicogram Quantities of Nucleic Acids. S. H. Hori ~, N. Kagiyama 1"2, S. Fujita z, M. Momiyama 2 and Y. Kondoh 2 ~Department of Zoology, Faculty of Science, Hokkaido University, Sapporo 060, Japan and 2AISIN Seiki Co. Ltd., Asahirnaehi, Aichi 448, Japan. Nucleic acid hybridization widely used in molecular biology usually necessitates the use of radioisotope-labelled probes. However, the usefulness of radiolabelled probes is limited by safety and disposal problems, short shelf life, radiodecay and duration of autoradiographic exposure. To circumvent these problems, we have synthesized a number of fluorochromes which can be used as substrates for alkaline phosphatase coupled to DNA probes, and tested for their usefulness. As a

Minisymposia

479

result, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate (HNPP) was found to be most suitable for detection of membrane-bound DNA. The target DNA was transferred to a membrane filter, hybridized with a biotin-labelled probe, treated with phosphatase-labelled antibody and then incubated with H N P P as a substrate. The fluorescent reaction product of H N P P was insoluble in buffer and precipitated on membrane filter. Under UV light (302 rim), a human single copy gene was detected on Southern blot hybridization. Sensitivity is compared with a chemiluminescent method using adamantyl1,2-dioxetane phosphate.

M17.6

Monoclonal Antibodies to Ornithine Decarboxylase: New Clues with Respect to the Active Site of the Enzyme. R. G. Schipper ~, R. G. J. Rutten ~, M. Sauerbeck 2, H. P. J. H. M. Adams 3, J. Kopitz 2, P. Bohley 2, A. A. J. Verhofstad t and G. Tesser 3 1Department of Pathology, University of Nijmegen, The Netherlands, 2Department of Physiol.-Chem., University of

M18.

HISTOCHEMISTRY

OF INTEGRINS

Tfibingen, FRG; 3Department of Organic Chemistry, University of Nijmegen, The Netherlands. The primary structure of rat ornithine decarboxylase, EC 4.1.1.17 (Ratus norvegicus) was used for the selection of an epitope by peptide structure calculations. The epitope, a hexadecapeptide representing ODC (34~Lys-36~ was synthesized by solid phase peptide synthesis (SPPS) using the Fmoc strategy and the p-alkoxybenzyl alcohol (Wang) resin as the support. A peptide-conjugate containing bovine serum albumin was used for the immunization of mice. Two hybridoma cettines, MPI6-2 and MP16-3, were selected by ELISA using a conjugate containing thyroglobulin. Further studies with ELISA, ODC-activity measurements and immunoblotting experiments revealed that MP16-2 and MP16-3 recognize the native enzyme but not the inhibited enzyme (i.e. ODC labelled with DFMO). These results indicate that the selected epitope represents (at least partly) the active site of the enzyme. Furthermore, MP16-2 shows affinity to purified native ODC as well as cytosotic ODC. On the contrary, MP16-3 recognized only cytosolic ODC indicating that (a) MP16-2 and MP16-3 react with different parts of the epitope and (b) the sequence recognized by MP16-3 is not available for antibody binding after purification of the enzyme.

AND EXTRACELLULAR MATRIX

M18.1 Immunoehemieal Localization of Integrin Molecules in Cutaneous Tissue during Tumor Promotion. 7". M. Oberyszyn and F. M. Robertson The Ohio State University Department of Surgery, Columbus OH 432t0, USA. Immunochemical localization techniques were used to examine the cutaneous compartments for expression of Intercellular adhesion molecule-1 (ICAM-1) following treatment of the dorsal epidermis of SENCAR mice with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate. At 24 h following exposure to 10~g TPA, the epidermal keratinocytes within the epidermis bound immunoreactive anti-ICAM-1 antibodies, however, there was no binding within the dermal compartment. Leukocytes within the dermis bound antibodies directed against Lymphocyte Function Associated antigen-I (LFA-1) but not ICAM-1 antibodies. Intravenous injection of antibodies directed against ICAM-1, LAF-I or the beta chain subunit of LFA-1 (CD-18) 2 h prior to exposure of the dorsal epidermis to TPA inhibited the leukocyte infiltration and the skin swelling normally observed at 24 h after TPA treatment, with the rank order of potency of inhibition of the antibodies anti-CD-18>anti-LFA>antiICAM-1. Taken together, these observations suggest that integrin and adhesion molecule interactions may play a pivotal role in the early stages of cutaneous inflammation induced by the potent tumor promoter TPA.

M18.2 Ultrastructural Localization of a6P4 and Laminin in Cell-Substrate and in Cell-Cell Contact Sites of a Transformed Mouse Keratinocyte Cell Line. J. Calafat, H. Janssen and A. Sonnenberg The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. The ~6P4 complex is a member of the integrin family of adhesion receptors. In a previous study we have shown that in human and in mouse epidermis the ad34 complex was localized in hemidesmosomes, and in the mouse it was also detected in small clusters on the surface of perineural myofibroblasts and Schwann cells. In all these tissues a6(/4 was always in contact with the basal membrane. This distribution is consistent with a matrix-adhesion function of this integrin. A possible ligand is laminin. However, a MoAb against the /34 subunit did not block the adhesion of ceils to proteolytic fragments of laminin. In this study we describe a transformed mouse keratinocyte ceil line RAC-11P that can be used as an in vitro model for the study of the function of a6/34. RAC-11P cells form colonies of stratified cells that resemble normal epidermis: a basal layer of epithelial cells, flattened suprabasal cells and cornified superficial cells. Using immuno-EM, a6/34 was localized on hemidesmosomes, also characterized by the labelling of the intermediate filaments with MoAb E59 against keratin. Moreover, some clusters of a6/34 were found in contact areas between two neighbouring cells and were also abundant on large filopodia. RAC-11P cells synthesize and secrete laminin. By immunoEM labelling, laminin was found in endoptasmic reticutum and, extracetlularly, on the basal side and in small clusters on the apical surface, in a pattern similar to that of ~6/34. Double

480 labelling for /34-subunit and laminin on RAC-11P (whole mounts), showed that both proteins were present in the same areas and often in the same clusters. This ultrastructural study suggests that laminin is the ligand responsible for aggregation of ad34 in clusters in cell-cell contact sites and on filopodia.

M18.3 Immunolocalization of Basement Membrane Components in Human Skeletal Muscle Cells In Vivo and In Vitro; Topographical Relation with Acetylcholine Receptors. T. van Kuppevelt, A. Benders and J. Veerkamp University of Nijmegen, Dept. Biochemistry, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

Human skeletal muscle cells are completely encompassed by a basement membrane (BM), which is specialized at the site of neuromuscular junction (the synaptic BM). Immunofluorescence shows that muscle cells in vivo are surrounded by a continuous layer of heparan sulfate proteoglycans (HSPGs), laminin, type IV collagen and fibronectin. HSPGs are distinctively concentrated at the synaptic BM and so are molecules reacting with isolectin B4 from Vicia villosa. Myotubes cultured on the serum substitute Ultroser G and brain extract show a continuous layer of HSPGs, laminin and type IV collagen. At sites of acetylcholine receptor clusters, HSPGs and lectin-positive molecules are distinctively concentrated, resembling the in vivo situation. In contrast, myotubes cultured on serum-containing media do not display these characteristics. Electron microscopy reveals that myotubes cultured on Ultroser/brain extract are surrounded by a continuous BM. Proteoglycans are present on the external site of the lamina densa, and associated in a regular fashion with collagen fibrils.

M18.4 Demonstration of Adhesion Molecules on Macrophages and Glial Cells during Acute and Chronic Experimental Allergic Encephalomyelitis. J. Bauer, C. D. Dijkstra, I. Huitinga and T. Sminia Dept. Cell Biology, Vrije Universiteit, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.

Several reports have described the expression of adhesion molecules on endothelial cells in the CNS and the role of these molecules in lymphocyte adherence and migration during experimental allergic encephalomyelitis (EAE). Hardly anything is known about the expression of adhesion molecules on glial cells during EAE. Therefore we investigated the presence of adhesion molecules in the brain parenchyma by immunocytochemistry at the light and ultrastructural level. The antibodies WTI (anti LFA-1/CD1 la), OX-42 (anti CR3/ CD1 lb) and an antibody against ICAM-1 were used. During the first attack, intensive staining for ICAM-1 and CD1 la was found on macrophages in the perivascular lesions and on activated microglial cells surrounding these lesions. During the relapse and remission phase of chronic and acute EAE respectively, staining for these adhesion molecules appeared less intense. Here, however, microglial cells all over the brain parenchyma were positively stained for ICAM-1 and LFA-1. OX-42 reactivity, which in the normal brain is present on

Minisymposia resting microglial, was enhanced during the first attack. During none of the phases of EAE were glial cells other than microglia found to express ICAM-1, CD1 la or CDI lb. From these results it can be concluded that microglial cells through their expression of ICAM-1, C D l l a and C D l l b are able to interact with each other and with cells of the immune system.

M18.5 Immuno-EM Localization of Beta-1 lntegrin in WetCleaved Fibroblasts. A. M. L. Meijne, D. M. Casey, C. A. Feltkamp and E. Roos Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

We have used immuno-EM to study the localization of/31integrin in chicken embryo fihroblasts that had spread for 3 h on fibronectin in serum-free medium. Cells were "wetcleaved" which yields a ventral substrate-associated membrane with associated cytoskeleton, that was sometimes removed with actin depolymerization buffer and high salt buffers to improve accessibility. Antibodies against the cytoplasmic domain of//1-integrin revealed/31 to be located in the cell periphery often close to talin. Furthermore, pl was abundant above fibronectin fibrils. Adhesion plaques contained /31. However, it was not distributed all over the plaque like vinculin and talin, but located in patches, in close proximity to the plaques. Results obtained after application of actin depolymerization and high salt buffers indicate that this observation is not caused by limited accessibility of integrins located under the adhesion plaque. Our observations indicate that /31-integrins are located at the periphery of adhesion plaques. If so, other membrane proteins should be responsible for matrix attachment of the center of the plaque.

M18.6 Internalization of the a6/34-Integrin from the Basal Membrane of Dispase-Detached Keratinocytes. Y. Poumay, M. Leclercq-Smekens, S. Grailly, I. Roland, A. Degen and R. Leloup Histologie-Embryologie, Faeultds Universitaires Notre-Dame de la Paix, Namur, Belgium.

The epidermal keratinocytes form a polarized tissue and can be cultured for the preparation of skin grafts which mimic features of the original tissue. Undissociated cultures sheets can be detached by incubation with dispase. In consequence of this detachment of basal cells, the culture area retracts rapidly and basal cells turn from a flattened to a columnar morphology. When these floating cultures are stored for several hours, the polarized spatial organization into superposed layers is progressively lost. Detachment in dispase at 4~ only prevents the culture retraction. Integrins are restricted to basal ceils of the epidermis and believed to determine the spatial localization of cells in this tissue. Using specific antibodies and immunofluorescent labelling, we show on cryostat sections that the integrin 06/34 is rapidly internalized from their basal membrane when basal cells retract within the first minutes following detachment, whereas

Minisymposia integrins of the VLA-subtype remain on the cell surface. Using EM and immunogold labelling of the a6 chain at 4~ on nonretracted detached cultures, we observe the label on hemidesmosome-like structures typical of keratinocyte

481 cultures. Incubation at 37~ then induces internalization of the label, concomitantly with retraction of the culture area. Studies using skin biopsies suggest a similar event in noncultured basal keratinocytes after dispase-detachment.

M19. O N C O G E N E S A N D T U M O U R S U P P R E S S O R GENES M19.1

P53 Protein Overexpression in Normal, Premalignant and Malignant Tissue of the Cervix Uteri. R. Holm ~, H. Skomedal 1, A. Helland 2, G. Kristensen 3, A.L. Borresen 2 and J. M. Nesland ~ Departments of Pathologj, Genetics2 and Gynecologj, The Norwegian Radium Hospital, Montebello, Oslo, Norway. Mutations in the p53 tumour suppressor gene play an important role in the development of many human malignancies. Because of the short half-life of normal p53 protein, only the mutated form seems to be detectable immunohistochemicaUy. The aim of the present study was to examine the overexpression of p53 protein in 238 benign and malignant cervical lesions in an attempt to define when elevated p53 protein occured in the development of cervical cancer pathogenesis. Immunohistochemicatly, no staining was seen in normal tissue, condyloma, dysplastic tissue and adenocarcinoma in situ, whereas p53 protein was identified in 7070 of squamous cell carcinomas in situ, 11070 of infiltrating adenocarcinomas and 6207o of infiltrating squamous cell carcinomas. In 9% of the cases 5070 to 50~ of tumour cells were immunoreactive for p53 protein, whereas the other positive specimens were characterized by only rare p53positive cells. In conclusion, our results demonstrated that overexpression of p53 protein is common in infiltrating cervix carcinomas, rare in carcinoma in situ and not found in dysplastic, condyloma and normal tissue, and therefore appears to be related to turnout progression.

M19.2

P53, EGFR and ERBB2 Expression in Prostatic Carcinoma. T. Visakorpi, O.-P. Kallioniemi, T. A. Koivula and J. J. Isola Tampere University Hospital, P.O. Box 2000, SF-33521 Tampere, Finland. We analyzed the expression of p53, EGFR and ERBB2 proto_n in paraffin-embedded primary prostatic carcinomas by immunohistochemistry using CM-1, MAb31G7 and MAbl antibodies for p53 (n = 137), EGFR (n = 147) and ERBB2 (n = 147), respectively. Accumulation of p53 protein was found in 17~ carcinomas, whereas normal and hyperplastic prostatic tissues were always negative. Six percent of carcinomas showed intense immunostaining in more than 20~ of carcinoma cells whereas 11% had lower levels of immunostaining. In 47o70 of carcinomas almost uniform EGFR immunoreactivity was detected, whereas in 39~ only some carcinoma cells were positive. Fourteen percent of carcinomas were EGFR-negative. All prostate hyperplasias (n = 17) tested, were strongly positive for EGFR. All prostatic carcinomas were ERBB2-negative. Both EGFR-

positivity and high level p53-immunostaining were associated with high cell proliferation activity (determined by flow cytometric S-phase analysis) and predicted poor 10-year progression-free (p<0.05) and prostatic carcinoma specific survival (p<0.01). Low level p53 immunoreactivity did not have any prognostic value. In conclusion, both EGFR and p53 -positivity were associated with high cell proliferation activity and poor prognosis suggesting that p53 and EGFR may contribute to the progression of prostatic carcinoma.

M19.3

Current and Future Prognostic Factors in Breast Cancer. A. K. Tandon BioGenex Laboratories, San Ramon, CA, USA. Axillary node-negative breast cancer accounts for about onehalf to two-thirds of new cases. While these lesions are apparently "cured" by initial surgery, up to 30% will eventually recur and ultimately kill the patient. Considerable effort is being made to reliably identify patients who are destined to recur in order to spare patients the side effects of unwarranted treatment. The identification of patients at low- and high-risk for recurrence involves the use of a variety of prognostic factors. Although many of these factors have been identified over the years, new ones continue to be discovered. Exploring both contributions and controversies, the majority of this presentation will focus on prognostic markers most commonly used in breast cancer analysis (e.g. estrogen receptor, progesterone receptor, c-erbB-2, cathepsin D) and examine the potential of several recently discovered markers (e.g. p53, Ki-67, PCNA, pS2, EGF Receptor). This presentation will highlight the use of various markers in breast cancer utilizing immunohistochemical approaches.

M19.4

hnmunohistochemistry of Myc, Ras and Ets Oneoproteins in Cervical Carcinomas and Premalignant Diseases. N. T. Raikhlin 1, S. V. PetroC and N. N. MazurenkC ~Cancer Research Center, Moscow, Russia; 2Kazan Medical Institute, Kazan, Russia. An immunohistochemical analysis of cervical carcinomas, displasias, methaplasias and nonmalignant tissues was made with specific antibodies against synthetic peptides of myc, ras and ets-2. Indirect immunoperoxidase and PAP methods were used with unfixed cryostat sections. In the nuclei of turnout cells, expression of myc oncoprotein was higher than in precancer cells. A high level of myc was observed in the cytoplasma of basal cells of normal ectocervix and in clusters

482 of malignant cells in invasive cancer (but not in all cases). The ets-2 antigen was expressed in the cells nuclei of intermediate layers of normal ectocervix and in the cytoplasma of metaplastic, premalignant and malignant epithelium. The highest level of ets-2 expression was revealed in cases of severe displasia in all ceils, while in early displasias ets-2 was expressed in basal cells. A marked ets-2 expression was observed in stromal ceils also.

M19.5 Ultrastructural Localisation of Rho Proteins (Members of the Ras Oncogene Superfamily) Following Micro-Injection of Epitope Tagged Plasmids. D. Robertson, H. F. Paterson and P. Adamson Institute of Cancer Research, Haddow Laboratories, 15 CotswoM Road, Sutton, Surrey SM2 5NG, England. Ras proteins occur in all mammalian cells and overexpression is known to be oncogenic. The three mammalian ras proteins associate specifically with the plasma membrane and this is essential for their biological activity. The location of the related rho proteins has not been fully elucidated. If plasmids coding for rho proteins (A, B or C), members of the ras superfamily, are micro-injected into the nucleus of MDCK cells, their expression causes gross morphological changes and eventual death. We have demonstrated these changes using scanning and transmission electron microscopy. In order to immunolocalise the products plasmids were constructed that had the sequence MEQKLIEEDL added to the N-terminus of the rho protein. This sequence is the epitope for the 9El0 antimyc monoclonal antibody. After allowing a suitable interval for plasmid expression ( 6 - 18 hours) micro-injected cells were fixed and processed by the progressive lowering of temperature method (PLT) into the low temperature resin Lowicryl HM20. Cells were sectioned en face and immuno-

Minisymposia labelled using PE10 and colloidal gold conjugates. The results are shown in conjunction with fluorescent labelling of similarly treated whole cells imaged using confocal microscopy.

M19.6 Expression of N-ras, C-myc Oncogenes and InsulinLike Growth Factor 2 (IGF-2) in Human Hepatoceilular Carcinoma of 55 Cases. J.-F. Zhang, Y.-F. Liu and Q. Su Department of Pathology, The Fourth Military Medical University, Xi'an, PR China. In order to study the role of oncogenes in human hepatocellular carcinoma (HCC), 55 paraffin-embedded sections of HCC were examined for insulin-like growth factor 2 (IGF-2) using immunocytochemical techniques. Twenty-one cases carrying N-ras and C-myc oncogenes were examined using in situ hybridization with biotinylated probes at the same time. Immunocytochemical staining showed that 87.3070 (48/55) of the cases and 88.5~ (23/26) of the peritumor liver tissue samples were positive for IGF-2. IGF-2 was predominantly localized in cytoplasm of both the carcinoma and the peritumor liver tissue. In the latter, the expression level was higher. In 21 sections, in situ hybridization demonstrated that 42.9070 (9/21) of the cases were C-myc positive, 4 of the 21 cases were N-ras positive. Of these, C-myc and N-ras cases were slightly positive in the peritumor liver tissues. The positive signal for C-myc and N-ras was localized within cytoplasm of the carcinoma and hepatocyte of the peritumor tissue. However, the positive signal in the carcinoma was much stronger than that in the peritumor liver tissue. It is suggested that IGF-2, C-myc and N-ras are overexpressed in HCC. They may interfere with signal transduction inside the cell. IGF-2, C-myc and N-ras may coordinatively maintain the malignant phenotype of HCC.

M20. STANDARDIZATION

M20.1 Requirements and Meaning of Standardization of Dyes, Stains, and Chromogenie Reagents for Use in the Histology Department. H. O. Lyon Dept. of Pathology, Hvidovre Hospital, University of Copenhagen, DK-2650 Hvidovre, Denmark. Standardization of dyes, stains, and chromogenic reagents includes specification of their physical and chemical characteristics. Minimum requirements include knowledge of the visible and ultraviolet absorption curve, molar extinction coefficient, and thin layer chromatographic or high performance liquid chromatographic data. However, only for a minority of these reagents do sufficiently precise data exist. This is due to the very few reagents, particularly dyes, so far produced in completely pure form. When a standard does exist, a candidate sample of the reagent must fulfil the specifications for the standard. To get reproducible staining results with these reagents, standardization must also include all other parts of the

staining solution. Similarly, one must standardize the whole procedure of slide preparation. To achieve maximum quality assurance of histological and cytological diagnosis, our goal might well be to circulate tissue and cell specimens. This could be by post or by electronic video transmission. The need for national and international rules of standardization in this field is therefore urgent. We must find ways to assure drafting of such rules and adherence to these.

M20.2 Standardization and External Quality Control of Dyes for Microscopy by National and International Institutions. E. K. W. Schulte Dept. of Anatomy, University of Munich, D-8000 Munich, Germany. In the past, various efforts have been made on both national and international levels to establish an external quality control

Minisymposia or scientific standards for dyes for microscopy. The US Biological Stain Commission (BSC) which was founded as an independent non-profit organization performs an external quality control of commercial dyes submitted to the BSC by the supplier on a volunteer basis. Representative samples of the dyes are tested for total dye content and staining performance and undergo thin layer chromatography to check for coloured impurities. Satisfactory dyes are certified by the BSC, non-satisfactory batches are rejected. The Subcommittee on Reference Materials for Tissue Stains (SRMTS) within the European Committee for Clinical Laboratory Standards has established scientific standards for dyes for microscopy on a physico-chemical basis: the standard describes the absorption characteristics of the pure dye, maximum amount of coloured and uncoloured contaminants permitted, stability on storage, and requirements for labelling of commercial batches. The data given in such a standard shall enable the user to check the quality of an actual batch and can be used for calibration of commercial samples. An external quality control is not performed by the SRMTS. The same procedure is applied by the German Bureau for Standardization on a national level~ The pros and cons of these two strategies and how they can be used to improve the quality of commercial dyes are discussed.

M20.3 Standardization of Dyes and Stains. A. P. de Leenheer, B. van Liedekerke and W. E. Lambert Laboratoria voor Medische Biochemie en voor Klinische Analyse, Universiteit Gent, B-9000 Gent, Belgium.

Standardization usually requires two steps. First, reference compounds should be available and second, a chromatographic process is necessary to separate the main compound from impurities or degradation products. Reference compounds can be obtained by collection of the separate peaks of an impure dye from a semi preparative high performance liquid chromatographic (HPLC) system. After crystallisation, these compounds have to be characterized by nuclear magnetic resonance (NMR), UV and IR spectrometry and by mass spectrometry. After structure elucidation these compounds can serve as reference material. Appearance of extra peaks in the chromatogram during the second step unequivocally demonstrates degradation or contamination. Classical thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) have been applied for standardization purposes. Automatic application of the sample combined with computerized densitometry not only allows quantitative work but also provides spectral data of the separated compounds. It is clear that the same preparation can be obtained by HPLC, while photodiode array detection also offers spectral characteristics of the eluting peaks. However, HPTLC has the advantage that a large number of samples can be analyzed simultaneously in one single run resulting in a much lower solvent consumption as compared to HPLC. On the other hand, from an HPLC column peaks can more easily be collected for further analysis by other techniques.

483

M20.4 Differences in the Staining Pattern Between the Standard Romanowsky-Giemsa (RG) Stain and NonStandardized Commercial Versions. D. H. Wittekind Anatomisches Institut II, Universitdt Freiburg, Germany.

This subject offers the opportunity to emphasize the necessity to define clearly the technical terms used in biological staining. Many commercial variants of the RG stain "in the strict sense" will give the typical RG staining pattern in cytology. " I n the strict sense" means the RG-working solution which is a mixture of the "proper" stock solution diluted with water and a good buffer (e.g. HEPES). The factors inherent in commercial RG type stains which can cause deviations from the standard will be presented and interpreted. The RG stain "in the broad sense" comprises the RG stain in the strict sense, preceded by "proper" fixation, "conditioning" of fixed samples for the RG stain - and in histology, also embedding and differentiation after staining are components of the RG stain which can profoundly alter the ultimate staining schedule. Examples will be given of how these components of the RG stain "in the broad sense" may change the histological RG staining pattern. It will be emphasized that it is necessary to discourage the use of nonstandardized versions of the RG stain because their staining performance will diverge qualitatively from the standard and will be less reliable, and so, less meaningful. The notions set in quotation marks will be explained in the text.

M20.5 Impure Stains are Troublesome, and such Trouble is Avoidable: but how do we Convince the W o / m a n down the Corridor? R. W. Horobin The Dept of Biomedical Science, University of Sheffield, Sheffield $10 2TN, UK.

Enthusiasts long since documented both the warning and the optimism of the title. They developed analytical methods for stains; produced pure reagents; devised standardised staining methods. But benchworkers don't know, don't believe or don't bother about this. The scholarly approach to telling the world of these issues having proved insufficient, how do we bring the good news to the wo/man down the corridor? The following possibilities will be discussed, together with their implications. Persuading editors in routine histopathology and biology journals to make recommendations to authors. Persistently writing on this topic for technical magazines and proceedings. Publishing low cost booklets on stain ira/purity. Persuading authors and editors of staining manuals to add 'purity tips' to their practical recipes. Producing new kinds of information sources - - eg databases and expert systems.

M20.6 Standards for Standards in Scientific Publications. P. J. Stoward Department of A natomy and Physiology, University of Dundee, UK.

Standards submitted for publication for the purity of

484

Minisymposia

histochemical reagents, including dyes, should themselves meet certain criteria if they are to be accepted as authoritative and definitive by the scientific community. First, the standard should be written in an easily understood and useful form, preferably in a structured format. Second, the title of the standard should be short and informative, and capable of being traceable in the abstract literature. Third, the standard should contain sufficient data (e.g physical and biological properties) for the claimed purity and identity to be readily confirmed. It is not the task of journals to prescribe the type of data required. Fourth, the permissible limits of impurities, and their chemical identities, should be included, together with explicit evidence as to whether the presence of such impurities has any significant effect on the staining properties of the principal dye. Most standards are drafted by a Committee. This can give rise to dull documents, which are then ignored by the scientific community. Submission of draft standards to one or more independent assessors, who would carry out an experimental confirmation of the data claimed, may help to avoid this ignominy.

M20,7 Quality Assurance in the Medical Laboratory. A. Uldall University of Copenhagen, Department of Clinical Chemistry, Herlev Hospital, DK-2730 Herlev, Denmark. The basic elements of quality assurance in the medical laboratory [1] consist of: (1) Defining the medical needs for quality. (2) Selection of methods and measurements procedures which fulfil the needs. (3) Documentation of all laboratory procedures so its elements are defined and can be monitored; this includes also the content and design of reports. (4) Internal quality control system, which involves all

M21. DEVELOPMENTAL

BIOLOGY

elements described under 3, but often focuses on measurement of control samples and making decisions of acceptance or rejection of each analytical run; long-term monitoring of accuracy of results is another main issue. A further aspect is implementation of rules for good laboratory practice (GLP) [2]. (5) External quality assessment (EQA) involves comparison of results obtained on the same material at different laboratories - and preferably a comparison with results of a reference measurement procedure. Regular EQAschemes for clinical chemistry have been working for more than 30 years [1]. EQA of differential counts of white blood cells on slides have been conducted for more than a decade [1]. EQA in cytology and histopathology have started recently in the UK and the US [4]. More recently an overall concept of quality management according to ISO-9004 part 2 (or European Standard, EN 45001) is being introduced in the medical laboratory. This involve that the total quality management system is established and described in a quality manual [3], so an internal or external inspector can check the reliability of the laboratory. This is an essential aspect of accreditation of laboratories according to the stringent EN 45001. The Clinical Pathology Accreditation system in the UK [5] seems more relaxed. 1. Uldall, A. Quality assurance in clinical chemistry. Scand. J. Clin. Lab. Invest. 1987; 47 suppl 1 8 7 : 1 - 9 6 . 2. The Royal College of Pathologists. Codes o f Practices f o r Pathology Departments. London 1989: 1 - 16. 3. Multiple authors. A Quality Manual f o r the Clinical Laboratory - Structure and Elements. Proposed guidelines prepared by a NORDKEM-project group 1992:1 - 2 9 . 4. DHSS. United Kingdom External Quality Assessment Schemes. Annual report for 1989; London 1990: 4 0 - 4 1 . 5. Advisory task force on standards to the Audit Steering Committee of the Royal College of Pathologists. Pathology department accreditation in the United Kingdom: a synopsis. J. Clin. Pathol. 1991; 44: 7 9 8 - 802.

AND AGING

M21.1 Early Embryonic Development of Transgenic Mice Blocked by a Truncated Retinoic Acid Receptor. S. H. Fromm% S. Reppe 2, J. A. Iversen% K. B. Andersson 3, H. K. Blomhoff3, B. Roald 4 and R. Blomhoff2 1Laboratory of Molecular Embryology and 2Institute for Nutrition Research, University of Oslo, 3The Radium Hospital, Institute for Cancer Research and 4UllevM Hospital, Department of Pathological Anatomy, Oslo, Norway. Interaction of PGFs and non-PGFs with plasmamembraneand nuclear receptors are key elements in embryonic induction leading to primary axes formation and regionalisation in vertebrates. Vitamin A derivates (RA) may play a central role in such processes indicated by experiments with exogenous RA. Effects of RA are probably mediated via the RA-nuclear receptors acting as ligand inducible transcription factors. We have blocked axes formation in transgenic mice around gastrulation using a construct for a truncated RARa receptor and a strong promotor, giving dominant negative mutations of RA-receptors and probably functional vitamin A deficiency. Out of 211 microinjected zygotes transferred to 22

pseudopregnant (PS) mice, 101 embryos developed and 38 were considered transgenic based on PCR and phenotype. PSmice were dissected at 8.5-15.5 dpc. Thirty-two small size swellings were observed containing decidua/embryos resembling control embryos at 6 . 5 - 7 . 0 dpc. Results were confirmed by histological examination of decidua with arrested embryos, and by gel shift analysis of HL-60 cells transfected with the construct. These investigations are now being extended using inducible and cell specific promotors with truncated constructs for RA- and PGF-systems in transgenic mice, supplemented with similar transgenes and sense and antisense mRNAs in the zebrafish (The 'Zebra.mus'-System) and combined with in situ hybridization and immunocytochemistry of 'whole-mounts' and sections.

M21.2

Interesting Endocrine Cell Distributions in the Developing Non-Human Primate Pancreas. S. Wolfe-Coote, C. Chapman and J. Louw Medical Research Council, Parow, Cape, RSA.

Minisymposia The gtucagon (G)- and pancreatic polypeptide (PP)-rich areas observed in a number of adult animal pancreases has been attributed to the organ's dual origin: the PP-rich area arising from the ventral bud and the G-rich area from the dorsal bud. Our finding of an unexpected homogeneous distribution of pancreatic endocrine cells in some baboon foetuses of indeterminate age (Wolfe-Coote et al, Med. Hypotheses 31: 313, 1990) led to the present study of pancreatic endocrine cell distributions at intervals throughout gestation in vervet monkey foetuses. Serial sections of ERL-embedded pancreatic tissue from foetuses of a range of gestational ages were immunolabelled for pancreatic peptides using avidin-biotin peroxidase or silver enhanced 1 nm gold markers. Specificity and method controls were included. The first peptides to be localized were somatostatin and PP in an eight week foetal pancreas in which the two primordial buds were unfused. This was much earlier than previously reported for both peptides and was surprising since glucagon has been considered to be the first pancreatic peptide to be produced and PP has only been observed very late in gestation and often only after birth. The pancreatic buds were closely juxtapositioned, but still separate, at nine weeks gestational age and in these and all older foetuses all four major pancreatic peptides were observed in both dorsal and ventral regions. G and PP were co-localized in a large number of cells, but rarely in equal proportions. There were areas of pancreas in which the expression of one peptide, in all of the cells displaying dual immunoreactivity, predominated over the other, resulting in glucagon rich and PP rich areas. These results would appear to refute the theory that all PP cells are supplied by the ventral bud in the developing pancreas. They also provide evidence for a possible common origin of G and PP cells.

M21.3

Prohormone Convertases PC1 and PC2: In Situ Hybridization in Mouse Hypophysis during Outogeny. M. Marcinkiewicz, R. Day, N. G. Seidah and M. Chr6tien IRC34, ]10 Pine Ave West, Montreal, PQ H2W IR7, Canada.

Cellular co-expression of either of the pro-hormone convertases PC1 or PC2 with POMC, demonstrated that these convertases cleave P O M C in a distinct fashion to produce the same products as those observed in vivo in either the anterior (AL) [PCI produces ACTH and/~LPHI or intermediate (tL) [PC2 produces c~MSH and/3endorphin] lobe of the pituitary (Proc. Natl. Acad. Sci. USA 88: 3564, 1991). These cleavage selectivity profiles of PC1 and PC2 are in agreement with our adult mouse pituitary in situ hybridization data showing the presence of PC1 mRNA in both AL and IL, and the high abundance of PC2 mRNA in IL vs AL. Therefore in the adult pituitary, PC1 is the major enzyme responsible for the production of A C T H in the corticotrophs, whereas in the melanotrophs in addition to PC1, the more abundant PC2 is responsible for the efficient production of ~MSH and pendorphin. Interestingly, during development a fraction of the eorticotrophs in AL can also produce aMSH. In order to define whether this variation in POMC processing during fetal development is due to changes in the relative gene expression of PC1 and PC2, we compared the ontogeny of these convertases to that of POMC. In situ hybridization showed that PC1 and PC2 mRNAs are found in the pituitary

485 primordium (Rathke's pocket) at the embryonic day 14/15 (E14/15). In comparable tissue sections we detected weak immunoreactivities for ACTH, aMSH and /3endorphin at El6. Like in the adult, in fetal mice the level of PCI hybridization was higher in AL vs IL and PC2 was more abundant in the IL vs AL. However, Northern blots of growing adenohypophysis revealed a transient but significant increase of PC2 transcripts during early post-natal weeks. It is therefore possible that the reported formation of ctMSH in some corticotrophs reflects transiently elevated levels of PC2 in these cells.

M21.4 Cytochemical and Molecular Studies on Insect Programmed Cell Death. L D. Bowen and J. K. Skelton Cell Biology, University of Wales College of Cardiff, P.O. Box 915, Cardiff CF1 3TL, UK.

The salivary gland and midgut cells of the blowfly Calliphora vomitoria larvae initiate and complete their own destruction in a programmed manner at the onset of metamorphosis (Bowen and Bowen, 1990). A cataclysmic cell death is initiated on day seven of development. These changes are preceded by the appearance of an autolytic extracisternal acid phosphatase activity at ribosomal sites. No lysosomal leakage occurs although there is an apparent elevation in autophagy. Subsequent to the hydrolytic changes there is a precipitate collapse of ATPase activity. These enzymatic changes were conveniently mapped by means of transmission electron cytochemistry and in the SEM by means of baekscattered electron imaging which provides atomic number contrast. Molecular evidence was obtained of de novo mRNA and protein synthesis as a prelude to cell death. In vitro translation of the mRNA revealed the appearance of a new 50 kD protein immediately before cell destruction. Bowen, I. D. and Bowen, S. M. (1990). Programmed Cell Death in Tumours and Tissues. 1-268. Chapman & Hall, London and New York.

M21.5 Neuronal Changes Involved in 'Programmed Cell Death' in Newborn Rats of Superior Cervical Ganglion. G. Diaz, M. D. Setzu, S. Sirigu and A. Diana Dept. of Cytomorphology, University of Cagliari, Cagliari, Italy. There is good agreement that 'programmed cell death' represents a natural expression of development and morphogenesis; in the rat superior cervical ganglion a massive loss of neurons occurs during first postnatal days. The purpose of our research was to distinguish the early stages of neuronal degeneration in neonate rats. Toluidine blue semithin sections showed the following patterns of neurons: 1.Neurons exhibiting individual nuclear chromatin textures and nuclear shapes variably convoluted. 2.Neurons with one or more chromatin clumps leaving the nuclear membrane. 3.Neuron-derived intensely basophilic bodies lacking of cytological details and dissociated from neighbouring tissue.

486 In consecutive sections stained for Feulgen reaction, the red neuronal profiles showed only a partial overlap with the condensed Toluidine-stained chromatin. Such finding suggests that nuclear degeneration starts involving a disarrangement of RNA -rather than DNA-associated regions. In no case were found signs of lytic necrosis nor of inflammatory reaction. These data were confirmed at ultrastructural level. It would be interesting to verify whether such model of neonatal neuronal death is similar to that occurring in neuronal disorders of aging.

M21.6 Decreased Expression of Calbindin and Parvalbumin in the Forebrain of Aged Rats is Influenced by Chronic Treatment with the Calcium Antagonist Nimodipine. G. I. de Jong, C. Nyakas, E. A. van der Zee and P. G. M. Luiten Univ. of Groningen, Dept. Animal Physiol, Haren, The Netherlands. In the central nervous system (CNS) of the rat both calcium binding proteins calbindin-D 28K (CB) and parvalbumin (PV) show distinct distribution patterns in the hippocampus and neocortex. CB is localized in some hippocampal and cortical pyramidal cells, and is highly present in dentate gyrus granular cells. Furthermore, a subpopulation of interneurons in the

Minisymposia cortex and hippocampus contain CB. PV selectively stains a subpopulation of GABA-ergic interneurons in both hippocampus and cortex. Since aging-related functional alterations are associated with an increased neuronal calcium influx we examined the immunoreactivity (IR) to CB and PV in the brains of young (3 months) and aged (30 months) Wistar rats. Moreover, the influence of long-term administration of the calcium antagonist nimodipine (24 - 30 months) on CB-IR and PV-IR was determined. Adjacent cryosections were routinely immuno-processed for CB and PV. The number of hippocampal and cortical interneurons and the dendritic density of PV-IR interneurons in the hippocampus was quantified. In aged rats the number of CB-IR and PV-IR interneurons was significantly reduced in the neocortex, but was not changed in the hippocampus. Despite the fact that the number of hippocampal PV-IR interneurons was not changed, we found a large decrease in the dendritic density of PV-IR interneurons in the hippocampus in aged animals. When aging animals were chronically treated with nimodipine, a significant increase in the number of cortical CB-IR interneurons was observed. However, no effect on cortical PV-IR interneurons was established. In contrast, nimodipine treatment significantly increased the density of PV-IR dendritic profiles in the hippocampus. These data show that the IR to calcium binding proteins in the neocortex and hippocampus alter during aging. Moreover, chronic administration of nimodipine selectively influence these agingrelated changes.

M22. H I S T O C H E M I S T R Y OF R E C E P T O R S M22.1 Ultrastructural Localization of Growth Hormone Receptor Messenger RNA (GH-R mRNA) in Liver and Pituitary Gland.

receptor in hepatocytes where the effects of GH are known, and in pituitary gland where an autocrine action of GH can be suggested.

H. Mertani, R. Jambou, D. Le Guellec and G. Morel CNRS URA 1454, Fac. Mddecine Lyon-sud, BP 12, 69600 Oullins, France. Growth hormone is synthesized by somatotrophs in pituitary gland and has physiological effects on various target tissues after binding with high affinity to cell surface receptors. It may also bind to prolactin receptors and simulate lactogenic effects or mediate the IGF-I synthesis. We investigated the tissue localization of the GH-R mRNA at the electron microscopic level using an in situ hybridization method. Liver and pituitary gland were fixed in 4% paraformaldehyde and frozen. Hybridization was performed on ultrathin frozen sections using biotinylated oligonucleotidic probes. Hybrid detection was performed using rabbit anti-biotin serum (ENZO) and then using anti-rabbit serum conjugated with gold particle (5, 10 or 15 nm) (BioCell). Sections were stained with uranyl acetate and embedded in methyl cellulose before observation with an electron microscope. Gold particles which represent GH-R mRNA were localized near the endoplasmic reticulum and in cytoplasmic matrix of hepatocytes. In anterior pituitary gland, the presence of GH-R mRNA could be observed in the somatotrophs. Ultrastructural localization was similar to that of hepatocytes, near the endoplasmic reticulum and in cytoplasmic matrix. These results showed the presence of GH-R mRNA and indicated the synthesis of OH

M22.2 Angiotensin II Receptors Localized in Rat Renal Medulla by Electron Microscopic Autoradiography. J. Zhuo ~, D. Alcor# and F. A. O. Mendelsohn 2 University of Melbourne, Department of Anatomy and 2Department of Medicine, Austin Hospital, Victoria3052, Australia. We have previously demonstrated a high density of Angiotensin II (Ang II) receptors in the inner stripe of the outer medulla by using an in vitro autoradiography technique (Mendelsohn et al,, Fed. Proc. 45, 1420-5, 1986). The present study was undertaken to localize Ang II receptors at the cellular level using electron microscopic autoradiography. Adult male Sprague-Dawley rats (100 g BW) were killed by pentabarbitone overdose (Nembutal, 100mg kg-~). The kidneys were removed and the inner stripe of the outer medulla cut into blocks 1 x 1 • 5 ram. Preincubation was in modified Kreb's buffer saturated with carbogen for 30 min followed by incubation in fresh buffer containing "o260 pM of ~2~I[SaP,IleS]Ang II for 1 hr. Non-specific binding was determined in the presence of an excess of Ang II (1 ~M) in a parallel incubation. Following preparation for electron microscopy, sections were positioned on celloidin-coated glass slides, coated in Ilford K5 emulsion, and exposed at 4~ for up to 60 days.

Minisymposia No detectable specific binding occured in the tissue incubated in the presence of 1/~M of unlabelled Ang II. Silver grains were detected specifically over interstitial cells located between the tubules and between the components of the vasa recta bundles. These interstitial cells had multiple cytoplasmic processes, cytoplasmic microtubules and microfilaments, but few lipid droplets and appeared to belong to the type 1 interstitial cells of the renal medulla. There was no specific binding over cells of the thick ascending limbs, the thin limbs, the collecting ducts, the vasa rectae, or other blood vessels. These findings indicate that the type 1 interstitial cells are the primary sites for the high density of Ang II receptors located in the inner stripe of the outer medulla of the rat kidney.

M22.3

lmmunohistochemistry of Androgen Receptors in Normal and Benign Hyperplastic Canine Prostates. P. A. Laroque*, J. D. Strandberg and E. R. Barrack The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. *Present address: MSD-Chibret Laboratories, Riom, France.

Our goal was to compare the distribution of androgen receptors (AR) in normal and benign hyperplastic (BPH) prostates. AR positive cells were identified by immunohistochemistry. Glandular BPH was characterized by multifocal to diffuse hyperplasia of the epithelium, with formation of Jntraluminal papillary projections. Complex BPH contained areas of glandular hyperplasia as well as areas of atrophy and cystic dilatation. In normal adult prostates and in most cases of glandular hyperplasia, the secretory epithelium, although known to be androgen-dependent, was virtually devoid of AR; in contrast some subcapsular alveoli and periurethral tubules contained AR positive epithelial cells. One of six cases of glandular BPH contained AR positive hyperpIastic epithelium. In complex BPH, atrophic tubules contained many AR positive epithelial cells, but the hyperplastic and cystic areas were AR negative. The stroma always contained many AR positive cells; this was the case for normal prostates, glandular BPH, and complex BPH. The apparent absence of AR in normal secretory epithelium and in most of the hyperplastic epithelium suggests that the maintenance of prostate size and secretion may be regulated by androgen dependent stromal factors (synthesized and secreted by AR positive stromal cells in response to androgen), or by epithelial AR present at a level below the limit of detection; alternatively, factors other than androgen may also play a role. Peripheral alveoli likely are growing points, and the presence of AR positive epithelial cells in this location suggests a potential role of AR in proliferation. The distribution of AR in BPH may resemble that in normal prostates because both were at steady state, rather than in the proliferative phase, when we studied them. The presence of AR in periurethral tubules suggests a special function of this area of the gland; one possibility is the formation of new glands, which in BPH assumes a more nodular pattern in the periurethral area (Grant DKI9300).

487

M22.4 Immunocytochemical Detection of Sex Steroid Hormone Receptors in Human Lung Cancer Cell Lines. U. Kaiser ~, P. Tuohimaa 2, J. Hofmann ~, E. Wollmer j, G. Aumfiller 2 and K. Havemann ~ IZentrum for Innere Medizin, Abt. H(t~latologie/Onkologie, 21nstitutffir Anatomie, BaMinger Strasse, D-3550:~larburg, Germany.

Sex is a major prognostic factor in small cell lung cancer (SCLC). Premenopausal women have a significantly longer survival time than men, independent of therapy. Prerequisite for a therapeutic influence of sex hormones or their antagonists is the expression of specific hormone receptors. To study the role of sex steroids in lung cancer, specific antipeptide polyclonal antibodies directed against the human androgen receptor (AR), the estrogen receptor (ER) and a monoclonal antibody against the progesteron receptor (PR) were used for immunocytological studies of 14 lung cancer cell lines. Specificity was demonstrated by immunoblotting as well as inhibition of immunoreaction after presaturation with corresponding peptide. The human breast cancer MCF-7 cell line and human prostate served as positive controls. Immunoreaction was strictly nuclear. A n t i - E R predominantly reacted with cell lines deriving from non small cell lung cancer (5/7) and infrequently with SCLC lines (3/7). Presence of AR was only rarely demonstrated while immunoreaction with a n t i - PR was only faint in 3 of 13 cell lines but prominent in the mesothelioma celt line MSTO 211 H. There was an obvious co-expression of all three steroid hormone receptors in four cell lines. Steroid receptor binding assays with Scatchard analysis confirmed immunologic results. Immunocytologic detection of steroid receptors may be applicable for screening lung cancers that are accessible to hormonal treatment.

M22.5

Flow Cytometric Steroid Receptor Analysis. B. Schutte*, H. M. E. Scheres, A. F. P. M. de Goeij and

F. C, S. Ramaekers* Departments of Molecular Cell Biology & Genetics* and Pathology, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands.

It has been proposed that receptor expression might be related to the cell cycle phase. To this aim, we developed a flow cytometric method for the simultaneous measurement of estrogen (ER) or progesteron receptor (PR) and cellular DNA content. The hormone receptor content of different breast carcinoma cell lines were expressed as an average relative fluorescence intensity and compared with the average number of hormone binding sites/cell, measured radiochemically on cytospin preparations of the same cells, ER and PR were localized exclusively in the nucleus when examined microscopically or by confocal scanning laser microscopy. For the progesteron receptor there was a strong linear correlation between the number of receptor binding sites and the relative fluorescence intensity (RFI) as measured by flow cytometry. Furthermore, there was no apparent cell cycle specific expression found for both ER and PR, either during exponential nor plateau phase growth.

488

M22.6 Use of Biotin-Labelled Growth Factors for Receptor Studies. M. O. DeJong, H. Rozemuller, J. G. J. Bauman and J. W. M. Visser Department of Molecular Pathology, Institute of Applied Radiobiology and Immunology, TNO, Rijswijk, The Netherlands. To study growth factor (GF) receptors on the cell surface of hemopoietic cells, a sensitive staining method is needed. By using biotinylated GF we combined the advantages of ~25I-labelled GF (labelling only functionally intact GF receptors), and antibodies against the receptor (viability, double-staining and sorting in a fluorescence activated cell sorter). Cells were incubated with biologically active biotinylated GF, and fluorescently labelled (strept) avidin. After amplification of the fluorescence signal with alternate layers

Minisymposia of biotinylated anti-(strept) avidin and fiuorescently labelled (strept) avidin, it was possible to analyse cells with 100 cell surface receptors in a flow cytometer. On several kinds of cells, we found differences between the fluorescence signals after labelling with antibodies, and with biotinylated, GF, e.g., subclones of the FDC-P1 cell line that did not respond to stem cell factor (SCF) in biological activity assays also did not bind biotinylated SCF, while the same cells were positive for an antibody against the SCF receptor. An SCF dependent subclone of this cell line showed a strong fluorescence signal both with biotinylated SCF and with the antibody. When stimulated peripheral blood lymphocytes were incubated with biotinylated interleukin-2 (bIL-2) and an antibody against the IL-2 receptor, part of the cells showed a high fluorescence signal for the antibody, but a low bIL-2 signal.

M 2 3 . C Y T O C H E M I S T R Y AT T H E E M L E V E L

M23.1 Detection of Nucleic Acids by In Situ Molecular Immunocytochemistry. M. Thiry Laboratory of Cell and Tissue Biology, University of Lidge, Belgium. Two new and very convenient methods for detecting nucleic acids in situ on semithin or ultrathin sections are described. They rely on the fact that during sectioning DNA and RNA ends appear at the surface of the sections. These DNA and RNA ends can be labelled in specific way using either exogenous terminal deoxynucleotidyl transferase or exogenous polyadenylate nucleotidyl transferase and different nucleotides. Subsequently, the labelled sites can be detected by a very sensitive immunogold labelling procedure. Besides their specificity, they are not time-consuming and offer high resolution unlike the autoradiographic techniques. But the greatest advantage is that they are compatible with usual fixation and embedding procedures and can be combined with cytochemical methods. These two new techniques have been applied on a great variety of biological materials allowing the detection of DNA or RNA, even in structures where they are present in very low amounts.

Breer, Cell Tissue Res. (1991) 266, 247) and 1F4 (Pixley & Menco, Chem. Senses (1991) 16, 568), while one of them, 1A6, labelled the apical microvilli of a special type of microvillous cell as seen with electron microscopy (Carr et al., Neuroscience (1991) 45, 433). To determine the structures binding the MAbs we rapidly froze, freeze-substituted and Lowicryl K11M embedded rat olfactory epithelia using either perfusion (transcardially with 4~ paraformaldehyde; after Van Lookeren Campagne et al., J. Histochem. Cytochem. (1991) 39, 1267) and cryoprotection with glycerol, or unfixed and unprotected tissue (Menco et al., Neuron (1992) 8, 441). Sections were subjected to ultrastructural post-embedding immunocytochemistry. 1F4 strongly labelled supporting cell microvilli in unfixed tissue, but weaker in fixed tissue. S10/8A6 only labelled the microvilli in fixed tissue. 1A6 labelled microvilli of a non-neuronal cell in fixed tissue and microtubules of microtubule-associated structures in unfixed tissue of receptor cells and their cilia. None of the MAbs labelled any other compartment of olfactory cilia. Also, none of them labelled microvilli of brush cells. This study showed that even mild fixation can be an important variable in electron microscopic cytochemical studies using freezesubstitution techniques. (Supported by grants from NSF (IBN-9109851 to BPhMM), NIH (DC-00347 to AIF and SKP) and DFG (Br 712/10-1 to HB and JS)).

M23.2 Monoclonal Antibody Recognition of Microvillous Cells is Affected by Fixation in Freeze-Substituted Rat Olfactory Epithelia.

M23.3 The Substructural Localization of High-Affinity CaATPase Activity in the Vertebrate Retina by Means of Energy-Filtering Transmission Electron Microscopy (EFTEM).

B. P. M. Menco ~, A. I. Farbman ~, S. K. Pixley 2, J. Strotmann 3 and H. Breer 3 1Department of Neurobiology/Physiol., Northwestern University, Evanston, IL 60208, USA; ~Department Anatomy~Cell Biology, University of Cincinnati Medical Center, Cincinnati, OH 45267, USA; 3Inst. Zoophysiologie, Univ. Hohenheim, W- 7000 Stuttgart, Germany. We have examined ultrastructural binding patterns of several monoclonal antibodies (Mabs) developed to characterize membrane properties of rat olfactory receptor cell cilia and supporting cell microvilli. With light microscopy two of these bound to olfactory epithelial surfaces, S 10/8A6 (Strotmann &

K.-H. K6rtje, D. K6rtje and H. Rahmann Institut ffir Zoologic, Universitat Stuttgart-Hohenheim, i)-7000 Stuttgart 70, Germany. The introduction of EFTEM into the field of biological research opened a variety of new technical approaches, especially for contrast enhancement and element microanalysis. Thus the detection of high-affinity Ca-ATPase activity became possible after enzyme activation with micromolar calcium concentrations, precipitation of phosphate with cerium ions and microanalysis of cerium using electron energy-loss spectroscopy (EELS) and electron

Minisymposia spectroscopic imaging (ESI) with the CEM 902 (Zeiss). Highaffinity Ca-ATPase activity in the fish retina was analysed after a characteristic distribution of cytochemically detectable endogenous calcium was observed with high accumulations of calcium inside synaptic vesicles of photoreceptor and bipolar cells. ATPase activity was not found in rod and cone photoreceptor cells, but in the processes of horizontal cells contacting cone pedicles. Ca-ATPase was observed in the inner plexiform layer in conventional synapses associated with pre-and postsynaptic membranes, but not in the presynaptic terminal of bipolar neurons. The high-affinity Ca-ATPase activity thus seems to be involved in the regulation of the calcium homeostasis in some retinal cell types, but in the calcium accumulating photoreceptor and bipolar cells other mechanisms prevail. M23.4

Ultrastructural Localization of Xanthine Oxidase Activity in Unfixed Cryostat Sections of Rat Duodenum. R. J. M. van den Munckhof, H. Vreeling-Sindelfirovd, J. P. M. Schellens and W. M. Frederiks Laboratory of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Meibergdreef 15, 1105 A Z Amsterdam, The Netherlands. In most studies for ultrastructural cytochemistry tissues are fixed to preserve their ultrastructure. However, most enzymes are (partially) inactivated during fixation which hampers histochemical studies on optimal enzyme activities. We have developed a method to detect optimal xanthine oxidase activity at the ultrastructural level in unfixed cryostal sections of rat duodenum. For this purpose sections of 20 ~m thickness were mounted on a semipermeable membrane which was stretched over a gelled incubation medium. This technique prevents leakage of enzyme molecules from the section and seems to have a positive effect on the preservation of the ultrastructure. The medium contained cerium ions as capture reagent for hydrogen peroxide and hypoxanthine as substrate. Sections were incubated for 30 minutes at 37~ After removal of the gelled incubation medium the sections were cut out, fixed immediately and processed for electron microscopic studies according to standard procedures. X-ray microanalysis of cryostat sections showed a localization of cerium throughout the epithelial cells of the villi. In the epithelium of the crypts and the submucosa less cerium was found and cerium was absent in the muscularis externa, the mucus of the goblet cells and the lumen of the duodenum. In ultrathin sections reaction product was exclusively present in the cytoplasmic matrix of enterocytes. The reaction was specific because this reaction product was not found when sections were incubated in the absence of substrate or sodium azide and in the presence of substrate and allopurinol, a specific inhibitor of xanthine oxidase. It is concluded that ultrastructural studies to detect activities of cytoplasmic matrix enzymes can be performed successfully when unfixed cryostat sections are incubated by the semipermeable membrane technique.

M23,5 Platinum-Diaminobenzidine Reaction for Quantitation of Cytochrome Oxidase Activity by Energy Dispersive X-Ray Analysis,

489 T. Hiraoka* and K. I. Hirai** *Emeritus Professor, Shiga University of Medical Science, Seta, Ohtsu City, 520-21 Japan. ** Department of Anatomy, Kanazawa Medical University, Uchinada, Ishikawa Prefecture, 920-02 Japan. The platinum-diaminobenzidine (Pt-DAB) reaction yielded a black, electron-dense, insoluble, Pt-containing reaction product at the active site of cytochrome oxidase without postosmification. It was sensitively inhibited by KCN and NaN~ and showed the mitochondrial localization of the oxidase equivalent to that shown by the standard DAB reaction followed by postosmification. In the energy dispersive X-ray spectrum obtained from a single Pt-DAB stained mitochondrion, both Mo and Lo~ peaks of Pt stood out against the continuum. The X-ray image analysis and the spot analysis also proved that the distribution of Pt corresponded with the localization of the reaction product. Thus, the reaction permitted us to quantitate the oxidase activity in an individual mitochondrion or in its constituent part by EDAX. To elucidate the gradient of the oxidase activity along the radial axis of the mouse liver lobule, a comparative analysis was carried out with each individual mitochondrion in the peripheral and the central hepatocytes. The mitochondria in the former cells showed the activity of 71.0 _+ 17.4, while those in the latter 48.8 _+ 17.6 in terms of relative counts/500 sec./unit area of the reactive site.

M23.6 Glucose-6-Phosphate Dehydrogenase Demonstrated by Copper Ferrocyanide Method in Rat Liver. T. Saito, T. Takizawa and T. Ishibashi Department of Anatomy, Jichi Medical School, Tochigi, Japan. The distribution of glucose-6-phosphate dehydrogenase (G6PD) activity has here been studied by a copper ferrocyanide method in order to elucidate the ultrastructural sites of this enzyme in normal liver cells of rats. The estimated optimum fixation conditions to demonstrate G6PD were a 1 . 0 - 2 . 0 % glutaraldehyde solution in a cacodylate buffer, pH 7.2, for 1 5 - 3 0 rnin at 4~ for immersion fixation, and 2.0% glutaraldehyde for 5 rain in perfusion fixation followed by immersion fixation for 25 min. The incubation medium was that of the copper ferrocyanide method with 0 . 5 - 1 . 0 mM phenazine methosulfate (PMS) as an electron carrier in the incubation medium. The site of the final reaction products, copper ferrocyanide, were at the cytoplasmic side of the r-ER, close to the ribosomes. The final reaction product in the ultrathin sections were analyzed by an energy dispersive Xray microanalyser. The weight percent of 100 sec of Fe Ka (6.398 keV) and Cu Ka (8.040 keV) were 28.57 and 71.43, and the atomic percent was 31.275 and 68.725 respectively. The ratio of F e / C u of atomic percent was 2.19, the elements being reasonably concluded to be those of copper ferrocyanide. No reaction appeared in the specimens incubated in the medium lacking a substrate, coenzyme, or PMS, containing pchloromercuribenzoate, and undergoing an after heat treatment. G6PD activity was also demonstrated on the rat liver fixed by rapid freeze substitution. The enzyme activity appeared positive around the r-ER which showed a beautiful parallel arrangement.

490

Minisymposia

M24. HISTOCHEMISTRY IN CARDIOVASCULAR RESEARCH M24.1 Histochemical Assessment of Lesion Development in the Rabbit Carotid Artery. J. E. Beesley, E. A. Jessup, ,L C. M. Hooper, L. C. Tilling, A. Honey, G. Fleetwood and J. F. Martin Wellcome Research Laboratories, Beckenham, Kent BR3 3BS, UK. Specific antibodies were used to identify macrophages, smooth muscle cells and proliferating cells during lesion formation in the collared carotid artery of rabbits fed either high cholesterol (HC) or normal (N) diets. Macrophages and foam cells were abundant in intimal lesions in animals fed HC. Few macrophages were identified in lesions in animals fed N. Electron immunocytochemistry indicated that the antibody against smooth muscle cells was specific for myofilaments. Immunolabelling was much higher therefore in contractile ceils than in synthetic cells. Immunohistochemical staining of smooth muscle ceils in the medial region was evenly dispersed in unoperated and contralateral sham-operated arteries. After 24 hr of collar placement it was patchy and condensed indicating the presence of synthetic cells. Smooth muscle cells in lesions of animals fed either HC or N were immunolabelled with this antibody. Foam cells were not imunolabelled. There were more proliferating cells in collared arteries than contralateral sham-operated arteries. In conclusion, the intimal lesion of animals maintained on HC consists of smooth muscle cells and macrophages. The lesion in animals fed N consists of smooth muscle cells. A phenotypic change from contractile to synthetic of the medial smooth muscle cells begins 24 hr after placement of the collar and, during lesion development, there is an increase in the number of proliferating cells.

M24.2 Smooth Muscle-Like Contractile Cells in Myocardial Scar Tissue. Morphologic and Functional Observations. G. E. Fazzi, M. G. Havenith and J. G. R. de Mey Dept. of Pharmacology and Pathology, University of Limburg, PO Box 616, 6200 MD Maastricht, The Netherlands. We evaluated whether, in analogy to peripheral healing wounds, a mechanically active smooth muscle (SM) like component is present in myocardial scar tissue. Acute myocardial necrosis was produced by ligation of the left anterior descending coronary artery in the rat. We examined healing lesions at intervals up to 24 weeks after injury. Tissue samples were examined by light microscopy using immunohistochemical methods. Elongated cells observed in the scar showed immunoreactivity for antibodies against contractile proteins SM a-actin and SM myosin heavy chains, eytoskeletal elements vimentin and desmin, and the actinassociated protein gelsolin. SM a-actin positive cells appeared during the first days after injury diffusely distributed between still viable myocardium surrounding the necrotic area. During the first week these cells seemed to migrate towards the developing scar. After the first week the number of SM like cells increased and were still present at the end of the study. Functional observations by use of myocardial scar strips

revealed mechanical responses to high potassium and serotonin. Contractile responses of lesser amplitude were also obtained upon exposure to angiotensin II, phenylephrine and vasopressin. From these observations, we conclude that a SM like contractile tissue participates in the repair of myocardial damage and could play an important role in the prevention of ventricular dilatation and progression of heart failure.

M24.3 - Immunolocalization of Lipoproteins in Human Arteriosclerotic Tissue.

In Sitn

B. Kaesberg and H. Robenek Institute for Arteriosclerosis Research, University of Mfinster, Domagkstr. 3, 4400 Mfinster, Germany. Although the concentration of serum lipoproteins, especially those of low-density lipoprotein (LDL) and high-density lipoprotein (HDL), are related to the pathogenesis of arteriosclerosis, there is a paucity of data concerning lipoprotein distribution in the human arteriosclerotic plaque. For the detection of these lipoproteins we performed immunogold labelling on ultrathin sections of fixed (407o formaldehyde) and embedded (Lowicryl K4M) human arteriosclerotic tissue. We used a panel of specific antibodies to different lipoproteins and their apolipoprotein constituents, namely LDL, fixed LDL, apolipoprotein B-100, HDL and fixed apolipoprotein A-I. We also applied antibodies to alphaactin and cathepsin D for a characterization of the cells and organelles involved in lipoprotein uptake and metabolism. Semiquantitative evaluation was carried out for a detailed comparison of the results obtained. Electron microscopic examination revealed that the majority of HDL and LDL in the pathologic tissue was localized intracellularly in macrophage-derived foam cells and also in smooth muscle cells, whereas only LDL was found in the extracellular matrix. In some cases we observed an accumulation of lipoproteins in electron dense vesicles, which appear to be of lysosomal origin, as shown by double-labelling with an antibody to cathepsin D. These vesicles were only present in macrophagederived foam ceils, which were localized in the necrotic core of an arteriosclerotic plaque, and could not be found in healthy tissue or early stages of arteriosclerotic disease. (Supported by DFG, SFB 310, A7, B3; Vo 386/1-1.)

M24.4 Nitric Oxide Synthase in Rat and Guinea Pig Cardiac Innervation. L. Klimaschewski ~, W. Kummer ~, B. Mayer 2 and C. Heym ~ ~lnstitute for Anatomy and Cell Biology, Rupreeht-KarlsUniversity, Im Neuenheimer FeM 307, W-6900 Heidelberg, FRG; 21nstitute of Pharmacology and Toxicology, University of Graz, Universit(itsplatz 2, A-8010 Graz, Austria. Nitric oxide synthase (NOS) is known to be present in neurons of the central as well as of the peripheral nervous system. The participation of nitric oxide (NO) in the autonomic innervation of rat and guinea pig heart was investigated applying the NADPH diaphorase-technique and immunohistochemistry with NOS antiserum, which were found to label identical structures.

Minisymposia In both species, NOS immunoreactivity was present in endothelial and endocardial cells. Positive nerve fibers were particularly numerous in the area of the sinuatrial node. Fibers and terminals surrounded the proximal course of coronary arteries. Few fibers were seen in the atrial and ventricutar myocardium. In cardiac ganglia some neuronal and paraganglionic cell bodies exhibited NOS immunoreactivity. Positive varicosities formed baskets around positive and negative perikarya. Double-labelling immunofluorescence revealed partial colocalization of NOS with neuropeptide Y or vasoactive intestinal polypeptide in nerve terminals. No coexistence with substance P or tyrosine hydroxylase was detected. Some NOS positive cardiac neurons were surrounded by fibers and terminals positive for neuropeptide Y, vasoactive intestinal polypeptide or substance P. The results indicate a role of NO in the regulation of heart rate, neurogenic coronary vasodilation, myocardial cell function and neural transmission in cardiac ganglia.

M24.5

DeveLopment of the Rat Heart Conduction System as Indicated by Anti-leu-7 Immunohistochemistry and Computer Graphics Reconstruction. N. Aoyama ~ and S. Yamashina 2 Department of ~lnternal Medicine and 2Anatomy, Kitasato University School of Medicine, 1-15-1, Kitasato, Sagamihara, 228 Japan. We have recognized that the anti-leu-7 antibody cross-reacts with the cells of the embryonic heart conduction system. Thus, the development of the rat conduction system using this antibody, and 3-dimensional reconstruction from serial section micrographs from 10 days in utero to immediately after birth was examined. The earliest immunoreactivity appeared in a special region between the bulbus cordis and ventricle, which extended to form a ring at 11 days. At this time, positive reaction was localized at the junction of the sinus venosus and atrium. At 12days in utero, the development o f sinoatrial (SA) and atrioventricular (AV) nodes, a bundle of His (HIS), and right and left bundle branches (RBB and LBB) were indicated by this reaction. SA and AV nodes could be seen connected by internodal fibers at 14 days. Three internodal fibers and communication of AV nodes with the HIS were evident at 16 days, however, immunoreactivity gradually began to disappear from RBB and then from LBB at 18 days. Immunoreactivity could no longer be detected at d a y 0 after birth. The 3-dirnensional development of the conduction system, as obse-ved in the present study, essentially confirmed the findings in previous

491 reports which used other markers. The earliest conduction system appearing at the junction of the bulbus cordis and ventricle at 10 days in utero indicates a special region which possibly functions as a pace maker of ventricular contraction. SA and AV nodes, development at 12 days followed by their communicating with each other through internodal fibers at I4 days, and the communicating of the AV node with the HIS at 16 days, gave rise to regulated contraction of the heart.

M24.6 Localization of the Inducible HSPT0 Protein in the Hypertrophied and Non-Hypertrophied Heart Following Heat-Shock, R. N. Cornelussen ~, L. H. Snoeckx ~, L. G. de Bruin ~, G. J. van der Vusse ~, R. S. Reneman ~ and L. Rappaport z 'Department of Physiology, Cardiovascular Research Institute Maastricht (CARIM), University of Limburg, Maastricht, The Netherlands; 2INSERM U-127, Paris, France. In cardiac tissue, early adaptation to heat stress is characterized by a rapid and transient increase in mRNAs of the inducible heat-shock protein HSP70. In the present study, the accumulation and localization pattern of the HSP70 protein during heat stress, was investigated. Heat-shock was induced by increasing body temperature to 42~ for 15 rain in 18-week-old anesthetized male Lewis rats, which had been aorta-banded (hypertrophied) or sham-operated (nonhypertrophied) 7 weeks earlier. Non-heated rats were used as controls. After 3 or 24 h the hearts were fixed in 2% paraformaldehyde and prepared for cryosectioning (5 ~m). The HSP70 protein was identified using a monoclonal antiHSP70 and a polyclonal peroxidase-linked anti-mouse IgG. Subsequently, the complex was stained with 3' ,3'-diaminobenzidine (DAB). In both non-heated hypertrophied and nonhypertrophied hearts, the HSP70 protein could only be localized in the subendocardial layers of the left ventricular wall. Three hours after heat-shock the HSP70 protein was clearly identified throughout the left and right ventricular wall, in both hypertrophied and non-hypertrophied hearts. The protein was localized in the myocyte nuclei, as well as in fibroblasts. No differences in protein localization were observed between hypertrophied and non-hypertrophied hearts. HSP70 was never observed in the coronary vessel wall. After 24 h the protein was still present, but only in the left ventricular wall. These results indicate that the cardiac myocyte and fibroblast are primary targets for the expression of this early gene product in both the hypertrophied and nonhypertrophied hearts, and that the HSP70 protein is stable at least 24 h after induction of heat-shock in the left ventricular wall.

492

Minisymposia

M25. IMMUNOHISTOCHEMICAL METHODS M25.1 The Impact of Tryptophan Blockade in the Immunohistochemical Demonstration of Growth Hormone, Prolactin and Lysozyme. D. L Segura, C. Montero and M. Gutierrez Department of Pathology, University Hospital "Virgen del Rocio" Sevilla, and Faculty of Medicine, Cddiz, Spain. The work of immunochemists on the intrinsic mechanism of the antigen-antibody reaction at the atomic level has concluded that at least three amino acids are essential in the antigen-antibody binding. As tryptophan is one of these essential amino acids we initiated a series of studies to evaluate its importance in the antigen-antibody reaction in immunohistochemistry. As a model system we used human pituitary and small intestine samples held by the Pathology Department of our institution. The antigens studied were: growth hormone (GH), prolactin (PRL) and lysozyme demonstrated by a biotin-streptavidin-peroxidase method. The tryptophan blockade was done with performic acid following the procedure of Toennies and Homiller (1.942). As primary antibody we used four different monoclonal antibodies for GH, two for PRL and two for lysozyme. We made extensive studies of the different parameters intervening in the reaction and the results obtained confirm the importance of tryptophan in the antigen-antibody reaction. The results obtained with the performic blockade of this amino acid depend on the antigen studied, the monoclonal antibodies and the fixative employed. Supported by a Grant-in-aid of Research from the Junta de Andaluc[a, Sevilla, Spain.

M25.2 One Step IGSS Method for the Light Microscopical Detection of Leukocyte Cell Surface Antigens. V. de Valck, W. Renmans, E. Segers and M. de Wade Dept. Hematology, AZ-VUB, Laarbeeklaan 101, B-1090 Brussels, Belgium.

A few years ago two step immunogold and immunogold-silver staining methods were described for the light microscopical detection of leukocyte cell surface antigens. We have now developed a one-step IGSS method for this purpose. Monoclonal antibodies directed against lymphocyte cell surface antigens (anti CD3, CD4, CD8 and CD19) and appropriate negative isotype controls (IgG1, IgG2a and IgG2b) were produced and purified by FPLC. The purified antibodies were coupled to gold particles of 5 tam diameter. These probes were incubated with peripheral blood mononuclear cell suspensions of healthy adults and of patients with different diseases. Cytospins were made and silver enhancement was performed. The preparations were counterstained with May-Griinwald-Giemsa and examined in light microscopy. This direct IGSS procedure was less time consuming than the indirect procedure. A dense immunostaining without background was obtained. Nearly identical numbers of lymphocytes were found as with an indirect IGSS method and with flow cytometry on the same cell suspensions. The immunostaining was stable and could be visualized with brightfield and epipolarization microscopy. In

conclusion, this direct IGSS method is a reliable tool for the light microscopical detection of leukocyte cell surface antigens in cell suspensions.

M25.3 Immunogold Labelling of Fatty Acids and Phosphatidylserine. An Approach of the Technical Problems. P. R. Compere ~*, L. Maneta-PeyreP, G. Goffinet 2 and C. Cassagne t ~Laboratory d'Etude de la Biogendse Membranaire, IBCN-CNRS, Universitff de Bordeaux 2, 1, rue Camille Saint-Sad'ns, F-33077 Bordeaux, France. 2Laboratory de Biologie Gdndrale et de Morphologie Ultrastructurale, University de Lidge, Belgium. *Recipient of a postdoctoral fellowship from EEC (contract N~ *CT005004). Recently, high specific anti-phosphatidylserine and anti-fatty acid serums were obtained in the laboratory. ELISA specificity tests performed on antigen coated microtitration plates show that the anti-phosphatidylserine serum does not recognize the other phospholipids except for a weak crossreaction with phosphatidic acid. The anti-fatty acid serum appeared to be specific to the fatty acyl chain but not able to discriminate between different chain lengths. In order to develop a suitable method and to determine the ability of these antibodies for in situ detection of phosphatidylserine at the ultrastructural level, pre-embedding and post-embedding approaches were investigated. In both ways, as for the previous methods of lipid detection, several difficulties are encountered. They were related to the fixation procedure, the amount and accessibility of lipids in the tissues. According to the recent study of Girod et al., 1991 (J. Histochem. Cytochem, 54, 136), conventional ultrathin sections of epoxy resin embedded material appear suitable for phospholipid enzyme-gold labelling with phospholipase As. Will it be the same for immunogold labelling with anti-fatty acid and anti-phosphatidylserine antibodies?

M25.4 Immunohistochemical Staining of Mitochondrial Enzyme after Chemical Fixation with Aid of Ultrasound. K. Yasuda ~, S. Yamashita 2, S. Aiso 3 and M. Shiozawa 3 ~Cosmo Research Institute, Satte, Saitama 340-01, 2Junior College of Nursing and 3Department of Anatomy School of Medicine, Keio University, Shinjuku, Tokyo, 160 Japan. The utilization of microwaves has opened the way for the introduction of the principles of physics into chemical fixation procedures. Fixation was accelerated by microwaves but the temperature of the fixatives and the tissue block was increased. The application of ultrasound, in place of microwaves, was examined with the aim of obtaining an increased fixative infiltration rate without an increase in temperature. Rat liver and kidney were cut into small blocks, immersed in chilled 4070 paraformaldehyde then immediately exposed to ultrasound (3.75 MHz for 10 min). Tissue blocks were

Minisymposia dehydrated and embedded in paraffin and/or epoxy resin. Both thin and ultrathin sections were reacted with antiglutamate dehydrogenase (GD). The structure of the cell was well preserved even in the electron microscopic specimens. No sign of cavitation was recognized in the tissue. The mitochondrial matrix appeared darker than that observed in routinely prepared specimens for electron microscopy. The gold particles, representing the site of GD, were seen in the mitochondrial matrix along the cristae. The temperature increase of the fixative was limited to 2-3~ The results suggested that ultrasound accelerates the fixative's infiltration rate with a minimum temperature increase and enables the enzyme antigenicity to remain high.

M25.5

Dehydration and lmmunohistoehemistry. P. Jaarsma Working-Committee on Immunohisto- and Cytochemistry, Region 4; The Netherlands.

The influence of four different dehydration methods on immunohistochemical staining was investigated. Thirteen Dutch departments of pathology participated in this experiment. All four methods (grading ethanol, grading methanol, grading acetone with and without xylene as a clearing agent) were compared to standard (institutional) dehydration procedures. Five monoclonal antibodies (directed against keratin, vimentin, desmin, LCA and EMA) and one polyclonal antiserum (S-100) were used to evaluate the effect on immunohistochemical staining. Although probably not significant, there were some differences in the interpretation of the results. Nevertheless, in our opinion, if there is a problem in the interpretation of immunohistochemical

493 reactivity, it is unlikely that the cause wilt be associated with the dehydration methods.

M25.6 Formaldehyde Fixation in Immunohistochemistry. E. M, C. A. de Bruijn Working-Committee on Immunohisto- and Cytochemistry, Region 3, The Netherlands.

We investigated the influence of a number of parameters, related to routine formaldehyde fixation. These parameters were formaldehyde and phosphate buffer concentration, pH, fixation time and temperature. Four monoclonal antibodies (directed against keratin, vimentin, desmin, LCA and EMA) and one polyclonal antiserum (S-100), generally known for their reactivity with formaldehyde fixed and paraffinembedded tissues, were used to evaluate the effect of the mentioned parameters. Formaldehyde concentrations, ranging from 1% to 8% and fixation time, ranging from 6 h to 4 weeks, appeared to have an inversely proportional effect on immunoreactivity. A formaldehyde concentration between 1% and 4%, with fixation times between 24 h and 1 week, turned out to be optimal conditions, depending on the antibody used. The phosphate buffer concentration showed optimal results between 0.01 M and 0.05 M. Tests with other buffer-systems revealed that the deleterious effect on immunoreactivity is mainly due to the phosphate buffer components. Fixation temperature was found to be optimal between 4~ and 25~ Ranges of pH from 4.0 to 7.0 did not have a perceptible effect on immunoreactivity. An alkaline pH however, proved to be disastrous. A slightly acidic pH was found to yield optimal results.

M26. F R E E R A D I C A L S A N D TISSUE D A M A G E M26.1 Histochemical Changes in Rat Heart after Ischemia and Reperfusion: A Two Step Process. C. Melchiorri, G. Melzi* and P. Chieco Institute of Oncology F. Addarii, Bologna; *Schiapparelli Ricerche, Corso Belgio 86, Torino, Italy.

This study provides a qualitative and quantitative description of histochemical changes in the ischemic area from 2 to 72 h after !5 rain of temporary occlusion of left coronary in rat heart. Midventricular and apical regions from 10 treated and 4 sham-operated animals were selected and rapidly frozen at 2, 4, 24, 48 and 72h after reperfusion. Cytophotometric and morphometric measurements showed that the percentage area of lesions was limited to 2% up to 24 h, then suddenly increased to 40% of the total heart section. The early step ( 2 - 24 h) involved subendocardial areas, showing an exclusive depletion of cytoplasmic enzymes (lactate, malate and isocitrate dehydrogenase). In the second step of injury after 4 8 - 7 2 h a large transmural region was identified by disappearance of both cytoplasmic and mitochondrial enzymes (succinate and NADH dehydrogenase, cytochrome oxidase); mitochondrial damage was also marked by the presence of patchy calcium deposits. These findings indicate

that cell membranes are the major target of early myocardial ischemic injury followed by other types of damage, particularly of mitochondria. Oil Red O demonstrated diffuse accumulations of triglycerides around lesions; their production may be involved in defence mechanisms against lipid peroxidation, conjugating fatty acids with glycerophosphate generated by enhanced anaerobic glycolysis.

M26.2 Assessment of Histochemical Procedures for the Study of Oxidant-Stress-Derived Carbonyls in Tissues and Isolated Cells. A. Pompella Ist.di Patologia Generale dell'Universita, Via Laterino 8, 53100 Siena, Italy.

Upon exposure to conditions of oxidative stress, accumulation of carbonyl functions in living cells can result from direct oxidation of amino acid residues in protein, and/or from lipid peroxidation, i.e. an oxidative degradation of membrane lipids, leading to production of reactive and toxic aldehyde species capable to bind to protein -SH groups. Previous work from our laboratory showed the efficiency of the direct

494 Schiff's reaction for detecting aldehyde functions in frozen liver sections from mice exposed in vivo to the pro-oxidant bromobenzene; microphotometric analysis of sections showed the possibility of obtaining semiquantitative information, correlated with biochemical parameters of lipid oxidation (tissue content of malonaldehyde). Further studies were based on the use of a modified 3-OH-naphthoic acid hydrazide/Fast Blue B (NAH/FBB) reaction, whose sensitivity and reliability were assessed by microphotometric evaluation of sections exposed to pro-oxidants in vitro. The use of N A H / F B B allowed the appraisal of the carbonyls originating in individual isolated, pro-oxidant-treated hepatocytes, as welt as of the carbonyls accumulating in several organs (liver, kidney and lung) of mice exposed to pro-oxidants in vivo. Recent studies have been carried out by using fluorescent hydrazoneforming reagents, whose efficiency in revealing carbonyl functions has been tested on isolated cells and tissue sections by means of confocal fluorescence microscopy. Data have been obtained indicating that this might represent in the future the approach of choice for the study of oxidative stress in situ, even at subcellular level.

M26.3 Toxicity Studies with Video Microscopy in Living Cells. J. F. Nagelkerke, P. Zoetewey, B. van de Water and H. J. G. M. de Bont Div. Toxicology, Center for Bio-Pharmaceutical Sciences, Sylvius Lab., P.O. Box 9503, 2300 RA Leiden, The Netherlands.

We study the effect of hepatotoxic and nephrotoxic compounds on the biochemistry of living, individual, hepatocytes or renal proximal tubular cells with video microscopy. The main components of the system are a Zeiss IM-35 inverted microscope with a 100 W mercury arc lamp, a Photometrics 200 series cooled CCD camera and Imagine image processing unit. Filter changers and shutters are computer controlled. Cells are loaded with indicators for e.g. intracellular free calcium, sodium, potassium, pH, mitochondrial membrane potential (MMP) or free radicals; preferably with a combination, which allows determination of a number of parameters in the same cell. Due to the high sensitivity of the camera, exposure times are not longer than 0.5 s. This is essential for our toxicity studies because we found that a few seconds of exposure already induced free radical formation in hepatocytes. A major research line is the effect of toxicants on intracellular free calcium levels. We found that addition of ATP to hepatocytes induced an increase in intracellular calcium and a decrease in the MMP, which ultimately led to cell death. Amelioration of the increase in intracellular free calcium prevented the loss of MMP and cell death completely, which suggests a relation between these processes. Addition of di-chlorovinyl cysteine to renal proximal tubular cells also induced an elevation of intracellular calcium and a decrease in MMP; in this case however, the onset of cell death was only delayed by prevention of the rise in calcium, indicating that additional effects must be involved. A second research line is the effect of changes in intracellular biochemical parameters on the cytoskeleton of hepatocytes. We found that modification of less than 3o70 of the intracellular free protein thiol groups was sufficient to destroy the microtubular

Minisymposia ultrastructure and to completely prevent the secretion of very low density lipoprotein from the cells.

M26.4 Protection against Cumene Hydroperoxide-Induced Cell Damage by Stimulation of the Peroxidase and Reductase Reactions in the Glutathione Redox Cycle of Rat Cardiomyocytes. C. T. Le, L. Hollaar, L. J. M. van der Valk and A. van der Laarse Department Cardiology, University Hospital, Leiden, The Netherlands. Cumene hydroperoxide (CHPO) is severely cytotoxic to cardiomyocytes by a mechanism currently known as 'oxidative stress'. We investigated whether myocytes could be protected against damage by CHPO-induced oxidative stress by accelerating the glutathione redox cycle turnover. Cultures of neonatal rat heart cells were incubated for 90 rain with 80/aM CHPO alone (C), with 80/aM CHPO in the presence of 160/aM Trolox-C (C + T) which is a water soluble analogue of vitamin E, with 80/aM CHPO in the presence of 10 mM glucose ( C + G ) , and with 80/aM CHPO in the presence of 160/aM Trolox-C plus 10 mM glucose (C + T + G). Lipid peroxidation was assessed by assay of malondialdehyde (MDA), and cell necrosis was quantified by measurement of enzyme release. We also assayed cellular glutathione (GSH), glutathione disulfide (GSSG) and NADPH. Resuks are expressed per culture (mean values _+ SD). GSH

GSH/GSSG NADPH

(nmol) no CHPO CHPO C+T C+G C+T+G

46.3 __.8.9 40.8 _+18.7 13.5 _+8.3 4.8 _+ 2.8 9.6_+1.3 2.3+ 1.1 31.7_+3.8 29.8_+ 9.0 41.1-+3.4" 29.3_+10.8

MDA

necrosis

(pmol)

(nmol)

(o7oof cells)

170_+ 9 31 _+ 3 82_+13 117+11 149-+18'

<0.1 2.4 +_0.8 0.7_+0.1 0.9_+0.2 0.2_+0.1'

1.8 _+ 0.5 60.5 _+ 9.9 35.2_+20.3 19.0_+ 6.5 2.6_+ 0.6*

*p
M26.5 The Role of Reactive Oxygen Species in Ischemic Cell Damage. IV. M. Frederiks, A. Kooij and C. J. F. van Noorden Laboratory of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, The Netherlands.

During the last ten years, evidence that oxygen free radicals are involved in the induction of tissue damage as a consequence of ischemia and reperfusion has grown. The first indications were derived from studies in which addition or administration of oxygen radical scavengers, such as

Minisymposia

495

superoxide dismutase, diminished the ischemia-induced damage. Recently, more direct evidence was obtained by administration of capturing agents for superoxide anions or hydrogen peroxide which resulted in the formation of an insoluble product visible at light a n d / o r electron microscopical level. Different sources for the production of reactive oxygen species were proposed. Much attention has been paid to the rote of xanthine oxidase in this process. The enzyme occurs under physiological conditions in a NAD-using form (Dform), whereas a reversible or irreversible transition into an oxygen-using form (O-form) can take place under different conditions such as ischemia and proteolysis. It was assumed that during ischemia the D-form transformed into the O-form resulting in the production of superoxide anions during reperfusion when oxygen and hypoxanthine, as product of ATP breakdown, were available as substrates. A second producer of oxygen radicals was assumed to be the potymorphonuclear leucocyte. As a consequence of ischemia, endothelial cells were changed in such a way that adherence of blood cells during reperfusion may occur. Activation of PMN's may lead to the formation of free radicals. Another cell type which may be involved in the production of reactive oxygen species in liver is the Kupffer cell. Only very recently, several data have made clear that these cells can be activated as a consequence of ischemia leading to potential radical formation. Less attention has been paid to the possibilities that mitochondria and endoplasmic reticulum can generate free radicals under physiological conditions. Moreover, enzymes such as cyclo-oxygenase and lipo-oxygenase and auto-oxidation of catecholamines may be responsible for the production of flee radicals. Finally, it is possible that ischemia may not induce an enhanced production of free radicals but decreases activity of one of the many antioxidant defense systems in cells.

M27.

IMAGE

AND

FLOW

M26.6 Detection of O2(1Ag) in Leukocytes. M. J. Steinbeck, A. U. Kahn and M. J. Karnovsky Harvard Medical School, Boston, MA 02115, USA. Our laboratory has previously reported methods for detecting H202, utilizing 3-3'-diaminobenzidine (DAB) and Ce 3§ as reagents, and a DAB-Mn 2+ method for superoxide, in stimulated leukocytes. This latter method is based on an initial reaction of superoxide and manganese (Mn 2+) which leads to the generation of Mn 3§ which oxidizes DAB to an insoluble osmiophilic polymer (Briggs, et at., 1986). We have developed a specific and sensitive method for the quantitafion of the highly reactive oxidant, singlet oxygen (O2(lAg)), using 9, 10-diphenylanthracene as a chemical trap, and have demonstrated that stimulated neutrophils produce O2(~Ag) (Steinbeck, et al., J. Biol. Chem., 1992), as well as monocytes (in preparation). To determine if O2(~Ag)might be directly reacting with and oxidizing DAB in the absence of Mn 2+, a pure chemical source of Oz(1Ag)was synthesized, 1,4-dimethylnaphthalene-l,4endoperoxide (DNE) which when warmed to 37~ thermally releases O2(tAg) and regenerates the nonreactive parent compound, 1,4-dimethyl-naphthalene (DMN). DAB, in phosphate-buffered saline, was added to tubes containing DNE, DMN, or nothing. All tubes were incubated in the dark to prevent the oxidation of DAB by room light. Only those tubes containing DNE and warmed to 37~ produced a visible brown precipitate. No color change was observed in any tube incubated at 4~ even after 72 hr. The specificity of the O2(~Ag)reaction with DAB was verified by near-IR spectrophotometry. O2CAg) specifically emits energy at 1268 nm, and an IR eroAssion spectrum with a 1268 nm emission peak was obtained when DNE was warmed to 37~ in the absence of DAB. When DAB was added, the O2(tt,g) emission intensity at 1268 nm was decreased and the kq was calculated to be in the range of 1.0 • 108-9. Based on these results, we are currently attempting to develop a modified DAB cytochemical medium to localize the production sites of O2(IAg) in stimulated leukocytes.

CYTOMETRY

M27.1 Automatic Classification Analyses on LeukaemiaRelated Morphological Features. S. Serbouti, H. Harms ~ U. Gunzer § R. Beuscart and J.-Y. Mary* CERIM, Facultd de Mddecine, Lille, France, ~ Processing Laboratory, *Department of Hematology, Wr Germany and *INSERM U263, Paris, France. Two morphologic parameters which are playing an increasingly important role in the identification of mononuclear cells are: (a)chromatin network arrangement and structure; ( b ) a n d basophil colour. The measurement and analysis of these parameters is essential if the computer is to detect pathologic conditions in the blood.

Moreover, the predescribed therapy depends on the correct recognition of the leukaemic cells. Such an analysis requires texture algorithms which are independent of size and shape. In first clinical tests, computer programs are being used to diagnose leukaemias. The statistical analysis performed on the blasts, shows that it can also afford additional information concerning the leukaemia type. The purpose of cluster analysis is to construct a classification, that is, to place patients into groups or clusters suggested by the data.

496 M27.2

Cytokinetic Analysis of Lung Cancer by In Vivo Bromodeoxyuridine Labelling. The Role of Cell Death in Tumor Growth. M. Tinnemans j, M.-H. Lenders ~, B. Schutte 2, G, ten Velde3, F. Ramaekers 2 and G. Blijham 4 IDept. of Internal Medicine, Academic Hospital Maastricht; ZDept. of Molecular Cell Biology & Genetics, University of Limburg; 3Dept. of Pulmonology, Academic Hospital Maastricht; 4Dept. of Internal Medicine, Academic Hospital Utrecht, The Netherlands. Fifty patients presenting with various types of lung cancer were labelled in vivo with the thymidine analogue bromodeoxyuridine (BrdU). Bronchial biopsy specimens were collected and nuclear suspensions prepared. The S-phase fraction and BrdU labelling index (LI) was measured flow cytometrically, allowing to calculate the S-phase transit time and potential tumor doubling time. Although the parameter values showed a broad range, S-phase fraction, LI and potential doubling time differed significantly between nonmalignant and malignant biopsies. Between small cell lung cancer (SCLC) and non small cell lung cancer (NSCLC), no significant differences were observed between the mean values of these cytokinetic parameters. Since SCLC is known to be clinically more aggressive than NSCLC, the former is believed to have a higher proliferative capacity. In our study however, we found no evidence for a shorter cell doubling time in SCLC. Furthermore, volume doubling times for lung tumors as observed clinically are generally substantially longer than the average of eight days observed in our study. This must imply a considerable cell loss in both tumor types, with average cell loss factors of approximately 50% and 90% for SCLC and NSCLC, respectively. In accord with these assumptions is the fact that we have noted non-BrdU labelled S-phase cells, both in tumor biopsies and in apparently normal tissue. The implications of cell death for tumor growth will be discussed in the light of these observations.

M27.3

Intracellular Calcium Responses to 1~ DMSO in Normal and Human Papillomavirus Type 16 (HPV16) Transfected Rat Myoblast (L6 Cells) Using Imaging Analysis. M. M. Ennaji ~'2, J.-L. Schwartz ~, M. Arella2, H. Jouishomme 1, A. Merzouki 2 and J. Phipps 1 1National Research Council of Canada, Ottawa, Ontario, Canada, KIA OR6. 21nstitut Armand-Frappier, Laval, Quebec, Canada, H7N 4Z3. Calcium ions (Ca z§ play a pivotal role in signal transduction events as a secondary messenger. Transmembrane movement and intracellular organelle sequestration of Ca 2+ have been recognized as critical regulatory mechanisms of all cellular activities. Drastic changes in intracellular free calcium levels (Ca2"~) often follow exposure to differentiation inducers in many cell systems. In this study Ca2§ changes upon exposure to 1%0 DMSO inducer were monitored in individual ceils of confluent cultures. Dual-wavelength fluorescence microscopy was used to monitor Ca2§ in live, normal and HPV16-transfected L6 cells. Cells were loaded with the Ca2+~ indicator fura-2 and the

Minisymposia calcium response was recorded and analyzed by optical imaging, The Ca2+~ response to DMSO of control myoblasts was biphasic: After a slow increase, Ca2+~ remained at a steady, moderate level over 30 minutes. In contrast the HPV16transfected cells exhibited a sharp transient peak of Ca~% More drastic even was the kinetics of the response. Control ceils acted in synchrony after 28 s exposure while the reaction lag among individual transfected cells ranged from 8 to 210 s. Methoxyverapamil (D600, 50 ~M), a general calcium channel blocker, did not alter the response, suggesting that Ca 2§ was released from intracellular stores. In separate experiments, we demonstrated that HPV16transfected L 6 cells displayed a higher level of PKC activity, as compared to the normal L6 cells. Therefore, it appears that the calcium and the PKC responses are related, as a result of a common phospholipase C pathway activation. The potential role of gap junctions in the temporal aspect of the response is discussed.

M27.4 3D Analysis of the Progression of DNA Replication in S-Phase Nuclei Labelled with Two Fluorescent DNA-Synthesis Markers. J. A. Aten, E. M. M. Manders, J. Stap and R. van Driel University of Amsterdam, Amsterdam, The Netherlands. An immunofluorescence staining method was developed that effectively distinguishes between IdUrd and CldUrd incorporated in the DNA of cell nuclei and chromosomes. IdUrd was stained with Texas Red and CldUrd with FITC. Adequate double labelling with these nucleosides required 2 min pulses only. Cells were given pulses of IdUrd and CldUrd at different periods in the S-phase. In these experiments the time interval between application of the two replication markers ranged from 15 rain to 5 h. A confocal microscope was used to record three dimensional dual colour images of the double labelled cell nuclei. The time dependent changes in the replication patterns were quantitatively investigated using 3D image analysis techniques. This combination of methods has allowed us to study, in much greater detail than was possible before, the progression during S-phase of DNA replication through the nucleus. The arrangement of the red and green fluorescence distributions observed in nuclei cultured with short intervals between the labelling pulses, suggests a mechanism by which chromatin is pushed through the replication machinery. Replication patterns in early, middle and late S-phase have a life time of less than one hour. The same types of patterns reappear in cells that, after labelling, hav~ gone through mitosis.

M27.5

Quantitative Immunohistochemical Evaluation of Metallothionein by Image Analysis in Copper-Loaded Rat Kidney. K. 14I. Schmid, J. M. Morgan, D. ()fner, A. Hittmair, M. E. Elmes, W. E. Evering, S. Haywood, W. B6cker and B. Jasani Department of Pathology, University of Mfinster, Germany. By means of an image analysing system (Olympus BH-2 microscope, Sony CCD video camera module, Macintosh Ilic

Minisymposia computer; software: Image Analyst) the area and the staining intensity of metallothionein (MT) immunoreactivity demonstrated with a directly peroxidase-conjugated anti-MT antibody was investigated in the proximal tubule cells of kidneys of copper-loaded rats fed with a high copper diet ( l g / kg) for 16 weeks and killed sequentially during this period. The image analysis data were compared with analytically determined tissue copper and MT concentrations. The analytical determined copper and MT tissue concentrations were significantly correlated with the MTstained area obtained by means of image analysis. The MTstaining intensity increased in copper-supplemented rats of week 1 to 6 and decreased in rats which received the diet for 8 to 16 weeks. Our results provide evidence that area of MT immunoreactivity corresponds significantly with tissue copper and MT concentrations. The observed rise in the staining intensity of MT to a maximum at 6 weeks which subsequently declined suggests a continuing redistribution of copper and MT after a maximum of copper and MT concentrations was reached in the tissue. Immunocytochemical demonstration of MT may therefore provide a sensitive and reliable method for the demonstration of tissue copper and tissue copper excess.

497 M27.6 Sensitive Fluorescence and Raman Scattering Detection in Flow Cytometry.

R. M. P. Doornbos, B. G. de Grooth and J. Greve Cell Characterization Group, University of Twente, Enschede, The Netherlands. Detection of very low immunofluorescence signals in flow cytometry is often obstructed by the presence of autofluorescence. This intrinsic fluorescence is believed to be originating from molecules naturally present in cells, such as NADH, flavins and other protein complexes. We investigate the origins of this effect in human leukocytes and investigate ways to improve the sensitivity of fluorescence detection with the flow cytometer. Shifting the excitation wavelength to the red region of the spectrum will reduce the autofluorescent background, but this necessitates the use of red sensitive labels (for instance APC). The possibilities of using Raman scattered light in flow cytometry will be discussed. The results of the first Raman experiments with yeast cells will be presented, showing a linear correlation between a cell constituent (beta-carotene) and Raman signals.

M28. HISTOCHEMISTRY OF THE NERVOUS SYSTEM M28.1 Histochemistry of NO-synthase in Rat Brain: Methodical Aspects and the Influence of Various Metals. I. Seidel, H.-G. Bernstein, G. Poeggel, K. Richter and M. Mfiller Institute for Neurobiology, Brenneckestr. 6, D-0-3090 Magdeburg, Germany. NADPH-diaphorase was earlier often used as a marker of certain neurons in mammalian central nervous system. Nowadays there is a considerable renewal of interest in this enzyme because of its ability to generate nitric oxide.We studied NO-synthase in rat brain structures using different histochemical methods. Ca 2§ and calmodulin were shown to slightly activate the enzyme on unfixed cryostat sections. This effect was not observed when fixed tissue was employed. A second series of experiments dealt with the influence of various metals on the activity of NO-synthase both in situ and after separation of the enzyme by gel electrophoresis. Only copper sulphate, manganous chloride and zinc chloride were able to cause an inhibition of enzyme activity when they were used in a preincubation step. Special attention was paid to aluminum due to its neurotoxic effects, but no influence on the enzyme was observed. Moreover, adult rats, which had received different concentrations of aluminum chloride with the drinking water for 12 weeks, did not show changes of the intensity or pattern of NO-synthase positive structures. Further, we have shown for the first time a histochemical localization of NADPH-diaphorase in nerve endings innervating larger vessels in the rat brain, which may play a role in vasodilation. Our results led us to speculate that the observed effect of aluminum on certain transport phenomena through cerebral endothelia is not due to an influence of the

metal on the cyclic GMP system. Supported by the DFG (Be 1336/1-1) and the Land SachsenAnhalt (080A0731).

M28.2 Localization of Rat Brain Potassium Channels - - an Immunoeytochemical Analysis with Monospecific Antibodies against Individual Members of the RCK Potassium Channel Family. R. W. Veh E, R. Lichtinghagen 2, S. Sewing 3 and O. Pongs 3 ~Institut ffir Anatomie, Ruhr-Universitdt Bochum, 21nstitutffir Klinische Chemie L Medizinische Hochschule Hannover, and 3Zentrum for Molekulare Neurobiologie, Hamburg, Germany. An important step towards understanding the molecular basis of the functional diversity of voltage-gated potassium channels in the mammalian nervous system has been the discovery of a family of genes, which are closely related to the Shaker locus of Drosophila, and encode rat brain K-channelforming (RCK; rat cortex K-channel) proteins. To obtain antibodies, differentiating between individual members of the RCK family, which show high sequence similarities, fusion proteins containing predominantly the (as deduced from cDNAs) subtypespecific carboxy terminals were obtained by standard molecular biological techniques, using at least two different expression systems for each channel. Polyclonal monospecific antibodies were obtained after removal of residual cross reactivities by solid phase absorption with the corresponding fusion protein and further affinity purified. These new and highly specific antibodies were used for the individual localization of RCK1, RCK2, RCK3, RCK4, and RCK5 channels in rat brain sections. Each of these potassium

498 channels shows a highly distinctive distribution in rat olfactory bulb, basal forebrain, cortex, hippocampus, cerebellum, mesencephalon and spinal cord.

Minisymposia immunocytochemistry supports the theory regarding the role of CGRP in regulation of the alfa-subunit of the nicotinic acetylcholine receptor. M28.5

M28.3 Probable Sensory Innervation of the Rat Anterior Pituitary.

Porcine Enteric Neurons Projecting to the Cranial Mesenteric Ganglion (CrMG), as Revealed by Retrograde Tracing Experiments.

T. Shioda and N. Iwakawa Department of Anatomy, Teikyo University School of Medicine, Tokyo, Japan. Our previous studies demonstrated that the dense network of terminal nerve fibers showing calcitonin gene-related peptide (CGRP)- and/or substance P(SP)-like immunoreactivity (LI) is predominant in the rat anterior pituitary, while nerve fibers immunoreactive for other peptides, vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), neuropeptide Y (NPY) are absent, or very scanty, if any, in this gland. In order to elucidate the function of these nerves, it was investigated whether neonatal capsaicin treatment leads to a selective depletion of CGRP- and SP-LI fibers in the rat anterior pituitary. Twenty newborn rats were injected subcutaneously with 50 mg/kg capsaicin on the second and third day of life respectively. A comparative number of control rats was pretreated in the same way as capsaicintreated rats, and they received equal components of control solutions. Capsaicin-treated rats were killed 23 days, 30 days or 48 days after treatment, and pituitaries were examined with immunofluorescence technique using antisera to CGRP, SP, VIP, and NPY. The marked depletion of CGRP-LI and SP-LI after capsaicin treatment was observed in the rat anterior pituitary, while immunoreactivity of the other peptides was unchanged. These results show that capsaicin-sensitive sensory nerves distribute to the rat anterior pituitary.

M. Barbiers, J.-P. Timmermans, W. Stach', D. Adriaensen, M. H. A. de Groodt-Lasseel and D. W. Scheuermann Laboratory of Cell Biology and Histology, Department of Morphology, University of Antwerp, Groenenborgerlaan 171, B2020 Antwerp, Belgium and Ilnstitute of Anatomy, University of Rostock, Germany The retrograde tracers Fluorogold and Fast Blue were used to investigate projections from porcine enteric neurons to the CrMG. Two to four weeks after injection of the tracer into the CrMG, labelled cells were found in the myenteric and outer submucous plexus of duodenal, jejunal, ileal, caecai and colonic segments. The cells were mainly located in ganglia lying on the side of the mesenteric attachment and had a multidendritic appearance. The highest number of labelled cells was seen in the colon. Part of these colonic neurons were immunoreactive for CGRP or serotonin. Compared to the data obtained for small laboratory animals, similarities but also significant differences seem to be present in these intestino-intestinal reflex pathways.

M28.4

Calcitonin Gene-Related Peptide in Neuromuscular Junctions. B. Csillik, E. Knyih~tr-CsiUik, E. Kukla-Dobi and J. Tajti Department of Anatomy, Albert Szent-Gydrgyi Medical University, H-6701 Szeged, Hungary. Calcitonin Gene-Related Peptide (CGRP) characterizes "en plaque" and "en grappe" motor end plates, both in tetanic and tonic amphibian muscles. In mammals, only a small part of skeletal motor end plates displays CGRP immunoreactivity while neuromuscular junctions in all the visceral striated muscles are strongly reactive. In the majority of mammalian skeletal muscles, nothing or only faint outlines of the neuromuscular junctions can be seen, similar to end-plate "ghosts" characterizing supramaximally stimulated muscles. Chronic bupivacaine treatment i.e. immobilization of the otherwise non-reacting muscles results in expression of CGRP by motor end plates; the same occurs also in the course of regeneration of neuromuscular junctions. Electron microscopic immunocytochemistry proves presynaptic localization of CGRP. In the course of Wailerian degeneration, CGRP rapidly disappears from the end plates. It is concluded that

M28.6

Ca|citonin Gene-Related Peptide (CGRP)-mRNA Expression in the Motoneurons of the Rat Spinal Cord is Increased After Intensive Motoric Activity. A. Kresse, D. M. Jacobowitz and G. Skofitsch Department of Zoology, Universitdtsplatz 2, A-8010 Graz, Austria and Section of Histopharmacology, NIMH, Bethesda, AID 20892, USA. In a previous in situ hybridisation histochemical study using a radiolabelled synthetic oligodeoxynucleotide probe complementary to rat preproCGRP-mRNA (259-315) we demonstrated the expression of CGRP-mRNA in most of the large motoneurons in the ventral horn of the cervical and lumbar rat spinal cord. Recently we investigated the influence of intensive motoric activity during repeated periods of swimming (30 min each time) on CGRP-mRNA production in alpha motor neurons at the cervical (C5) and the lumbar (L3 to L5) level of the rat spinal cord. Quantitative analysis of the intensity of staining with silver grains that indicates CGRP-mRNA expression in a certain neuron has been performed with computer assisted image analysis. In situ histochemical investigation has shown clearly that the increased motoric activity is related to an increase in CGRP gene expression in the spinal motoneurons particularly at the lumbar level. CGRP has been shown by other investigators to increase the number of acetylcholine receptors in the striated muscle during development. The results of the present study suggest that CGRP may also play an important role at the mature neuromuscular junction. This work was supported by the Austrian Scientific Research Fund grant number P7050M.

Minisymposia M29, IMMUNOHISTOCHEMISTRY

499 O F P L A N T CELLS

M29.1 Combined Immunocytochemical and In Situ Hybridisation Studies of Storage Protein Gene Expression in the Developing Barley Grain. or. T. Davies, P. R. Shewry* and N. Harris Department of Biological Sciences, University of Durham, Durham DHI 3LE; *Long Ashton Research Station, Long Ashton, Bristol BS18 OAF, U.K.

At maturity the barley grain comprises mostly endosperm - - a highly specialised tissue primarily engaged in the synthesis and accumulation of starch and storage protein. The storage proteins of the barley grain - - of which B-hordein constitutes the major prolamin - - are of great commercial import. To elucidate the precise spatial and temporal patterns of storage protein gene expression during the early stages of grain fill we have used in situ hybridisation alongside conventional immunocytochemical techniques. Double-stranded DNA probes derived from a B-hordein cDNA clone were labelled with digoxigenin. Following hybridisation to the hordein mRNA species, in aldehyde-fixed tissues representative of the many developmental stages, the bound probe was localised using an alkaline phosphataseconjugated anti-digoxigenin antibody, with NAMP/Fast Red TR substrates. The storage protein itself was localised using a polyclonal rabbit anti-B-hordein antibody in an equivalent manner.

Both the mRNAs encoding B-hordein and the protein product were restricted to the starchy endosperm and were notably absent from the surrounding aleurone layer. The temporal and spatial patterns of hordein deposition in the endosperm have been determined throughout early/mid grain fill and a full 3-D reconstruction of hordein distribution has been computed from serial transverse sections. We have used the techniques optimised in this study to investigate the expression of several other genes isolated from a subtractive cDNA library derived from developing endosperm tissue.

M29.2 Expression of 30 k D Cysteine Endoprotease B in Germinating Barley Seed. S. Macttila', I. Porali', T.-H. D. Ho 2 and A. Mikkonen j 1Biol. Dept., Univ. Jyvdskylft, POB 35, SF-40351 Jyvdskyld; Finland, eBioL Dept., Washington Univ., St. Louis, MO 63130, USA.

During germination of barley (Hordeum vulgare), acid proteinases degrade the insoluble storage proteins to soluble peptides. In this work, the expression of the 30 kD cysteine proteinase EP-B was studied by immunomicroscopy and in situ hybridization to clarify its role in germinating barley grain. At the very beginning of germination, the synthesis of EP-B mRNA was started in the scutellar epithelium and aleurone cells next to embryo. Later, the m R N A levels were highest in the aleurone layer proceeding to the distal end of the grain in the course of germination. These results support the immunomicroscopy studies showing that during the first day of germination, EP-B protein was strongly localized to the so called germ aleurone and scutellar epithelium from where the

secretion into the starchy endosperm began. Localization was also seen to proceed along the aleurone layer to the distal end. Subcellularly, EP-B was found in the scutellar protein bodies, and it was also detected in the cis and trans area of the Golgi apparatus. These results show that EP-B is differentially localized during germination, and that the induction signal for the de novo synthesis originally comes from the embryo.

M29.3 Immunodetection of Spectrin Antigens in Plant Cells. N. C. A. de Ruijter and A. M. C. Emons Department of Plant Cytology and Morphology, Wageningen Agricultural University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands,

Three polyclonal antibodies, raised against spectrin from human erythrocytes, chicken erythrocytes and calf brain cells (fodrin) were used to detect a spectrin-like protein in a variety of plant extracts and to localize this protein in cells and tissues. The 'in situ' immunodetection was done after various fixation and embedding procedures. Paraformaldehyde fixation proved to be better than glutaraldehyde fixation, which reduced the specific immunoreactivity. Protoplasts from potato and single cells and cell clusters from carrot suspensions were wall-digested, detergent extracted and labelled 'en bloc'. Various tissues from carrot and maize were embedded in dissolvable resins (PEG, BMM), or polymerized in plastics (LR White, Technovit 8100). Also cryo sectioning after sucrose infusion was done. Fluorescent labels (FITC, Bodipy 503/512) or enzyme conjugate (hrp) were used and optimal labelling conditions were selected, Different procedures gave comparable results. Labelling was not only found at the plasma-membrane, but also at the periphery of plastids, depending on the developmental stage, and nuclei of highly proliferating cells were positive. Western blots of plant material showed immunolabelling at 220 kDa, where spectrin is to be expected, but also bands at 85 kDa were stained. We conclude that a spectrin-like protein is present in these plant cells.

M29.4 Immunogold Localization in Virus-Infected Leaves of a Viral Protein Involved in Infection Spread. O. Rohfritsch, C. Stussi-Garaud and T. Godefroy-Colburn tnstitut de Biologie Moldculaire des Plantes du C.N.R.S., Universitg Louis Pasteur, 12 rue du Ggndral Zimmer, 67000 Strasbourg, France.

Plant viral infections are thought to spread through plasmodesmata (natural connections between cells) with the help of virally-encoded "movement proteins". The movement protein of alfalfa mosaic virus has recently been shown to be the 32 kDa non-structural protein, P3. We detected this protein in infected tobacco leaves by western blot and immunogold cytochemistry. By western blot it was found that P3 accumulated transiently in a cytoplasmic membrane fraction but more constantly in a cell-wall fraction. lmmunogold cytochemistry showed furthermore that it was mainly localized along the infection front (defined by leaf blot

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assay using a virus-specific antiserum, as the limit between heavily infected cells and uninfected cells). P3 was never found in cells which contained large amounts of virus. Some of it was detected in cytoplasmic vesicles or in association with membranes. However, most of the protein was more or less associated with the celt wall, especially at the periphery of plasmodesmata.

other hand were found predominantly in the stroma. In case of glycerinealdehyde-3-phosphate dehydrogenase the results were not conclusive since only a small amount of gold markers were visible on the cuts, not enough for statistical evaluation. The methods and the results are discussed.

M29.5 Electron-Microscopical Localization by Immunogold Labelling of Chloroplast Proteins on Cryoembedded Spinach Leaves.

F. Vogel, C. Wolff, R. Menzel, E. K/irgel and W.-H. Schunck Max Delbrfiek Center for Molecular Medicine, Berlin-Buch, Germany.

K. Adler, R. Manteuffel and K.-H. Siiss Institut fiir Pflanzengenetik und Kulturpflanzenforsehung, D-0-4325 Gatersleben, Germany.

Little information is available about the spatial distribution of chloroplast proteins with special regard to the soluble proteins in the matrix of plastids. Two factors complicate the experiments of protein localization by immunogold labelling, i.e. the ease of extraction of proteins from chloroplasts during the embedding process, and loss of immun-reactivity by denaturation of components in conventional embedded material. Therefore, we used a technique of cryosubstitution in combination with LR-White embedding at low temperature applying chemical polymerization. Using this method and monospecific antibodies raised against different enzymes we were able to distinguish between thylakoid-bound and stromalocalized enzymes. The coupling factor CF1 and ribulose-5phosphate kinase were found to reside on membranes. Ribulosebisphosphate carboxylase and chaperone 60 on the

M29.6 Progress in Yeast Immunocytochemistry.

Yeast cells are likely to serve as a valuable model to study many cell biological phenomena of the eukaryotic cell. The increasing importance of this cell model for the expression of molecular cloned genes, the traffic of cellular proteins and the biosynthesis of membranes offer a wide field for immunocytochemical studies. Presently available methods even low temperature resins, do not allow both a well preserved ultrastructure and an effective immunoreaction. We describe a new strategy of yeast cell fixation, preparation of ultrathin cryosections and demonstrate the localization of membrane proteins (cytochrome P-450) within the endoplasmic reticulum membrane of the yeast Candida maltosa. In a further example we will show the overexpression of genetically modified gene constructs in the yeast Saccharomyces cerevisiae and their site-directed localization and influence on the morphology of ER membranes. In a comparison between low temperature resin embedded and cryosectioned material will be shown the increased efficiency of immunolabelling when cryosections were used.

M30. I N S I T U H Y B R I D I Z A T I O N : T E C H N I C A L D E V E L O P M E N T S APPLICATION M30.1 Resolving Overlapping Cosmids by In Situ Hybridization. A. K. Raap and J. Wiegant Department of Cytoehemistry and Cytometry, Leiden University, The Netherlands.

Fluorescent in situ hybridization (FISH) has already shown its great potential in genome mapping at metaphase and interphase level, providing a maximum resolution of 5 0 - 100 kb as just separated spots. We now report the use of DNA halo preparations from human fibroblasts to further increase the resolution. FISH to such DNA halo preparations with chromosome library DNAs resulted in numerous stained loops of nuclear DNA protruding from the nuclear matrix. Alpha-satellite DNAs stained several loops specifically. The signals appear as beadson-a-string. Cosmids generate beaded lines of up to 10 ]m. Multicolor FISH allowed to visualize the overlap of two cosmids as alternating red and green beads, flanked on one side by red and the other by green beads. The range over which cosmids can be directly ordered is probably limited to 100-200 kb, because of lack of morphological definition of the string. The use of yeast artificial chromosomes spanning the entire region of interest may solve that problem.

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This research was supported in part by the Netherlands Organization for Scientific Research (NWO) grant no. 900534-060.

M30.2 Simultaneous Demonstration of Insulin by Immunohistochemistry and Insulin mRNA by In Situ Hybridization. M. B. Jensen and J. Rygaard Bartholin Instituttet, Kommunehospitalet, Copenhagen, Denmark. When production of peptide in a cell is abnormal, it is of interest to know whether it is due to a defect at the mRNA level or at the encoded protein level. We developed two methods - - one for fluorescence and one for light microscopy - - and applied them using rehydrated paraffin sections of formaldehyde-fixed BB rat pancreatic tissue. For demonstration of insulin mRNA by in situ hybridization we used a biotinylated synthetic 25-mer oligodeoxynucleotide. The biotin was visualized using an immunological A P A A P technique. The chromogen used for the visualization of the alkaline phosphatase was Fast Red TR. The reaction product gives a brilliant orange-red fluorescence with green light, as used for rhodamin, but can also be seen under the same

Minisymposia conditions as for FITC fluorescence. Insulin was detected by an indirect immunological FITC method. The two techniques used for detecting insulin mRNA and insulin were carried out in the named order. For light microscopy the order was reversed and a necessary acetylation step was introduced between the two stainings. The chromogens used were Fast Blue BB for the mRNA visualization and 3-amino-9ethylcarbazole (AEC) for the enzyme in an indirect peroxidase technique for insulin. Normally there is a large amount of insulin in the cytoplasm of beta cells, which can interfere with the demonstration of the insulin mRNA. It is possible to carry out the m R N A detection first, but the colours wilt be weaker. Both the fluorescence methods and those for light microscopy can also be of use in investigating D N A / m R N A and cell membrane molecules.

501 numerous neuropeptidergic mRNAs have been established. Moreover, the use of fluorescence microscopy allowed the simultaneous detection of different RNA sequences by the combined use of biotin, digoxigenin and fluorochrome labelled probes. We extended our study with cultured cells. Human cytomegalo virus (HCMV) Immediate early (IE) mRNA could be detected in a transfected rat fibroblast cell line. In addition to cytoplasmic mRNA also IE pre-mRNA was detected in the nucleus of cells as evidenced by the use of intron specific PCR generated probes. The nuclear hybridization signal showed a path-like distribution possibly showing a transport route of mRNA to the cytoplasm. In addition, small intron specific fluorescent spots were scattered around the path-like signal. The current sensitivity of the ISH technique allows reliable detection of HEF and G A P D H house keeping gene mRNAs in a variety of cell lines using direct fluorochromized probes.

M30.3

A Correlation between Cytopathology of Papanicolaou Smears, DNA Content by Image Cytometry and the Presence of Human Papillomavirus (HPV) as Determined by In Situ Hybridization. H. A. B. Multhaupt, E. Bruder, I. P. Rothblat and M. J. Warhol Department of Pathology, Pennsylvania Hospital, Philadelphia, PA 19107, USA. Multiple cervical smears were obtained from each of 70 patients. These smears were subsequently stained with: conventional Papanicolaou stain and classified according to the Bethesda Classification System; Feulgen stain for the generation of a DNA histogram using an Image Analysis System; and in situ hybridization with a cocktail of probes specific for HPV types 6, 11, 16 and 18. DNA histograms were classified as diploid, diploid proliferative, polyploid and aneuploid. Normal cytology was correlated with a diploid DNA histogram. Cytologically normal smears with bacterial or fungal infections showed a diploid proliferative DNA pattern. All smears classified cytologically as a high grade intraepithelial lesion had an aneuploid DNA content. HPV infection as detected by in situ hybridization had either polyploid or aneuploid DNA content. Routine cytology detected only 5 of 27 cases with HPV infection. In summary, our results indicate that high grade cervical intraepithelial neoplasia is associated with aneuploid DNA. Routine cytology is an insensitive method for the detection of HPV.

M30.4 Detection of mRNAs in Tissue Sections and Cultured Cells by Non-Radioactive In Situ Hybridization. R. W. Dirks, F. M. van de Rijke, M. van der Ploeg and A. K. Raap Dept. Cytochemistry & Cytometry, Leiden University, The Netherlands. To study methodological aspects of non-radioactive ISH for RNA detection we have used a number of different biological models. For the application on tissue sections, the neuropeptidergic system of the pond snail Lymnaea stagnalis proved to be a good model. After optimizing conditions for fixation and protease treatments, the expression patterns of

M30.5

Non-Radioactive In Sitn Transcription of Human T-Cell Receptor mRNA. R. de Weger, B. Perunovic, P. van Diemen and D. van Wichen Department of Pathology, University Hospital, P.O. Box 85.500, 3508 GA Utrecht, The Netherlands. Non-radioactive in situ hybridization with T-cell receptor (TCR; C0-region) probes on cytospin preparations of human T-cell lines (Jurkatt, HSB and H9) did not result in consistent colorimetric signals. This prompted us to investigate the detection of TCR mRNA using labelled oligonucleotides and in situ transcription. Synthetic oligo-dT15 nucleotides were tailed by biotin or digoxygenin labelled nucleotides. In situ hybridization with these labelled oligonucleotides directed to the poly-A tail of mRNA's resulted in a strong signal in all cell lines. When the non-tailed oligo-dT15 nucleotides were used as a primer for reverse transcriptase reaction on the cytospin preparations in the presence of biotinilated or digoxigenated nucleotides, a strong colorimetric signal was obtained. This indicated that reverse transcription of total RNA in these cell preparations resulted in sufficient labelling to obtain a colorimetric signal. Specific synthetic oligonucleotides complementary to mRNA of the C/3-region of the TCR, tailed with biotin or digoxygenin labelled nucleotides only gave weak signals. The use of two or three tailed-oligonucleotides complementary to different regions of TCR mRNA did not substantially improve these results. In situ transcription on cytospin preparations of Jurkatt and HSB T-cell lines using digoxigenated oligonucleotides resulted in a strong signal and good localization of the mRNA. Biotin labelling in in situ transcription gave less intense signals. H9 T-cell lines do not express TCR mRNA and were negative in the experiments using specific Cfl oligonucleotides and in situ transcription.

M30.6 In Situ Hybridization Used for Localization of

Transgenic Vasopressin Expression in the Testis. J. M. Funkhouser, Q. Zeng, F. N. Chih, D. Carter and D. Murphy Institute o f Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511.

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Two lines of transgenic rats have been generated bearing a construct consisting of 3Kb, upstream of the start of transcription, linked to the entire vasopressin coding sequences containing a/3 galactosidase (/3 gal) tag inserted into the third exon. Northern analysis shows the transgene to be expressed only in the testis. For in situ hybridization (ISH) localization of the transgene mRNA within the testis, rats were perfusion fixed. A bolus of phosphate buffered saline with heparin was used to wash the vascular system, followed by Zamboni's fixative (a neutralized picric acid paraformaldehyde, PAF). Part of the tissue was cryoprotected overnight in a heparinized, diethyl pyrocarbonate treated, 30~ (w/v) sucrose in phosphate buffer. The remainder of the tissue was left in P A F overnight and embedded in paraffin the following day. The probe used was a 48 mer oligonucleotide which

M31.

ENDOCYTOSIS,

EXOCYTOSIS

hybridizes to the/3 gal insert in the transgene. ISH using a 35S label on both the frozen and the paraffin sections showed localization within the testicular tubules primarily over the cell layers containing the late pachytene, diplotene, cap and golgi phase cells. The same probe on wild type Sprague Dawley rats showed no specific signal. The expression of the mRNA appeared highest in tubules of stage X through XIV with medium expression in stages VI, VII, and VIII. The early stages I - V had the lowest expression levels. Use of non-radioactive digoxigenin label on the same oligonucleotide showed the expression to be within the developing sperm cells of the late pachytene, diplotene, cap and golgi phases. The relationship between these transgene derived RNAs and the endogenous rat testis vasopressin gene derived RNAs is currently being investigated.

AND INTRACELLULAR TRANSPORT

M31.1

Internalization of Surfactant Protein A (SP-A) into Lamellar Bodies of Rat Alveolar Type II Cells In Vitro. M. Kalina, F. X. McCormack, H. Crowley, D. R. Voelker and R. J. Mason Histology and Cell Biology Dept. Medical School Tel-Aviv Uni. Israel; Department of Medicine National Jewish Center for Immunology and Respiratory Medicine Denver, Colorado.

Pulmonary surfactant is thought to be internalized and processed for reuse by alveolar type II cells. In the present study, we followed the internalization and intercellular trafficking of purified surfactant protein A (SP-A) by primary cultures of alveolar type II cells. Internalization of native rat SP-A was compared to that of recombinant rat and human SP-A. All SP-A species were conjugated with colloidal gold for visualization by electron microscopy. The gold conjugates were biologically active as demonstrated by inhibition of phospholipid secretion. The SP-A gold conjugates were internalized to lamellar bodies (LB) via the endosomal system, which included both electron lucent and dense multivesicular bodies. The labelling of LB was time dependent and after 7 h 3 0 - 4 0 % of these organelles were labelled. No qualitative differences in uptake were observed with the three forms of SP-A. The percentage of labelled LB was similar ( 3 0 - 4 0 % ) after 7 h of internalization with the 3 species of SP-A. The recombinant SP-A produced using a baculovirus vector lacked hydroxyproline and had an altered oligosaceharide but these features did not affect its internalization or rate of LB labelling. Internalization of the gold conjugated SP-A as well as endocytosis of the fluid phase marker Lucifer Yellow was related to the shape of type II cells. Both uptake of SP-A, which is receptor mediated, and fluid phase endocytosis were found to be less active in the flattened than in the rounded cells. Thus, cell shape and hence cytoskeletal organization may play an important role in SP-A recycling.

M31.2 Endocytotic Pathways in the Melanotroph. N. Brick, I. Virtanen and S. Soinila Department of Anatomy, University of Helsinki, Siltavuorenpenger 20A, SF-00170 Helsinki, Finland.

The internalization of the extracellular tracers horseradish peroxidase (HRP) and cationized ferritin (CF) by the melanotrophs of the intermediate lobe of the rat pituitary was studied. Freshly dissociated tissue fragments and enzymatically dispersed cells cultured for 5 days were used. After 1 - 2 rain of internalization tracer was found only in coated pits and coated vesicles at the cell membrane. Later both tracers were found in peripherally located rounded vacuoles. After 2 h of incubation the labelling pattern was different for the two tracers. CF was found in larger vacuoles of varying morphology, within the Golgi complex, and at the edge of forming secretory granules. HRP was found in an extensive array of tubulovesicular structures throughout the cytoplasm, but not seen in the Golgi complex. The study shows that the melanotroph sorts CF and HRP to diverting pathways, and indicates that granule membrane, and possibly functional membrane components, are recycled in these cells.

M31.3

Comparative Analysis of lntracellular Traffic of Mannose-6-Phosphate Receptor in Ras-Transformed and Control HBL100 Cells. S. A. Rudchenko, N. Sdiqui and A.-C. Roche Centre de Biophysique Moldculaire, CNRS, 1, rue Haute, 45071 Orldans, France.

The transfection of HBL100 cells by the E J/T24 Harvey-ras oncogene induces their tumorigenic conversion which leads to the modification of cell morphology in culture as well as capability to induce tumors in athymic mice [Lebeau J. et al. (1991) Oncogene, 6, 1125- 1132]. Using confocal microscopy and flow cytometry we compared the mannose-6-phosphate (Man6P) receptor-mediated uptake and the intracellular traffic of fluorescein-labeUed Man6P-substituted bovine serum albumin (F-Man6P-BSA) in ras-transformed and

Minisymposia control HBL100 cells. The specific uptake of F-Man6P-BSA was more effective in ras-HBLlO0 cells and the main part of the ligand was partly colocalized with Texas-red-labelled wheat germ agglufinin which labels preferentially the Golgi apparatus. With control HBL100 cells, Texas-red-labelled wheat germ agglutinin gave roughly the same labelling when cells were in exponential growth, but a very faint labelling when cells were confluent: F-Man6P-BSA appeared as spots remaining in vesicles such as endosomes or lysosomes.

M31.4

Analysis of Giycosylation Sites of Rat Gastric Surface Mucous Cells Using Five GaINAc Binding Lectius. K. Ihida, S. Tsuyama, N. Kashio, T. Takatsuka and F. Murata Department of Anatomy, Kagoshima University, Kagoshima, 890, Japan. The gtycosylation sites of rat gastric surface mucous cells were examined by five different GalNAc binding lectins: Bauhinia purpurea (BPA), Dolichos biflorus (DBA), Helix pomatia (HPA), Maclura pomifera (MPA) and Glycine max (SBA). Glycosylation sites were studied using preembedding and postembedding staining methods. By preembedding method, BPA stains cis and trans side of Golgi lamellae and mature granules weakly, while the intermediate Golgi cisternae are negative or negligible. DBA strongly binds only one to two cisternae of the cis side of the Golgi apparatus. H P A intensely labels the cisternal space of the nuclear envelope, the rough endoplasmic reticulum (rER) and cisternae of the cis side Golgi lamellae. MPA stains the cisternal space of the nuclear envelope, the rER and cisternae of the cis and trans Golgi side. SBA labels cis side Golgi and trans Golgi side. On the other hand, the results obtained by postembedding staining method are almost agreeable with those obtained from preembedding method. From our results, it is concluded that, in gastric surface mucous cells, initial glycosylation of GalNAc occurs at nuclear envelope space and rER, and then proceeds to cis side of Golgi lamellae and to trans Golgi side and granules. The staining patterns of these five GalNAc binding lectins differed respectively.

M31.5

Cytochemical Characterization of the Oligosaccharide Biosynthesis of Rat Acrosomal Glycoproteins. J. A. Martinez-Men~.rguez, M. Avil~s, M. T. Castells, J. F. Madrid and J. Baltesta Department of Cell Biology Medical School, University of Murcia, Spain. The glycoproteins have been classified into two families: Nand O-linked glycoproteins. The addition of sugar residues to proteins occurs in endoplasmic reticulum (ER) and Golgi apparatus (GA). Spermatids show an active synthesis of both types of glycoproteins. In the present study, lectin cytochemistry in combination with enzyme and chemical treatments and ultrastructural immunocytochemistry were applied to investigate the formation of acrosomal glycoproteins. For light microscopy, sections of rat testes were stained with peroxidase- or digoxigenin-labelled lectins. For

503 electron microscopy, the samples were fixed in 2~ glutaraldehyde and embedded in Lowicryl K4M. For detection of lectin binding sites, one-, two- and three-step methods were applied. The lectins used in the present study were SBA, HPA, ConA, GNA, AAA, LTA, UEA I, PNA, RCA I, DSA, WGA, LFA, MAA and SNA. Both paraffin and ultra-thin sections were pretreated with endo- (endoglycosidase F/ peptide N-glycosidase F and endoglycosidase H) and exoglycosidases (neuraminidase and a-fucosidase) and chemical deglycosilation procedures (/3-elimination and acid hydrolysis). In addition, the vesicles involved in glycoprotein traffic were investigated using a monoclonal antibody against clathrin. The results obtained in rat acrosomes suggest the occurrence of both high mannose and complex type N-linked oligosaccharides and mucin type O-linked oligosaccharides. In N-glycoproteins, Man residues are incorporated into the nascent oligosaccharide in the ER, Fuc residues of the inner core of the oligosaccharide in the cis region of GA, GIcNAc in medial cisternae of GA and Gal residues in the transmost cisternae of GA. Neu5Aca2, 3Gal/31, 4GIcNAc sequence was detected in medial and trans cisternae of GA but not in acrosomes. In O-linked glycoproteins, the addition of GalNAc occurs in cis and trans cisternae of GA. Gal/31, 3GalNAc sequence was detected in medial and trans cisternae of GA. Immunoreactivity to clathrin was observed in the transition zone between ER and GA and in vesicles of the trans side of GA. This work was supported by a grant from DGICYT PB 90-0310.

M31.6 An Ultrastructurai Cytochemical and Morphometric Study on the Dynamics of Secretory Granules in the Peptidergic Adipokinetic Cells of the African Migratory Locust, Locusta migratoria. J. H. B. Diederen, K. M. M~ikel, H. E. Sharp-Baker and H. G. B. Vullings Department of Experimental Zoology, University of Utrecht, Padualaan 8, NL-3584 CH Utrecht, The Netherlands. The adipokinetic cells within the glandular lobes of the locust corpus cardiacum synthesize adipokinetic hormones. These hormones are involved in the mobilization of fuel stores from the fat body to serve as the energy substrate for the flight muscles. Flight activity is the only known natural stimulus that induces the release of secretory granules containing the adipokinetic hormones. The adipokinetic cells keep large numbers of these granules in storage. Moreover, secretory granules are continuously being produced by the trans-Golgi network. The adipokinetic cells avidly endocytose the cytochemically demonstrable adsorptive endocytotic tracer WGA-HRP, which then appears in the trans-Golgi network and in secretory granules originating from it, thus distinguishing these young granules from the older, already existing ones. This offers the possibility to study the effects of secretory stimulation by flight on both categories of secretory granules separately. The ratio between the numbers of tracerlabelled and unlabelled secretory granules appeared to be significantly smaller in flight-stimulated than in nonstimulated adipokinetic cells. This indicates that flightstimulated adipokinetic cells preferentially release newly formed (= tracer-labelled) secretory granules over older (= unlabelled) ones.

Poster sessions

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POSTER SESSIONS

P1. HISTOCHEMISTRY IN EXPERIMENTAL PATHOLOGY PI.1 Calcium as a Signal in the Activation of Murine Macrophages after Cisplatin or Carboplatin Treatment. J. P. Palma and S. K. Aggarwal Department of Zoology, Michigan State University, East Lansing, MI, 48824-1115, U.S.A, In vitro and in vivo studies have demonstrated the ability of anticancer drugs cisplatin and its analogue carboplatin to enhance the immune system through an activation of macrophages. Macrophages aspirated from the peritoneal cavity of the mouse when exposed to cisplatin (9 ~g/ml) or carboplatin (50 ~g/ml) for two hours and co-incubated with tumor cells (S-180) show extension processes that can recognize tumor cells. There is also an increase in the lysosomes that are transferred to the tumor cells leading to their lysis. Calcium (4 mM CaCI2) alone is able to induce extension formation in the macrophages, however, it is not able to induce lysosomal increase nor tumor celt recognition. EDTA (2.5 raM) can inhibit the extension formation induced by cisplatin or carboplatin. Activation of macrophages is also achieved by co-incubation with tumor cells that have previously been treated with cisplatin or carboplatin. The lysosomal changes in the macrophages and their transfer to the tumor cells after cisplatin or carboplatin was studied using acridine orange as the probe under the ACAS 570 Interactive Laser Cytometer outfitted with a confocal microscope. Calcium seems to be the priming signal for extension formation in the macrophages while cisplatin and carboplatin are essential in the lysosomal increase, recognition and tumor cell lysis.

P1.2 Treatment of Mouse with Cytostatics and Ability o f Immunity Induction in the Treated Animal. G. Hoser, J. Kawiak and M. Kawalec Department of Cytophysiology, Medical Centre of Postgraduate Education, Warszawa, Poland. Treatment of leukemia in animals and patients with cytostatics is performed according to standard protocols. Agressive treatment is preferred in several protocols as it induces prolonged remission in patients. This, however, has several side-effects including those on the immunological system of treated animals. Previously, we described a method for the induction of immunity against mouse L1210 leukemia in animals. Immunity was induced by repeated immunizations with non-proliferating mafosfamide-treated L1210 leukemia cells and the effect was then tested with malignant L1210 leukemia cells. This system was used to test the influence of the treatment on animals with cytostatics according to different protocols on ability of animals to induce the immunity against leukemia. Bibliography: M. Kawalec, M. Jakobisiak, T. Skorski, J. Kawiak (1982). Immunogenicity of cyclophosphamide~ treated leukemia cells. Folia Hiolog. (Praha) 28, 3 3 4 - 43.

Skorski T, Kawalec M, Kawiak J. (1991) Early induction of immune resistance against leukemia in mice after lethal irradiation followed by syngeneic bone marrow transplantation and injection of syngeneic leukocytes. Transplantation 51, 843 - 47.

P1.3

Ultraviolet Radiation Activates Bullous Pemphigoid Antigen Gene in Mouse Ears. K. Kayashima ~, T. Koji 2, M. Nozawa 3, T. Ono ~ and P. K. Nakane 2 ~Department of Dermatology, Kumamoto University Medical School, Kumamoto 860, n; :Department of Anatomy Ill, Nagasaki University School of Medicine, Nagasaki 852, ~Daiichi Pure Chemicals, Tokyo 103, Japan. Bullous pemphigoid (BP) is an autoimmune blister disease and is known as a photoaggravated dermatosis, however the mechanism of aggravation is still unknown. Since there are recent reports that damage to DNA initiates transcription of some genes, we investigated the relationship between DNA damage by ultraviolet (UV) radiation and BP antigen gene activation. Albino male mice were irradiated with 254 nm wave length UV radiation for a total dose of 500Jm -2. At 0.5, 2, 24, 48 and 72 h after irradiation, mouse ears were cut off, frozen and sectioned. Pyrimidine dimers, as studied by immunohistochemistry, were observed in all epidermal cells at 0.5 h and were almost repaired at 72 h. DNA-nick positive cells as studied by in situ nick translation were observed in all layers at 0.5 h and signal intensity increased at 24 h. BP antigen mRNA as studied by in situ hybridization using thymidine dimerized DNA probes was detected in nuclei of basal cells at 0.5 h and was detected in both nuclei and cytoplasm at 2 h. BP antigen, as studied by immunohistochemistry, increased in the basement membrane as time passed after UV irradiation. It is suggested that UV radiation increased BP antigen synthesis through BP antigen gene activation. This DNA-damageinducible transcription may be one of factors which aggravate BP.

P1.4 (Immuno)histology of the Rat Thymus after Cyclosporin Treatment. E. J. de WaaP, C. F. Kuper l, R. Broekhuizen 2, H. van Loveren 1 and H. Schuurman ~'2 1National Institute of Public Health and Environmental Protection, PO Box 1, 3720 BA Bilthoven, and :University Hospital, Utrecht, The Netherlands. In immunotoxicology, Cyclosporin A (CsA) was given special interest as a model immunotoxic compound. We evaluated its effects on thymus (immuno)histology. After a two-week course of daily injections (subcutaneously, 15 mg kg -~, or intravenously, 7.5 mg kg -~, histology of the thymus showed the near disappearance of the medulla; the histological section only showed the impression of a thymus cortex. This

506 phenomenon was studied by immunohistochemistry for markers of lymphocytes and stroma including reticular epithelium and medullary interdigitating dendritic cells (IDC). Lymphocytes with a mature T-cell phenotype were almost absent. Also the microenvironment made by medulla-type epithelium and IDC (defined by their MHC-class II expression) was almost absent. The vasculature in the medulla manifested changes identified in laminin staining. The apparent disappearance of IDC (MHC-class II staining) was confirmed by electron microscopy. Only very few IDC's were left, showing some alterations at the ultrastructural level. After discontinuation of CsA treatment, a rapid recovery occurred, with reappearance of the medulla after 2 weeks. These areas differed from the normal medulla by the absence of medulla-type epithelium. This cell population recovered at its normal location in about 4 weeks. The reappearance of medullary IDC was associated with that of lymphocytes of mature T-immunophenotype. In the regenerating thymus, immunohistochemistry revealed the presence of 'holes' in the microenvironment that lacked epithelium and IDC, and that were filled by lymphocytes with an immature cortex phenotype. These changes in thymus (immuno)histology may be associated with changes in shaping the T-cell repertoire (defects in induction of self-tolerance), as can be concluded from the so-called 'syngeneic graft-versus-host disease' that occurs under special conditions after CsA treatment.

P1.5 Fluorescent Peroxidation Products in Rats with Iron Overload. Effect of Dietary Vitamin E. S. Parkkila ~, O. Niemel~i2, B. R. Bacon 3, R. Singh 3 and

R. S. Britton 3 Depts. Anatomf and Clinical ChemistrJ, University of Oulu, Finland. Div. of Gastroenterology and Hepatologf, St. Louis University, St. Louis, MO, U.S.A.

Chronic iron overload causes hepatic lipid peroxidation with an increase in hepatic malondialdehyde (MDA). A covalent binding of MDA to macromolecules may produce fluorescent products. In this study we have examined the formation of fluorescent products in the hepatocytes of rats with iron overload and determined the effect of dietary vitamin E on fluorescence intensity. Three groups of rats were fed different diets for 2 months. The iron-loaded group received chow diet (50 IU a-tocopheryl acetate/kg) containing 2.5% carbonyl iron, while the iron-loaded, E-supplemented group received the same diet containing an additional 200 IU a-tocopheryl acetate/kg. Control animals were fed chow diet. Autofluorescence of the liver sections was studied at rhodamine excitation/emission wavelengths using a confocal laser scanning microscope and quantitated by digital image analysis. The fluorescence index was 49 _+ 4 in iron overloaded rats, 15 +_. 5 in iron overloaded, vitamin E-supplemented rats and 5 _+ 3 in rats with normal diet. The iron-loaded group had increased amounts of fluorescent granules locating principally in the periportal zone. Using Pearls'Prussian blue staining the iron colocalizes with the fluorescent material.

Poster sessions

P1.6 Influence of Calcitonin and Calcitonin Gene-Related Peptide Biosynthesis on Cell Proliferation in a Spontaneous Rat Medullary Thyroid Carcinoma (MTC). F. Treilhou-Lahille t, G. E. Voile ~, E. Pidoux 2, A. F. L~ger ~

and M. S. Moukhtar z ~URA 1116 CNRS, UniversitdParis-X[ Orsay, 91405 ORSA Y Cddex France, and eU 349 INSERM, 6 rue Guy Patin, 75010 Paris, France.

Morphological steps of C cell tumourisation have been established from diffuse and nodular hyperplasia to malignant MTC when invaded by capillaries. In patients, turnout progression is regularly followed by radioimmunoassays of circulating calcitonin (CT). However, along with turnout growth, dedifferentiation of the secretory phenotype occurs, i.e. loss of intracellular calcitonin stores due to a dramatic decrease of the number of secretory vesicles; moreover, in many cases, calcitonin gene-related peptide (CGRP), the alternate peptide of the CALC-1 gene, is overexpressed, both events leading to discrepancies between the assays and turnout growth. As the principal manifestation of tumoural development is the abnormal enhanced cellular multiplication, it would be important to correlate proliferation and dedifferentiation, especially during the first steps of tumour development, i.e. nodular hyperplasia or microcarcinoma. The in situ informations on CT and CGRP biosynthesis are recent and nothing is known about hyperplastic and malignant cell proliferation. We thus developed a simultaneous detection of both peptides biosynthesis by immunocytochemistry (ICC) and in situ hybridization (ISH), and cell cycle by autoradiography of thyroids injected in vivo with tritiated thymidine. We show that diffuse hyperplastic C cells are characterized by high levels of both peptides with detectable amounts of CT mRNA alone, along with very few S premitotic phase. In the small nodules, the only change is the first appearance of detectable amounts of CGRP mRNA, proliferation level is still low. Actual MTCs looked heterogeneous with a periphery similar to micronodules aspect. The centre was devoided of CT immuno-reactivity but showed important amounts of both CT and CGRP mRNAS and numerous S phases. However, some highly proliferating regions were negative to all markers suggesting either a complete dedifferentiation of the cells or proliferation of still embryonic stem cells.

P1.7 Oil Red O-Positive Cells in the Rat lung. F. van Meir and D. W. Scheuermann Laboratory for Cell Biology and Histology, University of Antwerp (R UCA), Groenenborgerlaan 171, B-2020 Antwerp, Belgium.

The distribution of interstitial lipid containing cells and alveolar macrophages changes significantly during lung development and after vitamin A administration. For use in light microscopy, a simple method is described to stain lipid inclusions in the interstitial cells and macrophages of the lung. The lungs were removed from the thorax and injected with a mixture containing 1 part of the embedding medium O.C.T. compound (Tissue-Tek R) and 1 part PBS. Tissue blocks of the inflated lungs were quickly mounted on prechilled specimen

Poster sessions holders and plunged in liquid nitrogen. 20 ~m sections were stained with Oil Red O. Alveolar macrophages, harvested by bronchoalveolar lavage, were fixed in 4% buffered formaldehyde, pH 7.2, whereafter cytospin slides were prepared and air dried. Both cryosections and cytospin preparations were soaked for 2 rain in absolute propandiol, stained for 45 rain in 0.5~ Oil Red O solution, differentiated for 1 rain in 80% propandiol and rinsed in aqua bidest. Optionally, nuclear staining with Erhlich's haematoxylin for 3 0 - 4 0 sec may follow the Oil Red O-staining.

507 that diets enriched with ~23 PUFA's may be a promising contribution for cancer treatment. This work was supported by Jahres Fabrikker, Sandefjord, Norge by providing the ~23 PUFA's.

P1.9 In situ Detection of the Messenger RNAs Coding for

Somatostatin, Calcitonin and Calcitonin, GeneRelated Peptide in Rat Medullary Thyroid Carcinoma Cells.

P1.8 The Effects of Diets on Lipid Peroxidation and its Consequences for Cell Proliferation in Regenerating Rat Liver. L M. C. Vogels ~, K. S. Bosch ~, G. J. Romijn z,

R. J. A. Wanders 2 and C. J. F. van Noorden ~ ~Laboratory of Cell Biology and Histology, 2Department of Paediatrics, University of Amsterdam, The Netherlands.

Certain products of lipid peroxidation (LPO) inhibit cell proliferation and both in regenerating liver and in tumours, low levels of LPO have been found. LPO can be manipulated by composition of the diet. Therefore, we investigated regeneration of livers of partial hepatectomized rats on diets enriched with either ~23 or ~26 polyunsaturated fatty acids (PUFA's) and the regeneration of livers in rats on a low fat diet. The capacity of cell proliferation in the three groups was determined with immunohistochemical detection of incorporated BrdU. Metabolic changes in livers of the three groups of rats were analysed by histochemical determination of reduced glutathione and neutral fat content and G6PDH activity whereas acylCoA-oxidase activity was determined biochemical]y. There was effective suppression of cell proliferation of 50%0 after partial hepatectomy in rats fed a high ~3 fat diet and 27% in rats fed a high ~26 fat diet. ~23 fat diet also induced a strong reduction of G6PDH activity. Feeding ~3 fatty acids induced a 5 times higher activity of acylCoA-oxidase and no increase in intracellular lipid droplets, this in contrast with ~26 fatty acids or a low fatty acid diet after partial hepatectomy. Diets rich in PUFA's resulted in high levels of the antioxidant glutathione as compared with a low fat diet. It can be concluded that diets enriched with PUFA's affect the antioxidant system in a positive way whereas only diets enriched with ~23 PUFA's inhibit cell proliferation after partial hepatectomy. It may be concluded that, as a consequence of the high acylCoA-oxidase activity, the metabolic breakdown of lipids functions as a major source of free radicals and that LPO form important components of physiological cell regulatory processes. These results indicate

L. Ouazzani ~, G. E. Voile l, P. LeGuellec ~, E. Pidoux z, M. S. Moukhtar 2 and F. Treilhou-LahilW ~URA 1116 CNRS, UniversitdParis-XI Orsay, 91405 Orsay Cddex France, and 2U349 INSERM, 6 rue Guy Patin, 75010 Paris, France.

Medullary thyroid carcinoma is a malignant neoplasm which arises from mammalian endocrine C cells. Their main secretion is calcitonin, but they also contain small amounts of CGRP, the alternate peptide of the CALC-I gene. Besides, somatostatin has also been found in some normal C cells. Recently we have developed a rapid detection of CT mRNA in rat normal and tumoral C ceils, by in situ hybridization on paraffin sections with a synthetic oligodeoxyribonucleotide probe labelled with digoxigenin. Using the same method combined with immunocytochemistry, we compared the expression of the somatostatin gene with that of the two peptides encoded by the CALC-1 gene during the successive steps of MTC progression, in rat thyroid developing spontaneously this neoplasm. Both peptides of the CALC-1 gene were equally detectable in normal and diffuse hyperplastic C cells. Calcitonin mRNA was abundant but CGRP mRNA was hardly observable, probably due to its very low level. The first tumoural event is a multifocal nodular hyperplasia of enlarged C cells in several adjacent follicles. These cells displayed high levels of both peptides and mRNAs. During turnour progression, the follicles fuse to form a dense solid mass. Cells lose their intracellular calcitonin stores but we always observed high levels of CT mRNA. CGRP was often overexpressed in the well-developed tumours where MTC cells were particularly rich in CGRP mRNA. In normal thyroids, somatostatin immunoreactivity was scarce, only found in few intrafollicular cells gathered in small areas which always displayed high levels of somatostatin mRNA. Both peptide and mRNA were colocalized with the products of CALC-1 gene in tumours at all steps of development, somatostatin producing cells were located in areas where all the cells were positive with both ICC and ISH. However their importance was variable, from 0 to 100o70 of the tumour cells.

508

Poster sessions

P2. HISTOCHEMISTRY IN TOXICOLOGY P2.1 Immunocytochemical Demonstration of Metallothionein in Cultured Canine Proximal Tubular Cells using Monoclonal Antibody. T. Hamada, A. Tanimoto, H. Teramoto, S. Iwai and O. Koide University of Occupational and Environmental Health, Japan. Monoclonal antibody (MoAb) against metallothinein (MT) was obtained by immunization of mouse with rabbit MT-I conjugated bovine serum albumin as antigen. One (C2) of the MoAbs for MT-I (MoAb-MTI) was found to react with rabbit MT-II, rat MT-I and MT-II, and 10,000 xg supernatant of liver and kidney homogenate of Cd-exposed beagle by ELISA using the biotinized MoAb-MT-I. Canine proximal tubular cells were separated from renal cortex of a beagle by density gradient centrifugation. These cells (B131 cells) were proved to be derived from proximal tubules by characteristic dome formation, enzyme histochemical and electron microscopical studies. Immunocytochemical reaction was performed by indirect method on the B131 cells cultured in chamber slides with or without Cd exposure. Cd exposure was done with the medium containing 10 to 100microM Cd for 4 to 24hours. Metallothionein was demonstrated both in the nuclei and cytoplasm of the B131 cells. There was no remarkable differences in the staining intensity and localization pattern between Cd exposed and control cells. These results indicated that the B131 cells had a considerable amount of MT without induction by metal exposure. Therefore, MT induction by Cd exposure, even if it had happened, could not be detected. The MT occurring in the cultured cells may be associated with cell proliferation.

P2.2 Tamoxifen-Induced Hepatocellular Carcinoma in Rat Liver: an Enzyme Histochemical Study. P. Hirsim/iki ~, P. Kellokumpu-Lehtinen2, C. S6derstr6m 1, Y. Hirsim/iki3 and L. Nieminen 3 ~Turku University Central Hospital, Department of Pathology, SF20520, Turku, Finland; ~University of Tampere, Department of Radiotherapy, SF-33520 Tampere, Finland; ~Orion Corporation Farmos, PO Box 425, SF-20101, Turku, Finland. The effects of two triphenylethyl antioestrogens tamoxifen (TAM) and toremifene (TOR) were studied on rat liver enzyme histochemistry after one year treatment. Female Sprague-Dawley rats were administered orally for one year with equimolar dose levels: TAM 11.3 and 45 mg/kg, TOR 12 and 48 mg/kg (n = 5), controls received the suspension medium CMC. Reduced body weight gain was observed with both antioestrogens. Alopecia and aggressiveness were observed in the high TAM dose group. Specimens of the livers, observed lesions and tumours were taken. In autopsy after one year treatment, liver tumours were observed in all five rats treated with the high TAM dose. No tumours were observed in the TOR treated rats. At the high TAM dose level, eosinophilic and basophilic foci as well as hyperplastic areas were observed. In three of these rats hepatocellular carcinoma was also observed. Such changes were not observed in TOR

treated rats. In histochemical studies, liver glucose-6phosphatase (G-6P) staining remained similar in the control, TOR and TAM treated rats throughout the study except the slight increase of activity in the beginning of the study. However, tumours of the high TAM dose level had no or only slight G-6P activity. Liver ATPase activity was weak but increased slightly with both antioestrogens towards the end of the study. No or very weak gamma-glutamyltranspeptidase (GGT) activity was observed in the control and TOR treated rats. In the high TAM dose group, GGT-positive foci were observed in the livers after 26 and 52 weeks treatment. In the TAM tumours, moderate or strong activity was observed. The glucose-6-phosphatase dehydrogenase activity was weak or moderate in the controls, TOR and TAM groups. In the TAM tumours, strong activity was observed. This study confirmed the earlier finding that TAM induces hepatocellular carcinoma. The histochemical studies further convinced us that the tumours are malignant.

P2.3 The Effect of Cyclosporin A on Expression of Renin in the Rat Kidney. C. N. Kind and V. Gill Fisons plc, Pharmaceutical Division, Research & Development Labs, Bakewell Rd, Loughborough, Leics., LEll ORH, UK. Nephrotoxicity is a major side-effect of the immunosuppressant Cyclosporin A (CsA) in clinical use. Although the precise mechanism of CsA-induced renal dysfunction is not yet established, increasing evidence indicates that its pathogenesis may involve a disturbance of the reninangiotensin system (RAS). CsA is known to alter plasma renin activity and cause morphological changes to juxtaglomerular cells in both experimental animals and humans. However, less is known about its influence on renin expression within the kidney. The effect of CsA on renin mRNA distribution was investigated using formalin-fixed, paraffin-embedded kidney tissue taken from male Sprague-Dawley rats following daily oral dosing at 27 or 81mg CsA.kg -~ for 14 consecutive days. Tissue from vehicle-dosed control animals were examined for comparison. 5/am kidney sections were prepared and subjected to in-situ hybridisation histochemistry using Digoxigeninlabelled synthetic oligonucleotide probes complementary to rat renin mRNA. Additional sections were stained for glycoprotein using periodic acid-schiff reagent. Kidneys from CsA-dosed rats showed a dose-related increase in the number of juxtaglomerular cells with detectable levels of renin mRNA compared to the control group. Moreover, CsA-dosed animals showed a higher incidence of renin mRNA expression in arteriolar smooth muscle cells proximal to the juxtaglomerular apparatus. Both locations showed evidence of increased PAS staining, indicating the presence of renin peptide. These results suggest that CsA administration causes a marked upregulation of juxtaglomerular renin expression in the rat, together with a recruitment of additional cell populations not normally involved in renin production. Increased renin activity at these sites may directly relate to reduced renal functional capacity (evident in this study from concomitant increases in plasma urea) via systemic and local generation of the potent vasoconstrictor, angiotensin II.

Poster sessions

P2.4 In vitro Drug Toxicity Testing: a New Avenue for the Application of Quantitative Cytochemistry. O. T. Markovic 1, N. S. Markovic 2, L. Rakic 3, D. S. Young 3 and J. Roberts 4 ~University Clinical Center, Belgrade; 2Institute of Oncology, Sremska Kamenica, Yugoslavia; 3University of Pennsylvania, PA and 4Medical College of Pennsylvania, PA, USA. Recent development of antineoplastic drugs targeting metabolic enzymes initiated a new interest for measurement of intracellular enzyme activity either for screening of new compounds or calculation of internal tumor sensitivity to escalating doses of an effective agent. In late eighties, we developed an image analysis based cytochemical technology, MIMDECI R, that may be helpful to meet these new needs. We used this method (1) to search for drugs targeting intracellular enzymes (esterase), and (2) for chemo-sensifive tumors bearing a target enzyme (inosine monophosphate dehydrogenase, IMPDH, EC 1.1.1.205). We found that drugs with different pharmacologic action which bear a quinoline ring-like chemical structure such as quinine, chloroquine, primaquine, chlorpromazine, prometazine, ethopropazine, norfloxacine and ciprofloxacine commonly inhibit carboxylesterase (EC, 3.1.1.1) in a time and concentration dependent manner. The inhibition was measured by image analysis as a decrease of the final reaction product inside cells after their exposure to a certain drug both in vivo and in vitro. Comparable data were obtained from experiments using an enzyme purified from porcine liver and carboxylesterase inside human and mice leukocytes, bovine and mice hepatocyte and neuronal cells, K562 and Hep G2 cell line cells. We applied MIMDECI to measure the sensitivity of tumor cells (acute leukemia, colon cancer, lung cancer, thyroid cancer, breast cancer, K562 and HL60 cell lines) to IMPDH inhibitors such as tiazofurin, and ribavarin. We demonstrated drug-concentration dependent sensitive malignant cells on tumor tissue sections and cell smears. In these experiments, MIMDECI was found to be a sensitive, specific and less expensive method, usable for detection of patients sensitive to enzyme targeting drugs and for screening new substances as potential drugs.

P2.5 )'/6 T Cells and Human Skin Reactivity to Heavy Metals. K. Nordlind and S. Lid6n Department of Dermatology, Karolinska Hospital, Stockholm, Sweden. ),/d T cells might act as a first line of defense and respond to stress signals of the surrounding tissue. In addition, they might participate in the pathogenesis of autoimmune disorders. In the present investigation the occurrence of ),/6 T cells was studied in the human skin after application of different heavy metal salts by routine epicutaneous patch testing procedure. In positive reactions to gold chloride and mercuric chloride, ),/6 T cells were found in a frequency of up to 25~ of the mononuclear cells, in addition to showing epidermotropism. In contrast, very few of these cells were seen after application

509 of salts of nickel and silver, no cells being found in control tissue. The ),/6 T cells expressed the V62 and the )'V2(a) gene segments and by comparison of adjacent slides and double staining seemed to be CD 4-8-, indicating that they have the same phenotype as ),/6 T cells in the peripheral blood.

P2.6 Differentiation of Hypertrophy and Hyperplasia by Fully Automated TV-Image-Analysis in Series of Conventional, Feulgen Stained Liver-Sections. E. D. Wachsmuth Research Department, Pharmaceuticals Division, CIBA-GEIG Y Limited, CH 4002 Basel, Switzerland. The goal was to establish a microscopic method for the routine use in pharmacology and toxicology to quantify, in the liver, the gross cellular response to chemicals. The method must also he applicable to conventionally fixed and embedded tissue, and permit automated feeding of the measuring device, counting of nuclei, possibly differentiating hepatocytes, and thereby determining the frequencies of nuclei in series of sections. The morphometric results can then he compared with the liver weight on an individual basis. Nominally 3 ~m sections of formaldehyde-fixed, paraplast-embedded livers (archived blocks of 1-month toxicity studies; eight compounds with four dose groups; five rats per group) were Feulgen stained. A TAS-Leitz (Wetzlar, FRG) was used for image analysis (equipped: 100 W quartz iodine lamp; N2-filter for incident light; dry objective 40• low-light-level CCD camera [PC LL 1450, Proxitronic, Bensheim, FRG]; x,ymotor driven stage to house 12 slides; z-motor for focus control). An algorithm was developed: to move 12 slides in a preset x,y-position on the microscope stage, to focus on the fluorescent section, detect and count fluorescent nuclei and measure their fluorescence intensity. In up to 24 sections (2 per slide) the nuclei per section were serially counted (>3 mm 2, >100 fields of observation, >12500 nuclei). Hepatocytes and littoral cells were distinguished by their fluorescence intensity (DNA) and size. Compound specific, reproducible (r = 0.94) and statistically significant (p<0.001), systematic dosedependencies of hypertrophy or hyperplasia, or both were established. Mitoses were only observed in hyperplastic livers. Hypertrophy by morphometry was false positive by eye in >18~ and false negative in >28~ The morphometric data agreed well with the findings by electron microscopy.

P2.7 Environmental Toxicology: Glucagon Immunoreactivity and Ultrastructure of Endocrine Cells in Rat Duodenum after Herbicide Saprol Exposure. J. R. Ptashekas and M. R. Ptashekas Lithuanian Institute of Hygiene, Lab. Electron Microscopy, Bistrychios st. 19-1, 232055 Vilnius, Lithuania. Wistar rats 250 gr b.w. had been once tube fed with 0.1 LD50 water solution of piperasin derivate herbicide saprol (Celamerck, FRG), and immunohistochemically both by means of electron microscopy investigated 15 rain., and 1 hour after exposure. Glucagon immunoreactivity was visualized by means of immunoperoxidase and DAB techniques. Number of immunoreactivity showing cells and their relative

510 volume density, granulation index per field at X400 was estimated. Transmission electron microscopy of the samples enabled to verify mast both K, S types of neuroendocrine cells according to Lausanne 1981 Enteroendocrine Cells Classification, and statistically process their secretory granules morphometry data. Thus, 15 min. and 1 hour after chemical irritation glucagon immunoreactivity showing cells got decrease in their number and granulation index (40~ 580/o and 200/0; 21~ of control level respectively). Scattered glucagon cells located unevenly at the bottom part of duodenal glands by lamina propriae

Poster sessions mucosae. Ultrastructural analysis showed expressed vacuolization of cytoplasm matrix and secretory granules in K, S, mast cell types 15 rain. and 1 hour after saprol exposure. Number of secretory granules per 1 sq. mkm of cytoplasm decreased up to 16070, 15%, 59~ of control values respectively 15 min. after irritation. 1 hour after treatment number of secretory granules in S cells was 30~ and in mast cells 46o7oof control level. Average size of secretory granules varied from 102~ to 109~ of control figures. These data correspond to results of prevous studies on prime endocrine reaction after chemical irritation in digestive tract epithelia.

P3. P L A N T C Y T O C H E M I S T R Y P3.1

Ultrastructural Characterization of Electron-Dense Material in the Cell Wall-Plasmalemma Interface of Uninfected Cells of Nitrogen-Fixing Nodules of Peanut. A . K. Bal Department of Biology, Memorial University of Newfoundland, St. John's, NF, Canada, A 1B 3X9.

The nitrogen-fixing peanut root nodules have three layers of uninfected cells adjacent to the infected zone which are distinct from the other cortical cells due to the presence of prominent amyloplasts, lipid bodies (oleosomes), large microbodies and localized pockets of electron-dense material within invaginations of the plasmalemma at the cell wall interface. Samples fixed in aldehydes/osmium tetroxide or only glutaraldehyde and embedded in Spurr's medium have been used to analyse the cytochemical properties of the dense material. Several staining and extraction procedures have been followed, which indicate that the material is resistant to solublization by hexane and chloroform. Electron density was enhanced by en bloc staining with p-phenylenediamine. Aldehyde-fixed specimens could be lightly stained with ferric chloride, uranyl acetate and osmium tetroxide. Preliminary trials with iodine-potassium iodide/sulphuric acid pretreatment for silver proteinate staining have shown positive results indicating presence of suberin or lignin in the dense material. However its binding with heavy metals may be due to phenolic complexes. These cell wall deposits may be related to the presence of rhizobia or to the regulation of water transport in the nodule tissue. Supported by NSERC, Canada.

P3.2 In Situ Detection of Sucrose Synthase Distribution in Developing Maize Leaves. J, Brangeon, B. Nguyen-Quoc and A. Lecharny Laboratoire Structure et Mgtabolisme des Plantes, associg au CNRS (UA 1128), Bgt. 430, Universit~ Paris-Sud, 91405 Orsay Cedex France.

Immunogold labelling was used to detect the cellular and subcellular distribution of sucrose synthase along a developing maize leaf. The protein was detected in thin sections of tissue embedded in LR white acrylic resin by employing a polyclonal antibody preparation, active against the SS2 form of the enzyme. A developing maize leaf exhibits an acropetal lamina

base to tip differentiation; the leaf grows by the activity of a basal meristematic region and an adjacent elongation zone, resulting in a differentiation gradient along the leaf. A tissuespecific distribution related to developmental stages was detected in photosynthetic cells, stomatal cells and intervascular parenchyma elements of the phloem. The cytosol was immuno-reactive for SS in all dividing cells in the meristematic and elongating zones at the leaf base, and in differentiating mesophyll and bundle sheath cells of "juvenile" tissue. In fully differentiated leaf tissue, however, the protein was no longer detectable in photosynthetic ceils, but was present in stomatal guard cells and to a lesser extent in parenchyma ceils within the vascular bundles. The tissuespecific distribution of SS, a key enzyme in carbohydrate metabolism, is discussed as regards to the transition from a heterotrophic to an autotrophic status along the developing maize leaf.

P3.3 Immunolocalization of Isoprenoid Enzymes in Plastids: Chemical Fixation Versus Cryofixation. C. Cheniclet ~, F. Rafia ~, A. Verna 2 and J. P. Carde ~ ~Physiologie Cellulaire Vgggtale, URA CNRS 568, Universitd Bordeaux 1, F-33405 Talence, 2Neurocytochimie-Cytologie, URA CNRS 339, Universitd Bordeaux 2, F-33405 Talence.

The enzyme geranylgeranylpyrophosphate synthase (GGPPS), which controls the formation of diterpenes and carotenoids in plastids, was localized in chloroplasts and chromoplasts from green and red Capsicum fruits, respectively, by immunogold EM cytochemistry of Lowicryl embedded material after chemical aldehydic fixation or cryofixation by slam-freezing on a nitrogen cooled copper block (1) followed by freezesubstitution with pure acetone. In green fruits, where the GGPPS amount is low, the enzyme was nearly undetectable in chemically fixed material, whereas a precise labelling was observed in cryofixed chloroplasts, mainly in the plastid stroma and plastoglobuli. In chromoplasts of red fruits, which contain large amounts of GGPPS, a high density of labelling was observed in both chemically fixed and cryofixed materials. Cryofixation followed by freeze-substitution seems therefore the more reliable method to localize some antigens present in small amount in the tissues and/or highly modified or lost during chemical fixation and dehydration. (1) Verna A., Biol. Cell 49, 9 5 - 9 8 (1983).

P o s t e r sessions P3.4

Absence of N-Aeetyl-D-Giucosamine Residues in Secondary Cell Walls of the Dwarf Mistletoe Korthalsella lindsayi, J. Coetzee Electron Microscopy Unit, University of Pretoria, Pretoria, South Africa. It is known that WGA-colioidal gold labelling of secondary cell walls of plants reveal the presence of abundant amounts of non-chitin N-acetyl-D-glucosamine residues. However, in the green plant parasite Korthalsella lindsayi these residues do not occur in the secondary cell walls of the absorbing cells in the haustoriaI tissue. Secondary cell walls in the rest of the parasite body, as well as the tracheary transport cells in the haustorium, do react with the lectin marker. The absence of this wall component in the host/parasite absorptive interfacial tissues is probably related to the specific function of the haustorial tissues, where high rates of apoplastic transport of water and solutes are known to occur.

P3.5

Quantitative Histochemical Measurement of G6PD Activity in Shoot Apices by Image Analysis. M. Crevecoeu? and X. Albe 2 JPlant Physiol. Univ. Geneva, CH-1211, Geneva. 2Department of Pathology, Cytology, Geneva 51, Bd. Cluse, CH-1205, Geneva. Transition to flowering was induced in the long day plant Spinacia oleracea by the transfer of vegetative short days plants (8 h white light) to continuous light. The changes occurring in the meristematic shoot ceils during the first hours of this photoperiodic induction were previously investigated. The earliest modification so far observed was an approximate doubling of glucose-6-phosphate dehydrogenase (G6PD) activity, in the axial zone of the shoot apex, in plants transferred to continuous light for 15 to 17 h (1). The reaction was quantified with a Leitz MPV2 scanning integrating microdensitomer. We are now interested in quantifying the G6PD activity with a computerized image analyser (BIOCOM 200, Les Ullis, France) linked to a Leica laborlux S microscope. It consists of a CCD black and white video camera and a color video display monitor. Each picture was converted to a digital image 512 x 512 pixels with 256 grey levels per pixel. The measurements were made using an interferentiel filter (546nm). The histochemical G6PD reaction was continuously monitored on frozen sections. The images gene-rated were stored at 2 rain intervals over a 20 rain period. The grey levels were converted to optical densities and the mean integrated optical density calculated with time. A series of assays that verify the reliability and validity of the system are presented. (1) Auderset et al. (1980) Plant Sci. Lett. 20, 109-113.

P3.6 Immunolocalization of a Glycoprotein Component in Lupin Nodules. M. R. de Felipe, C. A. de Lorenzo, M. M. Lucas and M. M. Fernfindez-Pascual Centro de Ciencias Medioambientales, CSIC. Serrano 115 his. 28006 Madrid, Spain. In this work it is shown that the localization of a nodule protein is related to the oxygen diffusion barrier placed in the

511 cortex of legume nodules. The rate of oxygen diffusion to the central part of nodules depends on the regulation of this barrier, which can be affected by environmental factors. On pursuing the identification of this barrier, conventional electron microscope studies were used to examine the composition of the nodule cortex in Lupinus plants grown under control conditions and with nitrate applications. The lupin nodule cortex shows 2 - 3 layers of cells with extremely thick cell walls surrounding large intercellular spaces. These layers are located between the outer and inner cortex. Van den Bosch et al. (EMBO J. 8, 3 3 5 - 342, 1989) found a glycoprotein present in the intercellular spaces of pea and soybean nodule cortex by means of the monoclonal antibody MAC 236. We performed silver-enhanced immunolabelling with MAC 236 (kindly provided by Dr N. J. Brewin, John Innes Institute, UK) to the lupin nodule cortex, and observed that the intercellular spaces surrounded by the thick cell walls were the main sites of glycoprotein accumulation in lupin nodules. Nitrate caused a higher occlusion of the spaces after 4-days treatment. At the EM level, glycoprotein localization was observed on a thin matrix in the spaces mentioned above, either in the corners or occluding the whole space. Quantification of immunolabelling rendered a significant increase after 4 days of nitrate treatment. It is proposed that the thick cell walls and the glycoprotein occlusions are the components responsible for the oxygen diffusion barrier in lupin nodules.

P3.7

Histological Evidence for Peroxidase Activity on Isoorientin in Tissues of ChamaelauciBm uncinatum Schauer. P. CuriP, M. Dolci~, F. Mariani ~ and A. Mercuri ~ ~lstituto Sperimentale per la Floricoltura, Sanremo, Italy; 2DI. VA.P.R.A., Chimica Agraria, Universit~ di Torino, Italy. Chamelaucium uncinatum Schauer, a plant cultivated for flowering branches, contains large amounts of the uncommon flavone isoorientin, that however lacks in lignifled tissues. Since a high peroxidase activity was detected in the herbaceous tissues, a possible role of isoorientin as a substrate for peroxidase has been investigated. In in vitro experiments carried out in a solution of isoorientin 0.1 mM in phosphate buffer p H 6.0 using horseradish peroxidase in the presence of 0.05 mM hydrogen peroxide, a peroxidative reaction was evidenced by a colour change from pale yellow to deep yellow. The same reaction occurred when the previously described solution of isoorientin and hydrogen peroxide was applied to C. uneinatum sliced tissues without exogenously supplied peroxidase. In this way the cellular localization of endogenous peroxidase activity can be easily detected, because the applied solution gives a deep yellow colour only in the areas where peroxidase is present. This reaction, that is not seen when hydrogen peroxide is absent, proves its specificity for peroxidase detection. Peroxidation products of isoorientin are simple phenols, acids and polymers. Considering that isoorientin is exclusively present in the herbaceous tissues of the plant, this kind of reaction could be involved in the ligniflcation process of C. uncinatum tissues.

512

P3.8 Flow Cytometric Analysis of Polysomaty During Organ Development in Cucumber. L. IV. J. Gilissen, M. van Staveren, T. Creemers-Molenaar

and H. A. Verhoeven DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), P.O. Box 16, NL-6700 AA Wageningen, The Netherlands.

Several studies suggest that the nuclear DNA amount of protoplasts is an important parameter for their competence to cell division and plant regeneration. Therefore, the nuclear DNA content of cells from various organs of cucumber (Cucumis sativus), to be used as protoplast donor tissues, was determined by flow cytometry during the various developmental stages in vitro, i.e. from embryo to flowering plant. The results revealed the presence of organ specific distribution patterns of various ploidy levels in a given tissue (polysomaty), the maximum range being 2C to 64C. Endopolyploidization mainly occurred: 1) during tissue differentiation in the developing embryo, 2) during early stages of seed germination, 3) preceding elongation of the stem internode, 4) during flower maturation, and 5) during aging of the plant. In the germinating seed, rapid increase of polyploidy was observed, that originated in the root and passed through the hypocotyl to the tip of the cotyledons. Also in the youngest stem internode a rapid endopolyploidization was observed. The leaves and the petioles maintained the highest frequency (i.e. 60%) of diploid cells, as compared to all other organs. The results suggest, that endopolyploidy and polysomaty are essential for normal organ development and differentiation in cucumber. In order to find a possible relationship between ploidy level and competence of protoplasts for cell division and plant regeneration, the distribution of the cells with different DNA contents over various tissues is under investigation using confocal laser scanning microscopy.

P3.9 Outer Membrane Protein Gene Expression Traced by lmmunogold Labelling in Rhizobium Leguminosarum BV. Viciae Strain 248 In Vicia Sativa ssp. nigra. L. Goosen-De Roo*, R. A. de Maagd + and B. J. J.

Poster sessions the mature bacteroids. The young bacteroids were located in the invasion zone and in the early symbiotic zone whereas the mature bacteroids were located in the late symbiotic zone of the nodules. Therefore, the decrease of the MAb 38 epitopes is clearly correlated to a specific stage of differentiation of the bacteroids and to a certain site in the nodule. This indicates that the expression of the RopA protein is restricted to a specific stage of the symbiosis. (1) R.A. de Maagd et al. (1989) J. Bacterial. 171: 1136- 1142. (2) R.A. de Maagd et al. (1992) J. Bacterial. 174: 214-221.

P3.10 Microfilamental and Microtubular Cytoskeletons during Induction of Embryogenesis in Microspore Cultures of Brassica napus L. B. Hause, G. Hause and A. A. M. van Lammeren Agricultural University Wageningen, Department of Plant Cytology and Morphology, Arboretumlaan 4, NL-6703 BD Wageningen. Cytoskeletons were investigated from 0 till 24 h of culture at high (32~ and low embryogenic (25~ and in situ conditions using rhodamine-phalloidin staining for microfilaments (MFs) and indirect immunofluorescence for microtubules (MTs). Uninucleate and dividing microspores (MSs) in situ exhibited no MFs whereas comparable cells, cultured for 6 h showed clear MF configurations. In situ grown binucleate pollen had prominent MF arrays in a banding pattern whereas binucleate cells formed in culture showed random configurations of MFs, independent of culture temperature. After 24 h MSs divided symmetrically and showed fine MFnetworks in the daughter cells. The change in MF configuration is likely caused by manipulation rather than by differences in temperature. Directly after isolation, 300/0 of the MSs did not show MTs, 30% exhibited configurations as observed in situ, and 40% showed either numerous short and thick, or only some thin bundles of MTs. These cells likely become embryogenic. Preprophase bands were not observed but symmetrical divisions were preceded by a remarkable shift of 90 ~ in spindle orientation. Hereafter MTs were found parallel and adjacent to the division plane.

Lugtenberg* *Institute of Molecular Plant Sciences, Botanical Laboratory, Nonnensteeg 3, 2311 VJ Leiden, The Netherlands. +Present address: Plant Biology Laboratory, Sulk Institute for Biological Sciences, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.

A monoclonal antibody (MAb 38) recognizes an epitope of an outer membrane protein of Rhizobium legurninosarum by. viciae strain 248 (1). The gene (ropA) encoding this protein has been cloned and sequenced (2). The expression of ropA is repressed during symbiosis of Rhizobium legurninosarum bv. viciae in pea. MAb 38 was used to follow ultrastructurally the presence of the RopA protein in nodules of Vicia sativa spp. nigra. In order to detect the epitope on the RopA protein, fixation of the nodules, infiltration in LR White and polymerization had to be carried out at - 2 0 ~ Quantification of the gold labelling showed that young bacteroids have a gold particle density five times higher than

P3.11 Cytochemical Studies of Peroxisomes in Leguminous Plants. Y. Kaneko and H. Matsushima Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, 338, Japan.

At least three kinds of functionally specialized peroxisomes are observed in leguminous plant tissues. Glyoxysomes are involved in the storage lipid metabolism of germinating seeds. Leaf peroxisomes are taking part in photorespiration by processing glycolate in photosynthetic tissues. Finally, peroxisomes in ureide producing root nodules are known to be specialized for oxidation of urate. These types of peroxisomes contain malate synthase, glycolate oxidase and urate oxidase as marker enzymes, respectively.

Poster sessions As a step to elucidate development, occurrence and interrelation of various kinds of peroxisomes in the life cycle of leguminous plant at the ultrastructural level, cytochemical procedures were applied successfully to localize the marker enzymes of each type of peroxisome in various tissues. Malate synthase was detected by the ferricyanide reduction method. Glycolate oxidase and urate oxidase were localized using the indirect DAB method and the cerium method. The results of the ferricyanide reduction method and the cerium method were further confirmed by X-ray microanalysis.

P3.12

Are Histochemical Substrates for Non-Specific Acid Phosphatases Hydrolysed by Phytases in Plant Nutritional and Embryonic Tissues? Z. Nikolova and R. Gossrau Department of Anatomy, Free University of Berlin, Germany. Previous histochemical studies on plant seedlings have shown a dual localization for non-specific acid phosphatase (aP) in and outside globoids of protein bodies but not for all other acid hydrolases which are absent in the globoids. Since globoids are said to contain myoinositol phosphate (phytate)hydrolysing phytases the possibility of this enzyme in the participation of hydrolysing aP-substrates was analysed using naphthol AS compounds and mustard seedlings of day 2 of germination. All naphthol AS substrates were hydrolysed equally well. When the reaction was performed at the pH optimum of the phytases staining did not appear in the extragloboid areas but was present in the globoids of nutritional and embryonic cells. Inhibitors, which abolish or suppress aP activity, e.g. fluoride or cupric ions prevented staining outside but not in the globoids. Furthermore, phosphate groups could be visualized inside the globoids by substrate-free lead or cerium media. Therefore, histochemical substrates for aP may also be hydrolysed by phytases in nutritional and embryonic cells of plant seedlings.

P3.13

Immunoehemical Surface Labelling of Plant Protoplasts for Prefusion Aggregation and for Optical Selection of Heterokaryons. W. J. P. van Kesteren ~, T. Bakker Schut 2, W. Barlag 3 and M. J. Tempelaar ~ ~Dept. of Genetics, University of Groningen, PO Box 14, 9750 AA Haren; 2TUT, PO Box 217, 7500 AE Enschede; JAZG, PO Box 30.001, 9700 RB Groningen, The Netherlands. Biotinylation and subsequent incubation with avidin can effectively be applied on plant protoplasts in experiments on aggregation and optical selection. In mixtures of protoplasts, that were biotinylated only, with protoplasts that were both biotinylated and avidin saturated, extensive cell aggregation occurred. An increase in heterologous contact sites was observed, favoring formation of heterokaryons instead of hamokaryons after fusion. Both PEG fusion and electrofusion could be applied in these aggregates. Biotinylation offers, in addition to an expected increase in hybrid yields via prefusion aggregation, a universal procedure for attaching a wide range of labels to protoplasts, including colloidal gold, ferritin, enzymes and fluorochromes. Colours

513 of fluorochromes range from green via yellow and orange to red, and immunochemical surface labelling offers a multicolour staining procedure for plant protoplasts. For optical discrimination of heterokaryons, labelled and elecrofused protoplasts were evaluated by fluorescence microscopy and flowcytometry. It was concluded that immunochemical aggregation of protoplasts may raise heterokaryon yields, and that immunochemical staining may facilitate optical selection of these heterokaryons.

P3.14 Gene Mapping on Interphase Nuclei and in

Arabidopsis thaliana. P. van Oostveldf, S. Bauwens 2, G. Engler 2 and M. van Montagu 2 ILaboratorium voor Biochimie, Faculty of Agricultural Sciences, 2Laboratorium voor Genetica, Faculty of Sciences, State University Gent, Coupure Links 653, 9000 Gent, Belgium. Arabidopsis thaliana, a member of the mustard family, is recognized by classical geneticists as a good experimental organism and a useful model system for flowering plants. Recent progress is in the area of genome mapping and sequencing of specific genes amply demonstrate that Arabidopsis is useful to plant biology and possibly higher eukaryotes in general. The assembling of a physical map of the genome of Arabidopsis based on yeast artificial chromosomes (YACs) hybridized to different restriction fragment length polymorphisms (RFLPs) mapped in different linkage groups is not always straightforward due to the occurrence of dispersed repetitive sequences or the localization of some genes on different chromosomes. Although the chromosomes of Arabidopsis are too small to do high resolution cytogenetics on metaphase chromosomes, the technique of non-isotopic in situ hybridization (NISH) with different probes makes the localization of a library of YACs, cosmids or plasmids possible on interphase nuclei. A statistical analysis of the images can correlate the localization of genetic elements to particular locations along the chromosomes. We present the results of different NISH experiments with YACs and single genes with known or presumed localization. Our results illustrate the general utility of this approach and show the advantages of NISH for fast and accurate localization of cloned genome sequences.

P3.15 Effect of CaZ+-ATPase Inhibitors on Pollen Tube Growth and Intracellular Ca2+-distribution. J. Novoa, H. Quader and H.-D. Reiss Zellenlehre, University of Heidelberg, Germany. In the presence of inhibitors of the ATP-dependent Ca 2+pump of the ER, cyclopiazonic acid (CPA, 1-100 laM) or thapsigargin (1-100/aM), organeUe movement in growing pollen tubes of Lilium longiflorum slows down within 60 rain. At inhibitor concentrations >1 laM the tubes grow irregularly, at about 100/aM they begin to curl and growth proceeds in a spiral pattern, and at concentration >100/aM growth, stops immediately. Both the inhibitors cause in the tip region of the tubes a distinct increase in the cytosolic Ca2+-concentration as measured with the fluorescent CaZ+-indicator fluo-3 using a

514

Poster sessions

confocal laser scanning microscope (CLSM). However, the treated pollen tubes still show the tip-to-base calcium gradient characteristic of untreated tubes. The effect is thought to be due to the inhibition of the ATP-dependent Ca2+-pump of the smooth ER which is mainly observed in the tip region of pollen tubes. It may function as the major calcium-

P4. D E V E L O P M E N T A L

sequestering membrane system. Ultrastructural studies reveal that the zone of exocytosis (the very tip) is enlarged and, in some cases, shifted to the apical flanks. The results demonstrate that the fine regulation of cytosolic Ca2§ within the tip is particularly important for the regular growth of pollen tubes.

BIOLOGY AND AGING

P4.1 Changes in Liver Pigmentary Cells During Amphibia Hibernation. S. Barni, G. Bottiroli*, V. Bertone, A. C. Croce* and I. Freitas Dept. of Animal Biology and *CNR Center for Histochemistry, Pavia University, Italy. Morpho-histochemical changes of the liver parenchyma were found in Rana esculenta during natural hibernation (Barni et al. J. Exp. Zool. 258: 143, 1991). Liver pigmentary cells in Amphibia were shown to be derived from Kupffer cells and seem to play a protective role against oxidative free radicals; an inverse relationship between pigment storage and SOD activity was found (Sichel, Cell Biochem. Funct. 2 : 1 2 3 , 1987). In this work liver pigmentary components were investigated during the active (July) and hibernating (January) phases in Rana esculenta and Triturus alpestris with image analysis, electron microscopy and spectrofluorometry. In the two species the pigmentary components significantly increased, both in content and in the dispersion, during hibernation with respect to the activity. Electron microscopy revealed that the pigmented cells were hypertrophic and melanosomes had a more heterogeneous matrix during hibernation. Furthermore, a remarkable storage of moderately osmiophilic lipid droplets and residual bodies was found. Occasionally, pigment granules were observed also within hepatocytes, characterized by high numbers of cytosegresomes. Spectral analyses on both cryostatic sections and liver homogenates after extraction with solvents of different polarity, revealed the presence of 3 spectral components in the active animals. In the hibernated animals a broadening of the long-wavelength emission band along with the appearance of a new band in the short-wavelength region were detected. The latter may be ascribed to the residual bodies observed at ultrastructural level. The findings can be interpreted according to the role of pigments in the defense against uncontrolled lipid peroxidation. This role is enhanced during the metabolic quiescent period of the organ.

P4.2 Histochemical Study on Carbohydrates in Staged Human Embryos - - Special Reference to the Digestive Organs. K. Hashimoto, K. Tamura, A. Nakase, O. Tanaka*, H. Naora* and T. Yoneyama* Depts. of Surgery and Anatomy*, Shimane Med. Univ., Izumo, Japan. There are few reports of the histochemical study on carbohydrates in human embryos during organogenesis and

not the systematic study, all the embryonic periods round. In the present study, which was undertaken mainly for the purpose of studying the histochemistry of the development of the embryonic organs, 48 human embryos of Carnegie stages 13 - 2 3 (estimated postovulation age: 3 2 - 5 2 days) were used. Thirty six embryos were used for light microscopic study and serial sections were stained with PAS reaction, alcian blue, colloidal iron and toluidine blue. The other twelve embryos were used for electron microscopic study. To examine the localization of glycogen and other carbohydrates, ultrathin sections of the several regions were stained with periodic acidthiocarbohydrazide-silver protein reaction. At stage 14, the appearance of glycogen was as follows: general epidermis, surrounding mesenchyme of thyroid, epithelium of pharynx, hind gut and cloaca, notochord, eye lens, pancreatic epithelium, coelomic mesoblast and sinus endothelium of liver, heart muscles of auricle and ventricle, and mesonephric duct. At stage 15, mesenchyme of lung and otic vesicle and mid gut. At stage 16, hepatic coelomic epithelium, eye retina and precartilage, and stage 21, hepatic cells. The results of glycoprotein, acid and neutral mucopolysaccharides were almost similar to the findings of glycogen. There was the individual difference of appearance of carbohydrate.

P4.3 Chromogranin B Immunoreactivity in the Mouse Submandibular Salivary Glands During Postnatal Development. A. Letid-Gavrilovid, C. Radoji~i6*, A. Duji~* and K. Abe Department of Biochemistry, Faculty of Stomatology, University of Beograd, Beograd, Yugoslavia, Department of Oral Biochemistry, Fukuoka Dental College, Fukuoka Japan, and *Institute for Experimental Medicine, Military Medical Academy, Belgrade, Yugoslavia. Presence and distribution, developmental changes and molecular features of chromogranin B (Chr B) proteins within the submandibular salivary glands (SSg) of male mice were investigated by two-dimensional electrophoresis and PAP immunostaining. Chr B immunoreactivity was detected in the juxta-acinar (JA) cells of the SSg during the first week of postnatal development. During the second and third weeks, Chr B immunoreactivity reached the plateau level, while its reduction seemed to be occurred in correspondence to appearance of the granular convoluted tubular cells (GCT) from 3 up to 7 weeks of age. A few JA cells with Chr B retained even in the adult morphologically matured SSg. A predominant protein which can be revealed by twodimensional electrophoresis and by immunocytochemistry, was Chr A. Chr C was not detected in any cell type of the SSg of mice from early postnatal period up to 35 days of age.

P o s t e r sessions Physiological significance and possible function as well as an application of these findings were discussed.

P4.4 Developmental Maturation of Rat Small Intestine followed by Morphological, Histochemicai and Molecular Methods. F. Nobili, Y. Sambuy, C. Murgia, S. Gaetani and G. Perozzi Istituto Nazionale della Nutrizione, Rome, Italy. We have chosen the epithelium from rat intestine as a model system in cellular and molecular studies of the mechanisms of epithelial differentiation. The long term goal of this project involves the establishment of epithelial cell lines from rat intestine and the molecular isolation of genes specifically expressed in terminally differentiated cells. To choose the optimal developmental stages from which to isolate tissue for both approaches it was crucial to carry out a histological and histochemical characterization of intestinal development between the third week of pregnancy and the third week after birth. Tissue sections from rat small intestine were stained with a modified Mallory trichromic method to show the progressive maturation of the crypt-villous unit mad the appearance of the differentiated cell types characteristic of the intestinal mucosa. Functional maturation of the brush border was followed by histochemical methods. Immunofluorescence and in situ hybridization were applied to the study of the expression of stage-specific markers of intestinal development.

P4.5 Cytochemical and Ultrastructural Study of Sperm Dimorphism in D r o s o p h i l a S u b o b s c u r a , M. E. Perotti*, M. Pasini* and C. A. Redi** *Dept. of General Physiology & Biochemistry, University of IVlilano and **Dept. of Animal Biology, University of Pavia, Italy. In D. subobscura the male produces two classes of motile spermatozoa that differ in total length and nucleus length. The significance of this within-ejaculate polymegaly is obscure. We have carried out an ultrastructural and cytochemical analysis of both sperm to understand their possible role at fertilization. Short and tong spermatozoa have a similar architecture. The axoneme is of the classic insect type and together with the major mitochondrial derivative runs for almost the whole sperm length. The axoneme and the major mitochondrial derivative extend from the tail into the sperm head, flanking the nucleus and the acrosome. The axoneme ends just below the sperm apex with a centriole adjacent to the acrosome. Minor differences between the two types of sperm are related to acrosome length, nucleus morphology and relationship between nucleus and minor mitochondrial derivative. Labeling with FITC-conjugated Con A and PEA showed that, as in sperm of other Drosophila species phylogenetically near to D. subobscura, the area of the plasma membrane overlying the acrosome contains a-Mannose residues in both sperm types. Cytophotometric measurements of Feulgenstained samples indicated that long and short spermatozoa contain a similar amount of DNA.

515 Both short and long spermatozoa are transferred and stored in the female upon copulation. As they have similar ultrastructural and cytochemical characteristics, the two types are potentially functional in egg penetration and karyogamy.

P4.6 Morphological and Functional Changes in the Lumbar Vertebrae of Mice Following Prenatal Cyciophosphamide Administration. L Sauerbier and B. Decker Department of Anatomy, Hannover Medical School, Germany. Following exposure to cyclophosphamide during organogenesis, the skeletal system of mice is affected according to the dosage and the stage of gestation. Gross morphological abnormalities suggest disturbances in intrauterine growth. The general purpose of the present study was to assess the effect of a single dose of cyclophosphamide on prenatal ossification of the lumbar spine by means of histological, histochemicat and histomorphometric analyses. Pregnant Han: NMRI mice were injected intraperitoneally with cyclophosphamide (20, and 40 mg/kg) on day 12 of gestation. Control groups received an equivalent volume of the solvent. At 4, or 6 days later (i.e. on day 16, or 18 of gestation) the fetuses were removed, fixed in neutral formalin and embedded in paraffin. After serial sectioning along the sagittal plane (5 ~m), the following staining methods were used: haematoxylin-eosin, PAS/Alcian blue staining, 0.1% Alcian blue 8GX solution in sodium acetate puffer (pH 5.6) containing different molarities of MgC12, staining of mineralized cartilage and bone matrix according to yon Kossa. Cyclophosphamide-exposed litters showed a dosedependent reduction in size of the lumbar vertebrae associated with marked changes in the extracellular matrix, e.g. affinity to Alcian blue. Alterations in the different cell types were also seen, e.g. an increase in the size of the hypertrophic chondrocytes. The most striking findings revealed a general lack of ossification including the formation of a bony collar and was most pronounced in the high-dose group. There is evidence that cyclophosphamide strongly interferes with osteogenesis.

P4.7 Aging Changes of the Neurotensin and Galanin in the Paraventricular Nucleus of the Thalamus in the Rat. S. Shu, X. Bao and W. Wang Department of Neurobiology, The First Military Medical University, Pearl River Hospital, Guangzhou, China. Peptidergic neurons, such as neurotensin (NT), galanin (GAL) and substance P immunoreactive, were located densely in the paraventricular nucleus of thalamus. The aging changes of NT and G A L in the central aniygdaloid nucleus and basal forebrain were reported. However, the aging changes of NT and GAL in the PVT have not been well studied. The present study is to investigate the aging changes of the NT and GAL in the PVT by means of behavioral test combined with immunohistochemistry and imaging analyzer. 20 SD rats were divided into two groups, 10 young ( 3 - 4 months) and 10 old one ( 2 2 - 2 4 months). First, the ability of study and memory of two group rats were measured using Y-shaped maze, than 6

516 rats having obvious dysfunction were chosen to investigate NT and GAL immunohistochemistry with 6 normal young rats. Experiments were processed under same circumstances at the same time for reliable. Colchicine 100-120/~g was injected into the lateral ventricle 48 hours prior to perfusion. 4% paraformaldehyde in the phosphate buffer was perfused for fixation. Frozen sections (40 ~m) were used for NT and GAL immunohistochemical ABC method. A lot of NT and G A L positive cell bodies and dense immunoreactive fiber terminals were found in the anterior and posterior part of the PVT. In young rats, the number of cell bodies is more numerous and the staining is darker than those in the old rats. The area and grey level of NT and GAL positive cell bodies were measured using imaging analyser. The statistical results show that the area of GAL positive cell bodies and grey level of NT and GAL positive cell bodies in the PVT of old rats are remarkable decrease than those in the young rats. It indicates that the ageing changes also exist in the NT and GAL positive cell bodies of PVT, and may be concerned with the functional retard of study and memory.

P4.8 Immunohistochemical Study on Human Small Heat Shock Protein (HSP 28) in Human Embryos. O. Tanaka, M. Oguni, T. Hatta, R. Hashimoto, H. Shinohara* and K. Kato* Dept. of Anat., Shimane Med. Univ., Izumo and Dept. of Biochem. *, Inst. for Develop. Res., Aichi Pref. Colony, Kasugai, Japan. The exposure of cells to an increase in temperature results in the enhanced synthesis of several proteins, which have been referred to as heat shock proteins. In the present study, the immunohistochemical method was applied to the embryos, and the onset and localization of HSP28 were examined during organogenesis. The materials were 16 externally normal human embryos of Carnegie stages 13 - 2 3 (estimated postovulation age: 3 2 - 52 days). The specimens were fixed in Schmechel's ('80) and embedded in paraffin. 5/a-thick sections of the whole embryo were prepared and immunostained after ABC method of Hsu ('81). At stage 13, the notochord, skeletal muscles (including myotome), myocardium, eye lens, otic vesicle and surface ectoderm were already immunopositive. After stage 15, the distribution of HSP28 in the epithelium of the esophagus and gut were immunopositive. The mesonephric ducts were also positive at stage 15, and the tubules were positive after stage 18. These tubules are regarded as the mesonephric vesicles at stage 13 and 14, because the glomerulus is not formed at these stages. There were not observed the distributions in the cartilage centers, neural tube, otic vesicle and liver in all embryos. These findings suggest that HSP28 may relate to the development of the primordium of several organs in human embryos.

P o s t e r sessions

P4.9 Ultrastructural Modifications of the Nucleolus in the Course of Oogenesis in an Oviparous Teleost (Barbus Barbus L.). M. Thiry*, A. Lepoint*, P. Poncin** and G. Goessens* *Laboratory of Cell and Tissue Biology, **Laboratory of Animal Ethiology and Psychology, University of LiOge, Belgium. Several stages in nucleolar structure have been distinguished in the ovaries of adults: (1) a single and centrally situated nucleolus, with a fibrillar core and granular periphery, in the oogonial nuclei. (2) a single nucleolus adjacent to the nuclear envelope and surrounded by a layer of condensed chromatin, in the zygotene oocyte. (3) multiple nucleoli appearing in the cap of the pachytene oocyte. (4) in diplotene oocyte, while the cap gradually disintegrates, nucleoli enlarge and acquire an increasing number of vacuoles. (5) in the final previtellogenic stage, oocyte contains multiple spherical nucleoli with fibrillar core and granular cortex. (6) during vitellogenesis, multiple nucleoli are reticulated and contain fibrillar core and numerous granular strands. (7) at late vitellogenesis, fragmentation of fibrillar and granular components is observed. We show also that only fibrillar material of nucleoli contains silver-stainable material. Using the terminal transferase method for detecting DNA in situ, label is consistently found over the fibrillar material whereas the granular regions are devoid of gold particles. However, while vitellogenesis takes place, gold particles group progressively at the periphery of nucleoli.

P4.10 Plasma Membrane Carbohydrate Components of Embryonic Neuroepithelial Cells in Norm and Experiment: Cytochemical Study. V. R. Radeva, M. J. Velinova and W. A. Ovtscharoff Department of Defectology University "St. Kliment Ohridski" Sofia, Bulgaria; Department of Anatomy, Histology and Embryology Medical University o f Sofia, 1431 Bulgaria. Glucose and mannose residues on the plasmalemma of newt's embryo neuroepithelial cells by means of affinity reaction with Concanavaline A and Horseradish Peroxidase were studied ultrastructurally. The investigation was performed on the early neurula stages (14 and 15) in norm and after treatment with some neurotransmitters: Acetylcholine, Norepinephrine, Serotonin and Dopamine. The differences in the distribution of Concanavaline A binding sites at the experimental conditions were encountered as compared with the normal patterns. Our data suggest that the difference of the Concanavaline A binding sites is caused by the receptor interaction of the cell membrane glycoconjugates and the applied neurotransmitters.

Poster sessions

517

PS. IMMUNOHISTOCHEMISTRY PS.1 Application of Galactose Oxidase, Neuraminidase and Lectin Cytochemistry for Ultrastructural Detection of Penultimate Residues in Zona Pellucida and Acrosome of Rats. M. Avil6s, J. A. Martinez-Menfirguez, M. T. Castells, J. F. Madrid and J. Ballesta Department of Cell Biology, Medical School, University of Murcia, Murcia, Spain. Lectins isolated from soy beans (SBA) and peanuts (PNA) bind terminal residues of Gal and GalNAc respectively. Galactose oxidase (Gox) oxidizes the hydroxyl group at C-6 of these carbohydrates residues, blocking the binding of P N A and SBA. Neuraminidase (Neu) cleaves terminal residues of Neu5Ac; therefore, penultimate Gal or GalNAc residues may became accessible to the lectins. Combination of these lectins with the enzymes allows the discrimination between penultimate and terminal Gal and GalNAc residues at the ultrastructural level. Rat ovaries and testes were fixed in 10% formaldehyde and Bouin's solution, respectively, for light microscopy, or in 2% glutaraldehyde for electron microscopy. PNA-, SBA- and LFA-peroxidase labelled were used for light microscopy~ Two step method (LFA/fetuin-gold) and three step method (PNA- and SBA-digoxigenin (DIG)/anti-DIG/ anti-Ig-gold) were used for electron microscopy. Some sections were treated with the following sequences: Neu-PNA/ SBA, G o x - P N A / S B A and Gox-Neu-PNA/SBA. Zona pellucida was labelled by LFA, PNA and SBA. Acrosomes were reactive to PNA and SBA. The reactivity to PNA and SBA was weakly increased after neuraminidase digestion. Gox treatment abolished labelling by PNA and SBA. After Gox-Neu sequence, PNA-reactivity was decreased when compared with Neu-PNA. These results demonstrate that Neu5Ac residues are present in ZP but not in acrosome. Galfll-3GalNAc and GalNAc were detected in both areas studied. Neu5Ac-GalNAc/Galfll-3GalNAc were found only in ZP. Thus, this study suggests that Gox can oxidize penultimate Gal residues of the terminal trisaccharide Neu5Aca2-3Galfll-3GalNAc. In conclusion, the application of the Gox-Neu-PNA/SBA sequence is a useful technique for demonstration of penultimate carbohydrate residues in tissues with abundant terminal Gal and/or GalNAc residues. This work was supported by a grant from DGICYT PB 90-0310 (Spain).

P5.2 Urodilatin-like lmmunoreaetivity (URO-IR) and Urodilatin Gene Expression in Mammalian Kidney. A. Bub, M. Rose, H.-J. M~igert and W.-G. Forssmann Lower Saxony Institute for Peptide Research (IPF), Feodor-LynenStr. 31, D-3000 Hannover 61, Germany. Since the first characterization of a natriuretic-vasorelaxant polypepfide from the heart a number of analogous peptides of this family have been described. In addition to the myoendocrine cells of the atria some other organs contain these cardiac peptides. Recently, we have isolated and characterized urodilatin, a differentially processed peptide of

the C D D / A N P family (cardiodilatin/atrial natriuretic peptide) which is secreted into human urine. In comparison to the circulating C D D / A N P of 28 amino acids, urodilatin (CDD/-ANP-95-126) consists of 32 amino acids due to a Nterminal extension of CDD-ANP-99-126. Urodilatin specific antibodies were obtained by immunization with peptide fragments mainly containing the CDD/ANP-95-99 sequence (Thr-Ala-Pro-Arg-Leu-Ser-Arg-Lys-Lys, etc.). Polyclonal (rabbit) and monoclonal (mice) antibodies were applied for RIA and IH. Immunohistochemistry was carried out using the PAP and streptavidin biotin techniques. The gene expression for URO was demonstrated by extraction of kidney mRNA, subsequent cDNA synthesis, and amplification by the PCR method using two specific primers which correspond to the CDD/ANP-80-126 amino acid sequence. The results show that distal tubules of the kidney contain URO-IR in strongly variable concentrations. The extent of the URO-IR segment within the nephron is different in human, pig, guinea pig, and rat tissue. Especially the monoclonal antibody was adequate to show concentration differences. Gene expression was proved by cloning and sequencing of the PCR-amplified part of the cDNA corresponding to the CDD/ ANP molecule. Apparently in the kidney and in the heart comparable CDD/ANP messages are expressed. Therefore the kidney probably exhibits a cleavage enzyme different from that in the heart. This is in agreement with RIA results which confirm that URO is only present in urine but not in blood plasma. We conclude that URO is a candidate for a paracrine intrarenal system that is involved in the regulation of collecting duct function.

P5.3 Expression of a Mammary-Derived Growth Inhibitor in Normal Mouse Mammary Tissue and Explant Cultures. B. Erdmann and B. Binas Max Delbrfick Center for Molecular Medicine, R.-Rdssle-Str. 10, D-1115 Berlin-Buch, FRG. Mammary-derived growth inhibitor (MDGI), a 13-kD protein isolated from bovine mammary gland is known to inhibit mammary epithelial proliferation in cell culture. Dependent on the state of differentiation, MDGI expression could be detected in mouse mammary glands by immunogoldsilver staining. During pregnancy high MDGI levels became obvious mainly in epithelial cells of the branching alveoli, whereas large ducts were nearly free of label. In the latepregnant and lactating state a remarkable secretion of MDGI into the lumina occurred. In a serum-free explant culture system the hormonal requirements for in vitro-expression of MDGI and beta-casein were tested. Both proteins could be clearly induced in explants from virgin mice. Compared to normal tissue similar staining patterns of MDGI and casein were confirmed in the culture system. If explants of late-pregnant mice were cultivated with EGF and insulin, the lobuloalveaolar structure was still present whereas MDGI disappeared. The role of MDGI in association with functional differentiation of the normal mammary gland is discussed.

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P5.4 Evidence for Chronic Cytomegalovirus Infection in the Majority of Patients with Common Variable Immunodeficiency. E. Fietze ~, H. U. Simon 2, W. D. Doecke', C. Diener 2, G. Niedobitek 3, H. Stein 3 and H. D. Volk ~ tInst. Med. Immunol., CharitC HU Berlin; 2Inst. Clin. Immunol., FSU Jena; ~Inst. Path., FU Berlin, Germany. Common variable immunodeficiency (CVI), a heterogeneous syndrome characterized by acquired hypogammaglobulinemia, and recurrent bacterial infections is complex with regard to underlying immunological defects and is of unknown etiology. Although there exist only indirect evidences, a viral genesis of at least a part of the CVIs has been proposed. In agreement with several authors, in a high proportion of CVI patients (18/26) we could observe by cytofluorometric analysis increased numbers of CD3 + 8 + peripheral blood lymphocytes resulting in the decrease of CD4/8 ratio. The majority of these T cells expressed the CD57 antigen and activation antigens (HLA-DR). Similar alterations commonly occur in patients with systemic virus infections (e.g. CMV, HIV). In order to prove whether CVI patients suffer from subclinical chronic CMV infection, we investigated peripheral blood mononuclear cells (PBMNC) by means of molecularbiological and virological methods. In contrast to healthy donors, in the majority of CVI patients CMV-DNA (IE antigen) has been found by in situ hybridization (75070) and polymerase chain reaction (92%). The positive results of centrifugation culture (5/5) and the observation of cytopathic effects in conventional long-term co-cultures on fibroblasts (2 patients) support the existence of active CMV infection. However, none of the CVI patients had a history of clinically apparent CMV infection. The detection of CMV-DNA in PBMNC from the majority of CVI patients raised the question, whether the persistent CMV infection is a result of immunodeficiency or is a factor involved in the pathogenesis of CVI? In the latter case new therapeutic approaches in CVI would seem possible (e.g. antiviral agents or "biologic immune response modifiers" like IL-2 of interferon).

P5.5 Primary Infection in Spodoptera Exigua Larvae Using a Recombinant Autographa Californica Nuclear Polyhedrosis Virus. J. T. M. Flipsen, M. M. van Oers, J. M. Vlak and J. W. M. van Lent Dep. of Virology, Agricultural University, P.O.B. 8045/6700 EM Wageningen, Netherlands. In Lepidopteran larvae the primary infection of baculoviruses takes place in the midgut. In order to study the early events of baculovirus infection an Autographa californica nuclear polyhedrosis virus recombinant (AcMol6B) was constructed. This recombinant contained the bacterial/3-galactosidase gene under control of the Drosophila heat-shock promoter. This construct gives rise to p-galactosidase production as early as 4 h post infection (p.i.) in cultured Spodoptera frugiperda cells as detected by enzyme histochemistry. When this recombinant was used to infect Spodoptera exiqua larvae the

Poster sessions viral infection could be located in midgut columnar epithelial cells at 6 h p.i. This implies that viral infection can be detected a few hours before the start of viral DNA replication. For the detection of/3-galactosidase two substrates were used pre embedding, X-gal and Red-gal visible in light and electron microscopy. Light microscopically this detection of the virus was combined with immunofluorescence to demonstrate the membrane bound antigen gp64. Electron microscopically the immunogold technique was applied to detect gp64 and other viral antigens.

P5.6 A Study of B Cell Development in Human Fetal Liver. Z.-H. Ge, B.-H. Li and R.-Y. Wang Department of Pathology, Fujian Institute of Traditional Chinese Medicine, The People's Republic of China. A serise antibody and immunohistochemical technique have been used to observe the morphology and distribution of preB cells and the changes of antigen during the process from preB to B cell on frozen sections at different gestational fetal liver. The results revealed that at the early fetal stage ( 9 - 2 0 WK), the liver contains numerous pre-B cells, which were present in different size and shape, but their phenotypic expression was identical such as B A - 1, IgM, HLA-DR and TdT. Most Pre-B cells were scattered in extrasinusoidal space, a few of them displayed in sinusoid or around the blood vessel. After 13 WK, the IgD + and IgA + cells were appeared sequently, the numbers of OKB-2+ and L e u l 4 + cells were greatly increased. Meanwhile, the HLA-DR + , k a p p a + and L a m b d a + cells were corresponding to increase which indicated that the B cells further developed to a mature stage, but the B cells did not develop to plasma cells. In the fetal liver, the T cells were not required for B cell generation, even if the T cell was absent at early fetal liver, the B cell was still continuing to generate.

P5.7 Light and Electron Microscopic Demonstration of Acidic Glycoconjugates in Rat Gastrointestinal Epithelia using Cationic Colloidal Gold. N. Kashio, S. Tsuyama, K. Ihida, T. Takatsuka and F. Murata Department of Anatomy, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890, Japan. The cytochemical detection of acidic glycoconjugates located in rat gastrointestinal epithelia was performed using tissue sections embedded in Lowicryl K4M and cationic colloidal gold (CCG). CCG was prepared from poly-L-lysine and colloidal gold solution. The staining of CCG was amplified after photochemical silver reaction using silver acetate as an ion donor in the LM level. The staining solutions adjusted to pH 2.5 [CCG(2.5] and pH 1.0 [CCG (1.0)] were used for the characterization of carboxylated and sulfated glycoconjugates. In the LM level, CCG(2.5) stained several cell types of mucous cells, while CCG(1.0) stained selectively mucous cells in gastric pits, glandular pylorus, upper crypts of proximal colon and entire crypts of distal colon. In the EM level, both simple and double labelings were performed. Both CCG(1.0)

Poster sessions and CCG(2.5) labelled mucous droplets and trans side of the Gotgi apparatus in some mucous cells. Our present study revealed that CCG(2.5) and CCG(1.0) staining methods are useful and reliable postembedding staining procedures for the light and electron microscopic demonstration of intra- and extracellular acidic glycoconjugates. These results also indicated the sialylation and the sulfation may take place in the same cells and cell organellae.

P5.8 Alteration of Mesangiai Specific Proteins During Proliferative GIomerulonephritis Induced by Administration of Anti-Thymocyte Serum. V. Leardkamolkarn and S. Matsuo Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, Thailand and The Third Department of Internal Medicine, Nagoya University, School of Medicine, Japan. We induced mesangial proliferative glomerulonephritis into adult female rats by giving a single intravenous injection of anti-thymocyte serum (ATS). Kidney tissues from these rats were fixed, embedded and processed for immunogold labelling with two monoclonal antibodies (MAbs) against specific mesangial proteins (anti-OX-7 and anti-H49D6) at 4, 10, 20 and 30 days after the injection. Age-matched control rats were received normal rabbit serum and tissues were processed in parallel with the ATS rats. The results indicated that there was an alteration in mesangial surface protein (as detected by MAb-OX-7) as well as mesangial matrix protein (as detected by MAb-H49D6) during the process of disease. Four days after the injection, the labelling of MAb-OX-7 was dispersed among the injured mesangial cells whereas the labelling of MAb-H49D6 was decreased and scattered in the area of mesangial matrix. Ten to twenty days after the injection, the detected particles staining for OX-7 were increased and diffused more on the mesangial cell surface which was corresponding to proliferation of mesangial cells. The particles staining for H49D6, on the other hand, were atmost absent from the mesangial matrix. The screening for binding of both monoclonal antibodies on kidney of rats at 30 days after ATS injection showed more or less the same distribution as in the control.

519 Sections were stained with the Histostain-SP kit based on the streptavidin-biotin reaction. Immunoreactivity was found in sinusoidal endothelial cells and Kupffer cells and possibly also in sinusoidal stellate cells. The reaction was stronger in the liver samples from cows before calving than after calving. The hepatocytes were negative. The localization of the reaction corresponds with that reported for human liver (Haimoto, H. et al.: Lab. Invest. 1987: 57, 489-498).

PS.10 Ultrastructural and Immunocytochemical Investigation of the Cells Infected with Sindbis Virus. A. A. Manykin, I. N. Tjuchlova and N. V. Chutaretskaya The Ivanovski Institute of Virology, Rus. Acad. Med. Sci., Russia. BHK-cells, 22 hrs after infection with Sindbis virus SV, were fixed in 4070 paraformaldehyde-0,1% glutaraldehyde, dehydrated and embedded in Aratdit [Fluka] and Lowicryl K4M [Chemisch Werke Lowi]. Immunocytochemistry was performed by the standard procedure (Grigoriev. V. et al. 1989. Path. Res. Pract. 184, 494-497.); primary mouse polyclonal antibodies anti-SV (1 hr, 25C) and second antibodies goat antimouse IgG conjugated 10rim gold [Janssen] (1 hr, 25C) were used. Ultrastructural studies showed that infected cells contained typical cytopathic vacuoles (CPV), equipped with characteristic 50 nm light bulb-shaped membrane spherules and intact virus particals. We observed a lot of SV nucleocapsids on external site of CPV-membranes and on SV-particals, budding from cells' membranes. Some of nucleocapsids have typical light centre, and we suggest, that it is connected with the lack of virus RNA. Immunogold labels were found more often on SVnucleocapsids, located near CPV-membranes, and on SVenvelopes of budding viruses, than in other sites in cytoplasm. Possibly, many labels near CPV-membrane may be explained by transport of SV structural proteins from endoplasmic reticulum to late endosomes (CPV). (Froshauer. S. et al. 1988. J. Cell Biol. 107, 2075-2086).

PS.11

P5.9 S-100 Protein Immunoreactivity in Bovine Liver.

Detection of Protein Gene Product 9.5 ( P G P 9.5) Immunoreactivity in Epithelium from Genital Tract of Male Rats. A Quantitative Immunohistoehemieal Study,

T. Ukkola and L.-A. Lindberg Department of Anatomy and Embryology, College of Veterinary Medicine, Helsinki, Finland. The S-100 proteins are involved in calcium dependent biological reactions both in neural and extraneural tissues. The best-characterized S-100A and B polypeptides are apparently only two of several similar S-100-1ike proteins. We have used a commercial potyclonal antibody that reacts predominantly with bovine A and B proteins for a immunohistochemical study of liver biopsies from cows before and after calving. The biopsies were taken from 4 cows during 2 months before calving, and from 5 cows during 2 months after calving, The sampling interval was about 2 weeks. Small liver pieces were fixed in 10~ formalin and embedded in paraffin.

R. Martin, L. Santamaria and J. Codesal Department of Morphology, Faculty of Medicine, Autonomous University of Madrid, 28029-Madrid, Spain. Protein gene product 9.5 (PGP 9,5) is a soluble protein found in neuronal and neuroendocrine cells and also in some not nervous tissues (1,2). In this study, PGP 9.5 immunoreactivity was detected and quantified by immunohistochemical and morphometric methods in epithelial cells from several regions of rat epididymis and ventro-lateral prostatic glands (VLP). In proximal caput (Cl), PGP immunoreactive (IR) protein was observed in principal cells, being secreted to the lumen and linked to head of spermatozoa; in VLP also appears immunostaining in some epithelial cells and luminal secretion. In ductuli efferentes (DE), middle and distal caput (C2, C3) and distal cauda (DC), PGP-IR protein is not secreted to the

520 lumen. In these epididymal regions PGP immunoreactivity was only expressed in non ciliated cells from DE and apical and clear cells from C2, C3 and DC respectively, all those characterized for having endocytotic activity. PGP-IR protein detected in basal cells from C2, C3, could be related to the proliferating activity of these cells (2). No significant epithelial immunostaining to PGP was seen in other regions from epididymis and accessory male glands. References: 1-Wilson POG et al., Br J Exp Path 69 : 91, 1988. 2-Giambanco I e t al., FEBS 290 : 131, 1991.

P5.12 Mode of Expression of S-100 Protein Subunits in the Extraocular Muscles of Human Embryos and a Fetus. M. Oguni ~, T. Setogawa ~, O. Tanaka 2, H. Shinohara 3 and K. Kato 3 Depts of Ophthalmologf and Anatom) ?, Shimane Medical University, Izumo and Dept. of Biochemistrf , Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Japan. S-100 protein is a calcium-binding protein and has two subunits, S- 100a and S-100/3. Although the physiological roles remain unknown, S-100a and S-100/3 are predominantly present in the slow-twitch muscle fibers and nerve fibers, respectively, in the peripheral tissues. In the present study, the appearance of S-100a and S-100/3 was investigated immunohistochemically in the extraocular muscles of 15 human embryos (Carnegie stages 13 to 23) and a fetus (10 weeks of gestation). During stages 13 - 17, there were no immunoreactivities to S-100a and S-100/3 antibodies around the optic vesicle. At stage 18, S-100/3-immunoreactive nerve fibers appeared around the optic vesicle and they increased in number as the embryonic stage advanced. In the fetus, S-100a immunoreactivity was first observed in some muscle fibers. S-100/3-immunoreactive nerve fibers appeared around the optic vesicle at stage 18, the same stage when neuron-specific enolase, another marker of nerve fibers, appeared in the nerve fibers of the extraocular muscles (Oguni et al., 1991). Therefore, these findings suggest that the innervation of the extraocular muscle begins around stage 18. In addition, S-100a appears in a later stage of myogenesis than some muscle-specific markers, for example, muscle-specific enolase, muscle type of creatine kinase and glycogen phosphorylase (Oguni et al., 1991).

P5.13 Lectin-histochemical identification of glycoconjugates in the posterior intestine of the Rainbow Trout. B. Pajak and A. Danguy Universit~ Libre de Bruxelles, Facult~ des Sciences, Lab. de Biologic animale et ttistologie compar~e, Bruxelles, Belgium. In vertebrates, besides typical cells of the in immune system, cells of other systems also play an essential role in the immune function of the gut, e.g. goblet cells, enterocytes, enteroendocrine cells. In fish the posterior intestine is more particularly involved in the different lines of defense. Therefore the present work intended to take a close look at the distribution of glycoconjugates in this region of the trout (Salmo gairdneri) alimentary tract. A panel of 26 biotinylated lectins were used as probes and the avidin-biotin-peroxidase

Poster sessions complex technique was performed to assess extend of binding on Bouin's solution fixed paraffin-embedded specimens. Glycoconjugates of the enterocytes displayed a great variety of lectin reactivity. The supranuclear region of these cells were labelled with GSA-II, specific for terminal a,p-Nacetylglucosamie. Some goblet cells reacted with DBA and RCA-I indicating the presence of galactose and N-acetylgalactosamine. GNA affinity was localized to the entero-endocrine cells and macrophages indicating terminal mannose-a-l-3 mannose sequence. Besides mannose, galactose and sialic acid were found to be predominate sugar residues in macrophages. Two additional epithelial cell types were convincing detected. One type stained with DSL and PHA-E suggesting the presence of galactos/3(1-3,4)-Nacetytglucosamine residues; the other type contained terminal N-acetylglucosamine moieties as evidenced by STL and PSA labelling. These observations may probably be related to cytoprotective roles of glycoconjugates against potential pathogens in this part of the digestive tract. Supported by Eurogentec, S.A., Liege, Belgium.

P5.14 Lectin Binding Studies on Rat Submandibular Secretory Glycoproteins. G. B. Proctor, X. S. Zhang, J. R. Garrett, B. A, Schulte* and D. K, Shori Oral Pathology, K.C.S.M.D., The Rayne Institute, London, SE5 9NU & Pathology, *M. U.S,C., Charleston, South Carolina 29425. Oligosaccharide components of glycoproteins in saliva are important in determining functional characteristics. We have used labelled lectins to detect submandibular glycoproteins. Sympathetic saliva was collected from anaesthetized rats, then both stimulated and control glands were excised, and half of each was frozen. The rest was fixed in aqueous 3~ HgC12 in 1~ sodium acetate containing 0.2% glutaraldehyde, followed by dehydration, paraffin embedding and sectioning. Extracts of frozen tissues were obtained from homogenates by centrifugation. Proteins in saliva and tissue extracts were resolved in SDS gels then blotted onto nitrocellulose. Tissue sections and blots were incubated with lectin-horseradish peroxidase (HRP) or lectin-biotin (followed by avidin-HRP) and HRP activity localized using diaminobenzidine (DAB) or luminol-chemiluminescence and photographic development (Amersham). Luminol proved more sensitive and flexible than DAB for detection of blotted glycoproteins. Lectins from Dolichos biflorus, soyabean, Saphora japonica and wheat germ, bound to a mucin component in blots and bound to acinar cells which showed a reduced staining after sympathetic stimulation. Lectins from Lotus tetraglonobulus, Lens culinaris and Limax flavus bound to some granular tubule (GT) cells and showed bands (20 - 30 kD) associated with GTderived proteinases in blots. Peanut agglutinin also bound to GT cells and specifically to a 94 kD band in blots. The results indicate that lectins possess great potential for studying the glycoproteins in salivas and in their cells of origin and for studying factors which modify them in health and disease. Supported by King's RSF & NATO grant no. 0034/89.

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P5.15 Method of Serologic Differentiation of Brncella suis from Yersinia enterocolitica S e r o t y p e 9. C. L Schoilew, A. N. Kozarev and M. J. Velinova* Central Veterinary Research Institute, Sofia 1606; *Department of Anatomy, Histology and Embryology, Medical University of Sofia 1431, boul. G. Sofiiski 1, Bulgaria. There was prepared an immunsorbent from Yersinia enterocolitica serotype 9, which was used for the adsorbing of specific agglutinating antibodies against the mentioned serotype in the pigs blood. The adsorption of the antibodies was ensured for a short time at room temperature. It was determined that the received immunsorbent was with a high efficiency, combining the specifically agglutinating antibodies. The applied in the laboratory diagnostics immunsorbent allowed the quick serologic differentiation of yersiniosis from the brucellosis in pigs.

P5.16 Application of Recovery Schemes for Leukosis Farms Touched by Enzootic Leukosis. C. L Schoilew, V. A. Zuzumanski and M. J. Velinova* Central Veterinary Research Institute Sofia 1606 Bulgaria," *Department of Anatomy, Histology and Embryology, Medical University of Sofia 1431, boul. G. Sofiiski 1, Bulgaria. Practical possibilities for recovery of leukosis farms with 10%, 30% and more than 30% leukosis are investigated. It was established, that the recovery of the leukosis farms is successful and profitable when the seroagents are no more than 30%. It is necessary in the cattle farm with more than 10% leukosis to carry out serological investigations in short intervals 4 times in a month, yearly and obligatory keeping of the seroagents in isolators. For the cow farms with 29% to 39% leukosis are necessary 4 series serological investigations in 3 months intervals, keeping the leukosis animals in farms and insemination with bulls for meat production. In group with leukosis above 30% the recovery is to be carried out by creating of groups of healthy female calves, born by leukosis mothers, and on the 5th month should be serologically investigated 4 times on 1 month intervals and on the 7th month of pregnancy. The newborn female calves on the first day should be fed with collect colostrum and after that with pasteurized milk.

P5.17 Lectin Affinity-Histochemistry of Endothelium in Cancer Stroma. M, Shin and P. K. Nakane Department of Anatomy, Nagasaki University School of Medicine, Nagasaki, 852, Japan. Physiology and morphology of endothelial cells varies from organ to organ and functional state of the organ. The differences are reflected on the chemical compositions of the cells. In this study, we sought the differences among the stromata of gastric and mammary carcinomas by their ability to complex with various lectins. The tissue sections were reacted with UEA-1, GS-1, PNA, MPA, GS-2, DBA, WGA, Con A, BPA, SBA, Jacalin, Lotus and LBA. The reactivity

521 with stromata was graded as strongly positive (SP), positive (P), weakly positive (WP) and negative (N). The study revealed that UEA-1 which is a marker of endothelial cell was SP with almost all endothelial cells. GS-1 was P or SP with many blood vessels of both types of carcinomas. But one case of mammary carcinomas was N. P N A was P with some large vessels in gastric carcinoma whereas in mammary carcinoma, only a few microvessels were P. DBA and W G A were P or SP with most of vessels. But connective tissues of stroma were also WP or P. BPA was SP with many of large vessels but N with microvessels of gastric carcinoma and with all vessels of mammary carcinoma. SBA was SP with almost all vessels. GS-2, Con A, Lotus and LBA were N. M P A and Jacalin reacted generally with all types of ceils and stromata. Our results suggest that SBA in addition to UEA-1 is a useful general marker of endothelial cells and heterogeneity of lectin binding pattern is present between different tissues.

P5.18 Variations in Lectin-Binding Patterns of the Zona Pellucida of Follicular Oocytes of Different Mammalian Species. E. Skutelsky, E. Ranen and R. Shalgi Department of Pathology and Embryology, Sackler School of Medicine, Tel A viv University, Ramat A viv 69978, Israel. Carbohydrate residues of the zona pellucida (ZP) of mammalian oocytes are essential in sperm-oocyte interaction. In the present study we investigated the carbohydrate distribution patterns of the ZP in different mammalian species in an attempt to find their role in gamete interaction and prevention of interspecies fertilization. Ovaries isolated from different mammals, including mouse, rat, hamster rabbit, cat, dog and pig, were fixed with glutaraldehyde and embedded in paraffin. Five ~m sections were deparaffinized, rehydrated and labelled with 10 different biotinylated lectins as probes and avidin-biotin-peroxidase complex as visualant. The ZP of follicular oocytes exhibited species-specific differences in their lectin-binding patterns, whereas the cumulus cells and follicular fluids exhibited similar lectin-binding patterns in all species studied. Phylogenetically close species such as the rodents (mouse, rat and hamster) and rabbit, demonstrated high similarity in lectin-binding patterns, expressed in binding of succinylated wheat germ agglutinin and peanut agglutinin. Lectins such as Dolichos biflorus agglutinin, which bound only to mouse, Griffonia simplicifolia agglutinin-1 which bound to mouse and rat but not hamster and rabbit, and soybean agglutinin which bound only to the rodents reflect characteristic differences between closely related mammals. We suggest that differences in the expression of ZP carbohydrates, such as those demonstrated here, may be involved in species-specific control of fertilization.

5.19 Immunohistochemical Study of Bone Morphogenetic Protein in 30 Cases of Giant Cell Tumors of Bone. S.-X. Zhang, D.-F. Li and J. Liu The Fourth Military Medical University. Xian P.R. China. Paraffin sections from 30 cases of giant cell tumor (GCT) of bone were immunostained by ABC method using monoclonal

522 antibodies against bone morphogenetic protein (BMP-McAb). The result showed that monocytic stromal cells (MSC) are positive. We think (1) BMP in CGT is expressed by MSC (2) MSC are derived from osteoblast (3) the roentgenogram of

Poster sessions GCT is the display that MSC can attract osteoclast, on the other hand they can secrete BMP too (4) GCT and osteoblastoma may be different subtype or variant of same entity.

P6. CYTOCHEMISTRY OF FREE RADICALS P6.1 Ailoxan Toxicity is Highly Potentiated by Plasma Membrane-Associated Iron. H. Zhang and U. Brunk Department of Pathology, Faculty of Health Sciences, Link6ping University, S-581 85 Link6ping, Sweden. Alloxan is a prompt and potent experimental inducer of diabetes rnellitus due to its pronounced damaging effect on pancreatic islet 0-cells. Although the exact mechanisms for alloxan toxicity have not yet been completely elucidated, they are considered mediated through the formation and effects of reactive oxygen metabolites such as superoxide anion radical (O2-) hydrogen peroxide (H202) and hydroxyl radicals (OH.). In the present study we have further investigated the cytotoxic effects of alloxan by using a model system of cultured J-774 cells. Cell viability was estimated by the trypan blue dye exclusion test, the plasma membrane permeability by a modified fluorescein diacetate technique and lysosomal membrane stability by the lysosome tropic weak base acridine orange. Cells previously allowed to endocytose iron during culture (50 laM FeC13 for 6 h in complete F-10 medium followed by 1 h in F-10 medium without serum and iron) showed a greatly enhanced sensitivity to alloxan and cysteine exposure as compared to control cells. On the contrary cells which were initially exposed to desferrioxamine (100/aM, 1 h) were much less sensitive. Also cells which were briefly exposed to ferric iron (100 ~M, 20 min) in complete F-10 medium were easily damaged by alloxan and cysteine in low concentrations. Alloxan reacts with cysteine under the formation of dialuric acid. The latter compound auto-oxidizes spontaneously back to alloxan under the formation of 02- and H20~. H202 may diffuse through cellular membrane into the lysosomal vacuome and induce OH- production with ensuing membrane damage and leakage of lytic enzymes if not destroyed by the cellular antioxidant system, e.g. glutathione peroxidase. Thus low extracellular HE02 production (low All/Cys concentrations) will be harmless due to the antioxidative defense system, unless there is plasma membrane associated iron. In that case the combination of O2-, Fe 3+ and H202 will result in plasma membrane-associated OH- production with resultant hydrogen abstraction, peroxidation, fragmentation and destruction of plasma membrane integrity.

P6.2 Xanthine oxidoreductase Activity in Rat and Human Tissues in Relation with Ischemia-Reperfusion Injury. A. Kooij, M. Schijns and W. M. Frederiks Laboratory of Cell Biology and Histology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 A Z Amsterdam, The Netherlands. Localization and activity of xanthine oxidoreductase (XOR; dehydrogenase and oxidase forms) were studied in rat tissues

and in post-mortem material and biopsies of various human tissues with a histochemical method. Unfixed cryostat sections were used and the incubation medium contained hypoxanthine, poly(vinyl alcohol), 1-methoxyphenazine methosulphate and Tetranitro BT. High enzyme activity was found in epithelial cells, endothelial cells and connective tissue in rat, whereas in man activity was present in liver and small intestine only. In liver, enzyme activity was found both in sinusoidal cells and hepatocytes. In small intestine, enterocytes and goblet cells as well as the lamina propria underneath the epithelial lining showed activity. The results imply that a possible role of the enzyme in ischemia-reperfusion injury as proposed by Granger et al. (1981) may only occur in liver and small intestine. In the present study, liver tissue was investigated with respect to this phenomenon. Significant conversion of the dehydrogenase form could not be detected in ischemic rat livers or in post-mortem autolytic rat and human livers as determined biochemically. Upon reperfusion after in vivo ischemia for 60 min in rat liver the enzyme appeared immediately in the blood. XOR was found only in the oxidase form and not in the dehydrogenase form. Heterogeneously distributed areas in these reperfused livers showed diminished XOR activity. It is concluded that significant conversion of the dehydrogenase into the oxidase form of XOR after ischemia only occurs upon release into the blood during reperfusion. Granger et al. (1981) Gastroenterology 81: 2 2 - 2 9 .

P6.3 The Effects of Ischemia on the Localization of Xanthine Oxidase and Xanthine Oxidoreductase Activities in Rat Small Intestine. 17. Marx, W. M. Frederiks and A. Kooij Laboratory of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, The Netherlands. Effects of 60 and 120 min of in vitro ischemia on the localization of xanthine oxidase (O-form) and xanthine oxidoreductase (D- and O-form) activities were studied in rat small intestine. It was proposed that ischemia induced transition of the D- into the O-form giving rise to the production of oxygen radicals during reperfusion after ischemia. Xanthine oxidase and xanthine oxidoreductase were demonstrated in unfixed cryostat sections with histochemical methods. Xanthine oxidase activity was detected with a semipermeable technique with a gelled incubation medium in order to prevent leakage of the soluble enzyme. The incubation medium contained hypoxanthine as substrate, cerium ions which capture the enzyme product, hydrogen peroxide, and sodium azide to inhibit catalase and peroxidase activities. In a second step reaction, diaminobenzidine was polymerized in the presence of cobalt ions by decomposition of cerium perhydroxide. Xanthine oxidoreductase was

P o s t e r sessions demonstrated with the tetrazolium salt technique using a polyvinyl alcohol-containing incubation medium. A high xanthine oxidase activity was detected in the cytoplasm of enterocytes and goblet cells, whereas hardly any activity was found in the mucus of these cells. Xanthine oxidoreductase activity was localized in the cytoplasm of enterocytes and goblet cells. Moreover, activity was also present in the mucus and the lamina propria underneath the epithelial lining. Therefore, it was concluded that only the dehydrogenase form of the enzyme is found in mucus and the lamina propria. After 60 min in vitro ischemia at 37~ xanthine oxidase activity was localized at the basal part of the epithelial lining. A similar change in localization was observed for the activity of xanthinie oxidoreductase. It was concluded that ischemia did not induce conversion of the dehydrogenase into the oxidase form but that blebbing of the basal membranes of epithelial cells is responsible for increased activities of xanthine oxidase and xanthine oxidoreductase at the basal parts of the epithelial lining.

P6.4 Human PMN Produce Free Radicals from the CeilZymosan Interface During Phagocytosis and from the Plasma Membrane when Stimulated with PMA, FMLP and A23187. K. Moriguchi Dept. o f Anat., Kanazawa Med. Univ., Ishikawa 920-02, Japan. The production of free radicals, superoxide anions ( 0 2 , and hydrogen peroxide (HzO2) was histochemically investigated in human polymorphonuclear leukocytes (PMN) that were stimulated by either phagocytosis or the phorbol myristate acetate (PMA), formyl-methionyl-leucyl-phenylalanine (FMLP) and calcium ionophore A23187. To demonstrate O~, peripheral PMN from healthy donors were incubated at 37~ in a medium containing nitroblue tetrazolium and glucose in the presence of either opsonized zymosan A, PMA, F M L P a n d / o r A23187. To demonstrate H202, PMN pretreated with a stimulant for 10 min were washed and incubated in a cerium medium containing CeC13 and glucose in a Tris-maleate buffer. In cells engaged in phagocytosis, diformazan (for 02-) and cerium perhydroxide deposits (for H202) were restricted to the PMN-particle interface and on the inner surface of phagosomes. In the case of PMN stimulated with P M A and FMLP, the production of 02 and H202 was visualized at the cell-cell contact between the aggregated PMN. In the case of P M N stimulated with A23187, the production of O~ and H202 was visualized over the whole surface of the plasma membrane. The results showed that human P M N produce free radicals exocellularly and that the site of production varies with different stimuli.

523

P6.5 Quantification and Distribution Pattern of Corticotropin-Releasing Hormone Receptors at the Cellular Level in Normal and Adenomatous Human Pituitary. G. Smets ~'2, R. Abs 3, A. Beckers4, C. Mahler 7, A. Stevenaert 5, M. Reznik 6, M. Borgers ~ and G. KlOppel2 ~Dept. of Morphology, Life Sciences, Janssen Research Foundation, Beerse, 2Dept. of Experimental Pathology, Vrije Universiteit Brussel, 3Dept. of Endocrinology, University of Antwerp, Dept. of 4Endocrinology, SNeurosurgery, 6Pathology, University of Lidge, 7Dept. of Endocrinology, Middelheim Hospital, Antwerp, Belgium. Using autoradiography combined with immunocytochemistry (methodology described in Smets et al., JCEM, 73, 2, 268 - 274, 1991), we identified the target cells of corticotropinreleasing hormone (CRH), described the distribution of CRH receptors and quantified the receptor number at the cellular level in both normal pituitaries (autopsy specimens, n = 3; normal pituitary surrounding a gonadotroph adenoma, n = 1) and corticotroph adenomas (actively secreting tumors, n = 2; clinically silent tumors, n = 2). By computer-assisted image analysis the relative quantities of CRH receptor were determined per cell. In normal ACTH cells from the biopsy specimen as well as from normal pituitaries obtained at autopsy, (125I)Tyr~ was mostly found in cytoplasmic inclusions or at the cell periphery; ACTH tumor cells, in contrast, were faintly labelled at the cell periphery. The silent ACTH tumors, showed a smaller CRH receptor number as compared to the actively secreting ACTH tumors. Only in normal A C T H cells, CRH receptors seemed to be endocytosed. By receptor sequestration, a cell can adjust its sensitivity to the concentration of a stimulating ligand. As no internalized receptor was found in ACTH tumor cells, this form of desensitization seemed to be disturbed in corticotroph adenoma. Actually it was not possible to discriminate quantitatively between internalised and cell surface expressed CRH receptors. Although ACTH tumor cells had less CRH receptors, they might thus have more cell surface receptors than the normal cells, explaining the hyperstimulation of ACTH to exogenous administered CRH in Cushing's tumors. The down-regulation in the silent tumors is in agreement with a lower response to exogenous administered CRH as compared to the actively secreting ones. The results of the present study demonstrate in situ, that in ACTH tumor cells, CRH receptors were not endocytosed and were less numerous, but it is not yet clear if ACTH tumor cells express also less cell surface CRH receptors than do their normal counterparts.

P6.6 Immunohistochemicai Characterization of Radical and Lipid Peroxides Scavenging Systems in LEC (Hepatitis-Hepatomas Prone) Rat Livers. H. Utsunomiya, S. Satoh, S. Takekoshi, N. Komatsu, K. Watanabe, H. Suemizu, S. Yoshimura, T. Moriuchi and N. Takeichi Dept. o f Pathol., Cell Biol. Res. Lab. and Dept. of Cell Biol., Tokai Univ. Sch. of Med., Isehara Kanagawa, Japan 259-11 and Lab. Pathol., Cancer Inst., Hokkaido Univ., Sapporo 060 Japan. Long-Evans Cinnamon (LEC) rats are prone to develop fatty liver, hereditary hepatitis and hepatocellular carcinoma which may possibly be due to copper toxicity. Like as other metal

524

P o s t e r sessions

ions, e.g. ferric ion etc., copper ion (Cu §247 stimulates to generate oxygen radicals and lipid peroxidation in cells. In order to investigate the significance of those radicals and lipid peroxidation (LPO) as the pathogenesis of hepatic diseases mentioned above, the differences between control (LEA, Long-Evans Agouti) and hepatitis or hepatoma prone (LEC) rats (9 weeks of age) were immunohistochemically and biochemically studied on radicals and LPO scavenging enzymes, e.g. Cu, Zn-superoxide dismutase (SOD) and

P7. ULTRASTRUCTURAL

glutathione peroxidase (GSH-PO). Lipid peroxides were also measured by TBA method. As results, GSH-PO (effective LPO scavenger) was meaningfully lowered in LEC rat livers. SODs were roughly same in both rats. Being against expectations, fatty change, which was proved to be due to enhanced lipid peroxidation, was more prominent in LEA. The difference of LPO generating mechanism in hepatocytes of LEA and LEC is discussed.

CYTOCHEMISTRY

P7.1

Cell Membrane Calcium-ATPase Activity in the Rat Adenohypophysis: Combined enzyme- and Immunocytochemistry. E. Bdcsy, G. El-sherif and M. Szab6 Institute of Experimental Medicine, Szigony u. 43. H-1083 Budapest, Hungary. Calcium-ATPase activity was localized at the EM level in the rat adenohypophysis with a lead citrate technique while some of the parenchymal cells were identified by simultaneous immunocytochemical demonstration of anterior lobe hormones. After perfusion fixation with cold aldehydes in the presence of Ca 2+ 50/am chopper slices of the anterior lobes of young adult Wistar rats were incubated for Ca2+-ATPase activity (1, 2). The slices were embedded in Araldite, then ultrathin sections were mounted on nickel grids. Immunogold staining for three pituitary hormones: GH, PRL, and ACTH were then carried out. All immunological reagents were diluted in PBS saturated with lead phosphate in order to prevent losses of the enzyme reaction product. Lead phosphate reaction product was found between the cell membranes of most of the parenchymal and folliculo-stellate cells. Heavy precipitate was also found on the endothelial cells and the perivascular spaces of sinuses. High Ca2+-ATPase activity was seen at the surface of the GH cells. PRL cell membranes exhibited a considerably lower enzyme activity. The corticotrophs showed even less, sometimes no lead deposit at their surface. We suggest that, although Ca ions had been shown to be involved in hormone release by all the parenchymal cell types studied, significant differences in the mechanism o r / a n d rate of calcium dependent processes may be supposed among them. References: (1) Y. Takano et al., Cell Tissue Res. 243: 91 - 9 9 (1986). (2) G. El-sherif and E. B~icsy, Histochemistry 501 - 505 (1989).

P7.2 Cytochrome Oxidase Activity in Muscle Fiber Types of Extraocular Muscles - - Histochemical and Cytochemical Analysis. Y. Dake and T. Amemiya Department of Ophthalmology, Nagasaki University School of Medicine, Japan. Extraocular muscles are classified morphologically into two muscle fiber types. Our previous study showed that extraocular muscles of albino rats had two types of mitochondria,

which had significantly different cytochrome oxidase activity patterns. In this study we examined the differences of energy supply from mitochondria in the two types of extraocular muscle fibers. Materials and Methods: The caput of extraocular muscles of female Wistar albino rats was fixed with 2070 glutaraldehyde and cut into small pieces with a vibratome. With the diaminobenzidine (DAB) method of Seligman et al., specimens were incubated and examined by light and electron microscopy. The cytochrome oxidase activity was measured with a computed image analyzer, Zeiss IBAS, by a point-count method. On the basis of the classification of Noda and Kuwahara, mitochondria were classified into two types: Type A mitochondria with histochemically demonstrated reaction products in the inner membrane and outer compartments; Type B mitochondria with histochemically demonstrated reaction products in only the inner membrane of the cristae, or no reaction products at any site. Results: Light microscopy showed many dark brown granules indicating reaction products resulting from oxidation of DAB deposits in each muscle fiber. Muscle fibers were clearly differentiated into two types (type I and type II fibers) by the amount of reaction products on the mitochondria. Type I muscle fibers had more type A mitochondria than did type II fibers. In electron micrographs, two types of mitochondria were seen in type I and type II muscle fibers, and the percentage of type A mitochondria was not statistically significantly different in the two types of muscle fibers. Computed analysis showed high activity of cytochrome oxidase in type A mitochondria in both fibers and there was no difference in its activity level between the two types of muscle fibers. Conclusion: Type A and type B mitochondria are present in both types of extraocular muscle fibers, and there is no difference in energy supply from the mitochondria in the two types of extraocular muscle fibers. P7.3 Demonstration of Coexisting Catecholamine (Dopamine), Amino Acid (GABA) and Peptide (NPY) Involved in Inhibition of Melanotrope Cell Activity in Xenopus laevis. E. P. C. T. de Rijk, F. J. C. van Strien and E. W. Roubos Dept. of Animal Physiology, Faculty of Science, University of Nijmegen, Toernooiveld, 6525 El) Nijmegen, The Netherlands. This quantitative ultrastructural immunocytochemical study

Poster sessions demonstrates the coexistence of a catecholamine (dopamine; DA), an amino acid (GABA) and a neuropeptide Y (NPY) in axon varicosities innervating the pars intermedia of Xenopus laevis. The varicosities are assumed to control the pars intermedia melanotrope cells, which regulate skin colour during the physiological process of background adaptation. Varicosity profiles appear to abut melanotrope cells and folliculo-stellate cells, star-shaped cells that intimately contact the melanotropes. All varicosity profiles contain two morphological types of vesicle. Mono labeling studies on routinely fixed and freeze-substituted tissues show that the small, electron-lucent vesicles store GABA, whereas DA and NPY occur in larger electron-dense ones. Double and triple labelling experiments, in which the degree of immunoreactivity was quantified per varicosity profile and per vesicle, led to the conclusion that (1) DA, GABA and NPY coexist within almost all varicosity profiles, and (2) DA and NPY are costored within electron-dense vesicles. Varicosity profiles that abut melanotrope ceils show a much higher ratio between the numbers of electron-lucent and electron-dense vesicles than varicosities contacting folliculo-stellate cells (15.8 and 3.3, respectively). This differential distribution is in line with the previous demonstration that, in contrast to GABA, NPY does not act directly on the melanotrope cells but indirectly, by controlling the activity of the folliculo-stellate cells.

P7.4

Morphological and Cytochemical Characterization of the Interstitial Cells of Cajal (ICC) in the Intestinal Tract. M. Hasetdonckx, G. Nobels*, M. van de Ven, J. van Reempts and M. Borgers Janssen Research Foundation, Beerse, Belgium and *H. L Sint Lieven, Ghent, Belgium. The first description of interstitial cells in the intestinal smooth muscle wall was given about one century ago by Cajal who defined ICC as neuronal cells with small perikarya and long branching processes. However, at the present time there is still a lot of controversy about the anatomical localization and morphological aspect of these cells. Though, several investigators were able to attribute a physiological regulatory role of pacemaker cell to the so-called ICC. Thus far it could be derived from various animal and human studies that ICC are localized in the deeper layers and in septa of the circular muscle, as well as in between longitudinal and circular muscle layers. They could be morphologically differentiated by their elongated branching aspect and a close association with nerve fibers and blood vessels. Ultrastructurally they resembled either fibroblasts, glial cells, pericytes or smooth muscle cells. The present study aimed at further characterizing ICC in guinea pig ileum and colon. Cytochemical staining for purine nucleoside phosphorylase (PNP) revealed weakly positive cells with digitiform expansions in the deep circular muscle layer and its septa. The cells could be differentiated from strongly positive fibroblasts and non-reactive muscle cells. They formed gap junctions with identical neighbouring cells and with smooth muscle cells and they enwrapped at the same time nerve bundles and blood vessels. In the deep circular muscular layer certain cells contained myofilaments. It will be discussed whether the above described PNP-positive cells are ICC and how they can be differentiated from pericytes.

525

P7.5 Electron Microscopical Demonstration of Alkaline Phosphatase Activity with the Cerium-Based Method in a Citrate-Containing Medium at pH 9.3. C. E. Hulstaerf, K.-J. Halbhuber 2 and D. Kalicharan ~ tDepartment of Histology and Cell Biology, University of Groningen, The Netherlands; 2Institute of Anatomy, Department of Histochemistry, Friedrieh Schiller University Jena, Germany. A cerium-based incubation medium that was developed for the light microscopical demonstration of alkaline phosphatase activity l, was tried out for the electron microscopical demonstration of this enzyme activity in kidney and heart muscle of the rat. The medium is very stable; it consists of 14 mM CeC13, 11 mM Na-citrate, 4 mM MgCl2, I0 mM p-nitrophenyl phosphate, 0.18M glycine/NaOH buffer pH 9.3. The pH 9.3 of the medium is in the optimum range of the enzyme. Before incubation a preincubation was carried out without substrate to promote the penetration of cerium into vibratome sections, Other concentrations of cerium and citrate were tried out as well but 14 mM CeC13 and 11 mM Na-citrate gave the best results. A small amount of nonspecific reaction product was present in the nucleus but that could be largely avoided by postincubation rinsing in a ceriumcontaining buffer'. ~K.-J. Halbhuber, R. Gossrau, U. M611er, C. E. Hulstaert, N. Zimmerman, and H. Feuerstein (1988). Histochemistry 90: 289. 2E. C. M. Hoefsmit, C. E. Hulstaert, D. Kalicharan, and I. L. Eestermans (1986). Histochemistry 84: 329.

P7.6 Ultrastructural Demonstration of Succinate Dehydrogenase (SDH) Activity with the NiFerricyanide Method. Problems and Limitations. C. E. Hulstaerf, J. A. Oosterbaan ~ and M. J. Hardonk 2 1Department of Histology and Cell Biology; 2Department of Pathology, University of Groningen, The Netherlands. Vibratome sections of rat liver, fixed by perfusion with 0.5% glutaraldehyde (GA) and incubated according to the Cuferricyanide 1 method only contained reaction product outside mitochondria. Employment of nickel instead of copper as a capturing ion (complexed with citrate), resulted in reaction product inside mitochondria; however, the nuclei contained reaction product as well and so did the mitochondria in controls. Ultimately, fixation with 2% formaldehyde + 0.05% GA for 5 rain yielded the best results. Some mitochondria contained a high amount of reaction product, some contained little and some contained none. These differences could either reflect a difference in permeability of the inner mitochondrial membrane for the capturing ion complex or a real difference in enzyme activity. When fixed for 3 rain the ultrastructural morphology was too poor for an exact localization of the reaction product. When fixed for 7 rain inactivation occurred and controls appeared to contain substantial amounts of reaction product probably because of the presence of reducing groups introduced into the mitochondria by fixation with GA. For each tissue the optimal conditions with respect to fixation and retention of activity have to be established before determination of SDH is possible. ~S. Kerpel-Fronius and F. Hajds (1968). Histochemie 14: 343.

526 P7.7 Immunoelectron Microscopy Localization of Different Phosphoinositidase C Isoforms. iV. M. Maraldi, N. Zini, A. Valmori, A. M. Martelli ~ and L. Cocco ~ Inst. Citomorfologia CNR, c/o IOR, Bologna; ~ Anatomia, University of Bologna, Italy. It has been previously reported that nuclear polyphosphoinositides undergo a rapid decrease in mass in Swiss 3T3 fibroblasts after simulation with IGF 1 (1 - 2 ) , concomitantly with an increase of the amount of nuclear diacylglycerol (2), conceivably due to an increased nuclear phosphoinositidase C activity. An ultrastructural immunocytochemical study has been performed by using monoclonal antibodies against different phosphoinositidase C (PIC) isozymes In 3T3 cells the PIC form [3 is almost exclusively localized inside the nucleus, mainly in the interchromatin and nucleolar domains, whilst the PIC y is much more present at the cytoplasmic level; PIC 6 isoform is absent in any cell compartment. This degrees with Western blot analysis on isolated cell subfraction and indicates that the PIC fl is not randomly distributed throughout the nucleus but is localized in peculiar nuclear domains. The presence in the nucleus of a specific phosphoinositidase C and its activation by growth factors could represent one of the early nuclear events in the signal transduction between the plasma membrane and the nucleus itself. 1. Cocco L. et al. Biochem. Biophys. Res. Comm. 159, 7 2 0 - 725, 1989. 2. Divecha N. et al. EMBO J. 110, 3207-3214, 1991.

P7.8 Fresh Frozen Sections Mounted on Semipermeable Membrane: Recovery and Subsequent Loss of Fine Structure. P. J. McMillan, R. L. Schultz, S. Manoonkitiwongsa and E. Whitter Anatomy Department, Loma Linda University, Loma Linda, CA 92350 USA. Fresh frozen sections mounted on semipermeable membrane have been used for the light microscopic demonstration of enzyme activities. It is assumed, however, that ice crystal formation prevents using this method for studies of fine structure. It is shown in this report that fine structure is remarkably restored when a frozen section is thawed on a semipermeable membrane and immediately fixed. On the other hand fine structure is rapidly lost when such sections are incubated at 37~ Frozen sections (16/~m) of rat liver, kidney, heart and pancreas quenched in liquid propane are picked up on semipermeable membranes and transferred, section up, to a petri dish containing filter paper which is glistening wet with either fixative (2.5% glutaraldehyde and 2% formaldehyde in 0.1 M phosphate buffer, pH 7.3) for the control or incubation medium. Experimental sections are incubated for 45 min at 37~ before being fixed and processed for electron microscopy. Fine structure of controls was similar to perfusion fixed material. Experimental sections had severe disruption of RER with the appearance of large aggregates of smooth membranes. Mitochondria are greatly swollen and broken and Golgi is not recognizable. Nuclear chromatin loses its density. Muscle tissue is less effected; sarr are still

Poster sessions recognizable. When ATP is added to the medium autolysis is almost complete. Much of the autolysis is prevented by a five minute treatment with fixative diluted 100 fold.

P7.9 Effect of Mixture of Paraphenylenediamine and Imidazole on the Extraction of Lipid Droplets during Electron Microscopy Staining. S. Morii, I. Nakao, N. Fujita and N. Shikata Department of Pathology, Kansai Medical University, Moriguchi, Osaka 570, Japan. It had been reported by us that staining tissue sections with a highly alkaline lead solution induces markedly reduced electron opacity in cytoplasmic lipid droplets, and that electron probe X-ray microanalysis (EPXM) indicates removal of osmium in lipid droplets following to the extraction of osmium-labelled lipids. To prevent the extraction during a double staining procedure for electron microscopy, the tissue slices, double fixed with glutaraldehyde and osmium tetroxide to preserve lipid droplets in the cytoplasm, were immersed for 2 hr in veronal buffer, pH 9.0, containing 0.5% paraphenylenediamine and 0.5% imidazole after osmium postfixation, and then dehydrated, embedded and sectioned. The stained ultrathin sections of the immersed tissue slices showed blackend, well circumscribed lipid droplets similar to those in corresponding unstained sections. EPXM proved no osmium removal even in the stained sections. Moreover, highly contrasted features of the cellular architecture could be visualized with the double stained sections, as well as routinely prepared ones. As the examples, various states of male rat adrenal cortices, early stages of ethionine-induced fatty livers in fasted female rats, and microvesicular fatty livers induced by repeated ip injections of 4-pentenoic acid in male rats are exhibited.

P7.10 A New Cytochemical Method for Peroxidase Yielding An Osmiophilic Blue Dye. K.-I. Ohkawa, H. Chang and P. J. Goldblatt* Chest Disease Research Institute, Kyoto University, Kyoto, Japan and *Department o f Pathology, Medical College of Ohio, Toledo, Ohio 4369-0008 U.S.A. Pararosaniline base (Aldrich) and 2,3-dichlorophenol were used for cytochemical detection of peroxidase both at the levels of light and electron microscope. For light microscopy smears of adult rat peripheral blood were used after being fixed in 10%0 formalin at least for 10 minutes. For electron microscopy fluffy coats prepared from adult rat peripheral blood were fixed in cold 1.25% glutaraldehyde containing 6~ sucrose for 40 minutes. Twenty-thirty micrometres thick frozen sections were cut from those fluffy coats. Smears and the sections were incubated in the following reaction medium; pararosaniline base 5 mg, 2,3-dichlorophenol 5 mg, dimethyl sulfoxide 1 ml, 0.05 M phosphate buffer (pH 7.2) 9 ml, 0.1% H202 0.1 ml. Incubation time was 5 - 1 5 minutes. After incubation the smears were dehydrated in ethanol and xylol, and finally mounted in Canada Balsam. The end-product was blue. Frozen sections cut from fluffy coats were, after incubation, post-fixed in phosphate buffered 1% OsO4 and

P o s t e r sessions embedded in Luveak. The blue colored end-product was osmiophilic and non-diffusible. It is electron-opaque enough for electron microscopy. New fuchsin and basic fuchsin were also applicable.

P7.11

Electron Microscopical Application of TAM Methods for Peroxidase and Catalase. K.-L Ohkawa ~ H. Chang ~ K. Sasabe ~176 and P. J. Goldblatt* ~ Disease Research Institute, Kyoto University, Kyoto, Japan. ~176 Division, Nakalai Tesque Corp., Muko Shi, Kyoto, Japan and *Department of Pathology, Medical College of Ohio, Toledo, Ohio 4369-0008 U.S.A.

TAM methods for peroxidase and catalase, giving rise to red end-product, were applied for electron microscopical detection of the intracellular sites of the enzymes. For peroxidase fluffy coats of peripheral blood fixed in 2.5~ glutaraldehyde (buffered at pH 7.2 with 0. I M phosphate buffer) were used. Livers and Kidneys fixed in 2.5~ glutaraldehyde containing 0.005 M MnCI2 (buffered at pH 7.2 with 0.05 M Tris-HC1 buffer) were used in case of catalase. Composition of the reaction media was as follows: TAM (Tris (4-aminophenyl) methane) 5 mg, aniline 5 rag, Dimethyl sulfoxide 1 ml, 0.1 M Phosphate buffer, pH 7.2, 9 ml, 0.1% HzO2 0.1 ml. Incubation time ranged from 5 - 15 rain. In case of peroxidase, and for catalase, 40 - 90 mix. After incubation the sections were washed in Tris-HCl buffer and postfixed in phosphate buffered 1% OsO4. Then, they were washed, dehydrated and embedded. The red end-product yielded cytochemically were electron opaque enough for observing under an electron microscope. It was not diffusible and the localizations were clear-cut. In stead of aniline, dichtorophenols, dibromophenots, dichloroanilines and dibromoanilines were also applicable, except phenol.

P7.12

Sub-Cellular Location of a Mouse Glandular Kallikrein and its Precursor mRNA in Salivary Duct

527 basolateral surface of the cells. At the apical pole of SD ceils, secretory granules were present which also contained immunoreactive kallikrein, apparently secreted into the lumen of the duct. In ED cells, kallikrein was in small apical granules which appeared to be secreted from both poles of the cells. From this study, we conclude that kallikrein encoded by mGK-6 is synthesized on endoplasmic reticulum in the basolateral region of SD cells and is secreted via constitutive and regulated pathways. Secretion is effected from apical and basolateral surfaces of SD cells and ED cells. ~Penschow, J. D. et al., Mol. Cell Endocrinol. (1991), 81, 135.

P7.13

Investigation of Prokaryotic-Eukaryotic Cell Junction Between Spiral-Shaped Bacteria and Epithelium of the Guinea Pig by In Situ Nick - Translation Reaction on Ultrastructural Level. M. Raygoza-Anaya, A. A. Manykin and V. M. Bondarenko The Institute of Social Maintenance, Mexico, The Ivanovsky Institute of Virology and The Gamaleya Institute of Epidemiology and Microbiology Rus. Acad. Med. Sci., Russia. Samples of guinea pig mucosa were fixed 4% paraformaldehyde 0,1 O7o glutaraldehyde embedded in Lowicryl K4M and nick - translation reaction were carried out on ultrathin sections by standard method [Rigby, P. W. J. et al., 1977, J. Mol. Biol. v l t 3 , 2 3 7 ] - 3 7 C, 0,5 hr. The reaction solution had one of four deoxytriphosphates with biotin label. We used antibiotin antibodies conjugated 10 nm gold colloidal particals for detection of cell sites including labels. Typical ultrastructure of junction was observed [MoraGalindo, J., 1987, Cell Tissue Res. v250,475] :helical bacteria attached to the epithelium by one of the ends into the space between microvilli without damaging epithelial cell. Gold particles were found in nucleus and mitochondria in different epithelial cells, in zone of bacterial nucleus and, possibly, its indicate active DNA form of interacting prokaryoticeukaryotic cells [Reeves, R., 1984, B.B.A.v.782,343].

Ceils. Z D. Penschow and J. P. Coghlan The Howard Florey Institute of Experimental Physiology and Medicine, University of Melbourne, Parkville, Vie., 3052, Australia,

P7.14 Incubation of Unfixed Cryostat Sections for Ultrastructural Demonstration of D-Amino-Acid Oxidase Activity in Rat Liver.

Glandular kallikreins are serine protease enzymes encoded in mouse by 12 closely homologous genes. One of these genes, mGK-6, is expressed in striated (SD) and excretory duct (ED) cells of the major salivary glands as well as in other tissues 1. We have used a 3H-labelled oligonucleotide probe specific for mGK-6 transcripts and hybridization histochemistry at the ultrastructurat level to identify the sub-cellular location of mGK-6 mRNA in SD cells. The protein translated from mGK6 mRNA was located by immunocytochemistry, in SD cells and ED cells, using a polyclonal primary antibody and a 1 nm gold-conjugated second antibody enhanced by silver. The mRNA encoded by mGK-6 was on endoptasmic reticulum, in the basolateral region of SD cells. Immunoreactive kallikrein was present near the basolateral pole of SD cells and is apparently secreted by the constitutive pathway from the

J. p. M. Schellens, W. M. Frederiks, C. J. F. Van Noorden, H. Vreeling-Sindel~irov~ and F. Marx Laboratory of Cell Biology and Histology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands and Paul J. McMillan, Department of Anatomy, School of Medicine, Loma Linda University, Loma Linda, California 92350, USA. Unfixed cryostat sections of rat liver were incubated to demonstrate D-amino-acid oxidase activity at the ultrastructural level. Incubation was performed by mounting the sections on a semipermeable membrane which was stretched over a gelled incubation medium. This technique prevents leakage of proteins from the sections and may contribute also to preservation of ultrastructure. The incubation medium contained D-proline as substrate and

528 cerium ions as capture reagent for hydrogen peroxide. After incubation for 30 rain at 37~ membrane parts with adhering cryostat sections were cut out, fixed and routinely processed for electron microscopy. Ultrastructural morphology of hepatocytes was rather well preserved. Reaction product was localized in peroxisomes, the core of which was free from precipitate. In the absence of substrate the peroxisomes remained unstained. It is concluded that the semipermeable membrane technique permits ultra-structural study after enzyme histochemical incubation of unfixed cryostat sections. In this way optimal demonstration of enzyme activity is not limited by inhibition during fixation, while acceptable morphology is retained.

P7.15

Study on the Formation of Mono-Membrane and BiMembrane Vesicles During Mouse Sperm Acrosome Reaction. J. Sha, Z. Zhou and Z. Hu Shi Zhang Dept. of Histology, Nanjing Medical College, 210029 Nanjing, P. R. of China. The location of mono-membrane and bi-membrane vesicles of mouse sperm was identified using Con A in conjunction with the colloidal gold. The observation showed that both the mono-membrane vesicles and the outer layer of bi-membrane vesicles came from the outer acrosome membrane. The inner layer of the bi-membrane vesical and remain membrane which distributed among the vesicles consisted of the plasmalemma. It suggested that the outer acrosome membrane did not fuse with the plasmalemma during mouse sperm acrosome reaction and that both the mono-membrane and the bi-membrane vesicles of mouse sperm were formed due to winding of the outer acrosome membrane.

Poster sessions portion of the pellicle, with gold particles also on the electron lucent middle layer. Some gold particles were occasionally seen also on the intracystic bodies.

P7.17

Use of a Physical Developer Containing Crownether for Electron Microscopic Histochemical Methods for the Detection of Glycoconjugates. H. Kitamura, K. Ina, M. Nakamura and K. Yamada* Department of Anatomy, Medical University of Oita, Oita 879-56, and *Laboratory of Histochemistry, Department of Anatomy, Nagoya City University Medical School Nagoya 467, Japan. A new physical developer containing crownether has been derived and has successfully been applied to a couple of electron microscopic histochemical methods using silver enhancement procedures for the detection of glycoconjugates. The new developer contained four components; crownether instead of gum arabic, hydroquinone, silver nitrate and citrate buffer. The most favorable property of this physical developer lay in (1) an unusually low sensitivity to light, (2) easy and simple procedures of preparation and (3) rare background staining due to contaminations in tissue sections. Using the new physical developer, postembedding periodic acid-thiocarbohydrazide-silver protein-physical development (PATCH-SP-PD) and high or low iron diamine (HID or LID)TCH-SP-PD methods have successfully been performed upon Lowicryl K4M embedded ultrathin sections of buffered glutaraldehyde-formaldehyde fixed rat kidney and aorta. The results obtained by these methods were either superior or comparable to those yielded by corresponding techniques using physical developers without crownether but with gum arabic.

Ultrastructural Localisation of Cyst Specific Antigen on Pneumoeystis earinii.

P7.18 A Postembedding Method for Histochemical Demonstration of Acidic Glycoconjugates by Electron Microscopy.

A. Sukura, T. Ukkola and L.-A. Lindberg Department of Anatomy and Embryology, Collegeof Veterinary Medicine, Helsinki, Finland. Pneumocystis carinii (PC) is an eukaryotic extracellular organism which causes pneumonia to immunocompromised individuals. PC has three different morphological stages: the trophozoite, the precyst and the cyst with intracystic bodies. A monoclonal antibody from a commercially available diagnotic kit was used for ultrastructural antigen localisation on a rat originated PC. The PC -organisms were from Wistar rats with induced PC -pneumonia by injecting methylprednisolone (16 mg/kg once a week for several weeks). The lung tissue samples were fixed in 6% paraformaldehyde and 0.1% glutaraldehyde, infiltrated with sucrose, frozen in liquid nitrogen and sectioned with a RMC MT 6000 XL ultramicrotome with a CR 21 cryoattachment. The secondary antibody was conjugated with 15 nm gold particles. Although the trophotzoites were abundant in the tissue sections the gold particles were exclusively located on both the outer and inner layer of the cyst pellicle. The labelling was however more pronounced on the outer layer. Distinct labelling was seen on the thickened

K. Yamada, H. Ueda and H. Kitamura* Laboratory of Histochemistry, Department of Anatomy, Nagoya City University Medical School, Nagoya 467, and *Departmentof Anatomy, Medical University of Oita, Oita 879-56, Japan. We have established an efficient postembedding method for the histochemical demonstration of acidic glycoconjugates by electron microscopy, using high or low iron diamine thiocarbohydrazide-silver protein (HID or LID-TCH-SP) sequence followed by a physical development (PD) procedure. As a result of the present experimental studies on phosphate buffered 0.5% glutaraldehyde-4% paraformaldehyde fixed and LR-White embedded ultrathin sections of the rat sublingual and submandibular glands, spleen and colon, the efficiency and specificity of the newly established method were shown to be sufficient. In the rat tissues examined, sulfated and non-sulfated acidic glycoconjugates were visualized by extremely electron dense reaction products consisting of granules of different sizes. Comparison of the present method with the HID or LID-TCH-SP method hitherto used has substantiated the fact that the former method results in apparently higher electron density viz. visibility of reaction products than the latter.

P7.16

Poster sessions P7.19

Study of Enzyme Structure. J. Q. Zhang, X. Y. Lu and Q. Z. Ye Zhongshan University, 510275, Guangzhou, P.R. China. To determine Enz molecule structures with electron microscopy, the Enz specimens will be stained by heavy atoms to enhance the scattering contrast. The pitfalls in using the stain are the difficulty in the data interpretation of the highresolution structural detail. The most fundamental limitation in obtaining high-resolution structural information of unstained Enz molecules is the specimen damage by the irradiating electrons and vacuum. In order to surmounting these difficulties, following methods would be used: (1) Phase contrast; (2) The agueos medium is replaced by glucose; (3) Low dose electron microscopy. However, a low dose image will not display the projected structure of a molecule directly.

529 Because contrast and S/N are very low. Nevertheless, provided that the object is comprised of a large enough number of molecules and that the periodicities are precisely maintained, all the information need to reconstruct the "average" molecule will be available by computer. In order to reconstruct the object, one need to determine the Fourier amplitude and phase for each reflection. The Fourier phase can obtain from the Fourier transform the low-dose images and for calculating the Fourier amplitude is to measure the intensity of the reflection spots in an electron diffraction pattern. The micrographs and diffraction pattern were scanned with an automatic microdesitometer. The areas recorded consisted of 2048 x 2048 arrays of optical density readings. We initially processed a 1024 x 1024 region from the array on an IBM 286 computer using Fourier transform.

P8. H I S T O C H E M I S T R Y OF O N C O P R O T E I N S PS.1 Expression of p62 ~'mycOncoprotein in Breast Tumour Tissue. S. Frkovid-Grazio, S. Grazio, T. (~abrijan and K. Pavelid ~ "Sestre milosrdnice" Clinical Hospital and IRudjer Bo)kovid Institute, Zagreb, Croatia. The expression of the c-myc protooncogene product (p62 ~-myc) was examined by use of immunohistochemistry in 44 ductal invasive breast carcinomas, 16 fibroadenomas and 20 specimens of fibrocystic breast disease. Fresh frozen tissue sections were subsequently AMeX (Acetone, Methyl bensoate, Xylene) processed, embedded in paraffin and submitted to standard PAP immunohistochemical method. The intensity of immunostaining and percentage of positive epithelial cells were assessed and scored using semiquantitative method for reporting histochemical assay result (c-myc score). Positive staining was observed in all samples regardless pathohistological diagnosis but with considerable heterogeneity between different tumours as well as within individual tumors. Higher level of p62 c'myc expression was found in ductal invasive carcinoma sections comparing to fibroadenomas (P<0.01) but not comparing to fibrocystic breast disease sections. There were no differences of p62 c-myc expression between grade I and grade II carcinoma sections. Use of AMeX procedure in previously fresh frozen tissue resulted in predominantly nuclear immunostaining of p62 cmyc, which is its normal subcellular localization.

P8.2 Expression of c-erbB-2 in Paget's Disease of the Nipple and Underlying Lesions, 7'. Haerslev and G. K. Jacobsen Department of Pathology, University Hospital of Gentofte, 2900 Hellerup, Denmark. The expression of c-erbB-2-oncoprotein in Paget's disease of the nipple (PD) and underlying intraductal carcinoma and/or invasive carcinoma was studied using the indirect immuno-

ABC-technique with a monoclonal antibody, anti-c-erbB-2 (Biogenex). In 26 women with PD positive staining was present in the PD cells in 23 (88%). Nineteen of the patients had an underlying intraductal carcinoma and 18 of these (95~ showed positive staining reaction within the carcinoma cells. Eleven had an underlying invasive ductal carcinoma, and 10 of these (91%) were positively stained. In three patients the PD cells as well as the underlying lesions were negative. The c-erbB-2 staining in PD was compared with staining for cytokeratin (CK) and epithelial membrane antigen (EMA). C-erbB-2 was found to be a helpful diagnostic marker of the Paget's cells, especially in conjunction with CK. Previous studies have shown that the expression of c-erbB2-protein in primary breast cancers is correlated to shorter relapse free period and poorer short-time survival. This is under investigation in the present study. The high percentage of c-erbB-2-oncoprotein expression in PD and underlying lesions is noteworthy. Previous studies have demonstrated c-erbB-2-oncoprotein expression in only 1 0 - 3 0 % of invasive ductal carcinomas and in 6 0 - 62% of intraductal carcinomas (1,2). ~Lovekin, C. et al. Br. J. Cancer, 6 3 , 4 3 9 - 4 4 3 , 1991. 20'ReiUy, S. M. et al. Br. J. Cancer, 6 3 , 4 4 4 - 4 4 6 , 1991.

P8.3 Immunohistochemical Characterization of Rapidly Proliferating Breast Cancers. J. J. lsola, T. Visakorpi and O.-P. Kallioniemi* Dept. Biomed. Sciences, University of Tampere, Box 607, SF-33101 Tampere, Finland. Rapid proliferation rate as defined by flow cytometric (FCM) s-phase fraction (SPF) is the most important prognostic parameter in node-negative breast cancer. We studied how well steroid receptor (by ER, PR) and oncoprotein mediated (c-erbB-2, EGFR, p53) growth explain total cell proliferation rate and patient survival. In 289 node-negative patients, a significant association was found between high SPF and low ER and PR content, high EGFR immunoreactivity and c-erbB-2 over-expression. Stepwise logistic regression analysis

530

P o s t e r sessions

showed that hormone receptors and c-erbB-2 were independent determinants of SPF classifying proliferation status (SPF <12%0 vs. >12%) correctly in 75% of the tumors. Although ER, p53 and c-erbB-2 were significant predictors of poor outcome in univariate analysis, they lacked independent prognostic value in Cox multivariate analysis when S-phase fraction was included in the multivariate model. This suggests that the prognostic value of ER, c-erbB-2 and p53 is mainly due to their close association with increased cell proliferation.

Amplification of c-erbB-2 oncoprotein is generally correlated with estrogen receptor negative tumors (18%0) and well expressed with high levels of CA 15.3 and CEA glycoprotein in the serum, two parameters suggesting progression of the disease, recurrence of malignancy and spreading of metastases.

P8.4 erbB-20ncogene Expression in Breast Cancer Correlation with Serum CA-15.3 and CEA.

P8.5 Prognostic Significance of p53 Cytoplasmic Expression in Colorectal Adenocarcinomas.

N. Livni, P. Cohen, V. Barak and E. Rosenmann Department of Pathology and Oncology, Hadassah Medical Organization, Jerusalem, Israel.

X.-F. Sun, J. M. Carstensen, O. StM, H. Zhang* S. Wingren, T. Hatschek and B. Nordenskj61d Departments of Oncology and Pathology H* University Hospital, S-581 85 Link6ping, Sweden.

The c-erbB-2 oncogene is closely related to the epidermal growth factor receptor. This gene has been shown to be amplified in human breast cancer cell lines and encodes an 190 K protein. In this study 70 primary breast tumors were investigated by immunoperoxidase methods on formalinfixed, paraffin-embedded tissue, c-erbB-2 oncoprotein was expressed in 25 cases (350?0) and showed an intense membrane associated staining of tumor cells, evaluated visually. 38 cases, (36 infiltrating carcinoma; 2 intraduct carcinoma) were evaluated histologically for several parameters amongst them nuclear grade and mitotic activity. The 11 cases positive for erbB-2 showed moderate to high nuclear grade and elevated mitotic activity. In addition these data were correlated to more currently used prognostic factors including hormonal receptor status, CA-15.3 and CEA levels in serum. Results: ER+ erbB-2+ erbB-2-

ER-

serum values

12 (17~ 13 (18%) CA 15.3 36 (51070) 9 (13070) CEA

erbB-2+ erbB-229.6 (9) 6.75(6)

16.5 (16) 1.21 (14)

p53 is inactivated by mutation, deletion and complexing to other proteins in many different types of tumor. Inactivity often results in overexpression and thus detectability of p53 protein by immunohistochemistry, p53 oncogene expression was estimated in 293 human colorectal adenocarcinomas with standard immunohistochemical technique, p53 expression was classified as nuclear pattern, cytoplasmic pattern, as well as both nuclear and cytoplasmic pattern. Nuclear or cytoplasmic expression showed positive correlations between primary tumors and their lymph node metastases (p<0.0001 and p<0.01, respectively). The frequency of positive staining in the cytoplasm increased gradually from Dukes' stage A to D tumors (p = 0.0001). In multivariate survival analysis, p53 cytoplasmic expression indicated worse prognosis, independent of nuclear staining, grade of differentiation and Dukes' stage (p = 0.0021). We conclude that the cytoplasmic expression of p53 oncogene may be an important biological marker for determining prognosis in colorectal adenocarcinomas.

P9. C Y T O C H E M I S T R Y OF T H E N U C L E U S P9.1 DNase I Digestion as a Tool for the Quantitative Evaluation of C-Heterochromatic-DNA In Situ. M. G. Bottone Dept. Biologia Animale of the University and CNR Ctr Studio lstochimica, Piazza Botta 10, 1-27100 Pavia, Italy.

The amount of DNA resisting the C-banding pre-treatments (C-heterochromatic-DNA) was found to account for the interspecific differences of genome size in different Primate groups. The evaluation of this parameter is therefore of great interest in cytotaxonomy. In this work, DNase I digestion was used instead of C-banding, in an attempt to set up a suitable method for the quantitative evaluation of C-heterochromatic DNA in both metaphase chromosomes and interphase chromatin. In fact DNase I is known to preferentially digest "active or potentially active" chromatin, and the highly repetitive and inactive DNA in C-heterochromatin should characteristically resist DNase I cleavage. As a model system, differently fixed mouse splenocytes were treated with DNase I for various times, and the digestion was monitored by flow cytometry after propidium iodide staining. In addition, mouse

methaphase preparations from lymphocyte cultures were also digested with DNase I, and the amount of residual DNA was evaluated by static microfluorometry. Under controlled conditions of fixation, enzyme concentration, time and temperature, the same limit-digest can be obtained in both interphase nuclei and metaphases, which corresponds to the amount of residual DNA after C-banding and has a C-banding-like pattern in chromosomes.

P9.2 Interphase DNA Accessibility to In Situ Endonuclease Digestion in 3T3 Cells. C. Cinti ~, L. M. Neri 2, R. del Coco ~, N. Baidini 3, E. LaI1P and N. M. Maraldi ~ tlstituto di Citomorfologia Normale e Patologica, C.N.R. Bologna; 21stituto di Anatomia Umana, Ferrara; ~Istituto di Oncologia L O.R. Bologna; 4Istituto di Immunotogia e Genetica L O.R. Bologna, Italy. Cytological experiments were undertaken in order to characterize the action of three restriction enzymes on

Poster sessions synchronized 3T3 cell nuclei. Fixed cells were treated with endonucleases (AluI, HaeIII, EcoRI) for time ranging from 0.5 to 5 hours, stained with DAPI and subsequently analyzed by cytofluorimetry. After 3 hours every endonuclease reached its maximal activity. Fluorescence intensity was proportionally reduced corresponding to the DNA fragment length: the 180bp cutting frequency of AluI produced a reduction of nearly 50~ of fluorescence intensity after 30 min, while the 3200bp cutting frequency of EcoRI reduced the fluorescence measurements of 9~ In situ treatment with AluI and HaeIII produced a remarkable digestion of interchromatin domains, while EcoRI removed the heterochromatic regions A-T rich, which were hardly detectable after 180 rain digestion. In cells synchronized in G1 phase, the specificity of EcoRI for heterochromatic regions A-T rich in interphase nuclei is maintained and the accessibility to these regions is highly conserved. These data should be carefully taken into account by using restriction enzymes to produce single stranded DNA to study chromatin accessibility and organization.

P9.3

6-Iodoacetamidofluorescein Labelling to Study the State of Sulfhydryl Groups after Thermal Stabilization of Isolated Nuclei. A. M. Martelli, L. M. Neri *§ C. Cinti*, L. ZamaF, L. Manzoli ~ and L. Cocco Istituto di Anatomia Umana, Bologna," +Istituto di Citomorfologia del C.N.R., c/o LO.R., Bologna; *Istituto di Anatomia Umana, Ferrara; %aboratorio di Biologia Cellulare, c/o L O.R., Bologna; ~ di Morfologia Umana, Chieti, Italy.

It is well known that a brief exposure of isolated nuclei to the temperature of 37~ can "stabilize" the nuclear matrix, i.e. the high salt and nuclease resistant domain which is thought to serve as a nucleoskeleton. However, the molecular mechanisms underlying this phenomenon are completely unknown. Some authors have claimed that the formation of disulfide bonds could be responsible for the insolubilization of nuclear proteins, while others have been unable to confirm such an observation. Using the specific probe 6-iodoacetamidofluorescein we have performed a multiparameter study, involving flow cytometry, polyacrylamide gel electrophoresis and confocal microscopy analysis, to analyze the state of sulfhydryl groups in isolated murine erythroleukemia nuclei following a 37~ incubation. All the techniques employed failed to reveal significant differences between heat exposed nuclei and those kept at 0~ Thus we conclude that the formation of disulfide bonds cannot be considered a major factor which leads to the stabilization of the nuclear matrix induced by heat exposure.

P9.4 Modifications of DNA with Aldehyde Blocking Agents and their Use for DNA Light and E M Cytochemistry. E. A. Erenpreisa and T. Freivalds Latvian Institute of Experimental and Clinical Medicine, Latvia.

Depurinized DNA can be modified after linking with various aldehyde blocking agents. This approach has been used to develop some cytochemical reactions: I) enhancing acidic

531 properties of DNA (sodium bisulfite, hydroxylamine) to provide an attenuated DNA reaction with uranylacetate in ultrathin sections; to provide anisotropic and metachromatic reaction with toluidine blue, which may be used to estimate packaging regularity of the chromatin; 2) blocking accessibility of DNA to any ionic dye (phenylhydrazine) to obtain specific negative EM contrast of DNA-containing structures; 3) binding with dinitrophenylhydrazine to provide specific, stereochemical, orthochromatic, and virtually independent of the chromatin proteins staining with cationic dyes. Light-optical variants of the use of aldehyde blocking agents in couple with toluidine blue staining of DNA have been characterized by their absorption spectra, their dependence on the packaging of the chromatin and validity for TV image analysis of the chromatin compartments in various kinds of cell nuclei.

P9.5 The Centrosome as a Point of Reference in Nuclear Architecture. R. Hulspas, P.-J. Krijtenburg, A. B. Houtsmuller and

J. G. J. Bauman ITRI-TNO, Dept. Molecular Pathology, P.O. Box 5815, 2280 HV Rijswijk, The Netherlands.

It has been hypothesized that gene expression is related to the nuclear architecture of a cell. To be able to test this hypothesis, a precise description of the nuclear architecture is needed. In our studies we try to give a description of the topological DNA distribution within interphase cells. Visualization of specific DNA sequences is done by means of FISH on paraformaldehyde fixed cells in suspension. In order to measure intranuclear distances a topological point of reference was needed. We chose the centrosome to act as such a point, and developed a method to combine the visualization of specific DNA sequences and the centrosome. The doublelabelled cells were detected by means of confocal scanning laser microscopy. Bio Rad and SCILL software packages were used to make three dimensional reconstructions of cells and to interactively measure distances in three dimensions. Distances were normalized relative to the nuclear membrane or to the centrosome.

P9.6 Electron Microscope Cis-Platinum Staining of Chromosomes Digested with Restriction Enzymes. R. Del Coco, L. Stuppia, C. Cinti and N. M. Maraldi Inst. Citomorfologia C.N.R., Chieti and Bologna, Italy.

Restriction endonucleases have been used to digest fixed mammalian chromosome preparations. These enzymes cleave DNA at specific nucleotide sequences and induce characteristic banding patterns (Lima de Faria et al., 1980; Mezzanotte et al., 1983, Miller et aL, 1983) revealed by Giemsa staining or other DNA- specific dyes. The banding patterns reflect the frequency and distribution of restriction sites in relationship to regional variation in the higher-order chromatin organization. We examine ultrastrural alterations produced by digestion with three different endonucleases, by using the staining with

532 cis-dichloro-diammine platinum, an antitumor agent which reacts with nucleic acids, carried out on slide before to remove chromosomal spreads by a formvar film covering. In this way we observed morphological modifications produced by HaeIII, AsnI and Kpnt, not visible without staining or with staining with uranyl acetate. The Giemsa banding pattern of HaeIII digestion was compared with ultrastructural images to correlate the reduced density regions to restriction enzyme sites, Chromosome spreads digested with AsnI showed large weak regions irregularly localized. KpnI digestion produced a uniform decondensation of the chromatin, conceivably related to a release of chromatin loop coiling.

P9.7 Human Sperm Chromatin Structure after Treatment with Chemical and Physical Agents. M. Zuccotti*, C. A. Redi*, H. Katayose**, G. Bottiroli*, R. Ramponi*, A. C. Croce* and R. Yanagimachi** *Dipartimento di Biologia Animale and Centro di Studio per l'Istochimica del C.N.R., University of Pavia (Italy) -**Department of Anatomy and Reproductive Biology, University of Hawaii at Manoa, Honolulu (U.S.A.). When sperm were treated with different chemical or physical agents (H20, sonication, ETOH, 60-90-100~ EDTA DNase, U.V.), and then injected into mature hamster oocytes, some of them, according to the substance the sperm have been treated with, retained their ability to decondense (100~ DNase, U.V. were not able to decondense; EDTA and 90~ some nuclei can develop into pronuclei). The occurrence of sperm deeondensation does not necessarily mean a further normal development of the embryo, moreover it does not give any information on the changes the sperm chromatin went through after the treatments. With this in mind, we reconsidered these experiments and studied the changes in the sperm chromatin structure each of the treatments brought about, by the use of two very sensitive cytofluorometric techniques: Energy transfer and Image Analysis. These techniques give us an insight on the sperm chromatin structure in terms of the spatial relationship existing between DNA and protamines, before and after treatment. The rate of alterations shown by these analyses explains the failure of decondensation and pronucleus formation of some of the sperm microinjected inside the eggs. Moreover, the new inputs came from these data helped us to better tune up our knowledge on a model for the structure of chromatin in Mammalian sperm suggested by Balhorn (J. Cell Biol. 93: 2 9 8 - 305, 1982) and reanalyzed by Ward and Coffey (Biol. Reprod. 44: 5 6 9 - 574, 1991).

P9.8 Visualization of EGF-Receptor mRNA in A431 Nuclei. O. C. Sibon, F. F. Cremers and A. J. Verkley Department of Molecular Cell Biology, Padualaan 8 3584 CH Utrecht, The Netherlands. A lot of biochemical research has been done to investigate premRNA processing in the nucleus. These studies suggest that the nuclear matrix is involved in RNA packaging, processing and nuclear transport. For a better understanding of how

P o s t e r sessions RNA metabolism is integrated in the nuclear structure it is necessary to know the precise location of precursor mRNA in the nucleus. Non-radioactive in situ hybridization can be of great help to investigate this because with this technique it is possible to localize specific RNA sequences with a high resolution within individual cells. In our study we have localized the mRNA of the epidermal growth factor receptor (EGF-Receptor) in the nucleus of A431 cells. We use this as a model system because the EGF-receptor mRNA is present in a large amount in A431 cells. After hybridization with a digoxigenin labelled eDNA probe, detection was done via an antidigoxigenin antibody, directly coupled to alkaline phosphatase, followed by the alkaline phosphatase colouring reaction. We provide evidence that the EGF-receptor mRNA is associated with the nuclear matrix and is localized in restricted nuclear regions displaying a dotted or curvilinear shape. The regions were found preferentially localized around nucleoli.

P9.9 Counting of Centromeres in Interphase Nuclei of Tissue Sections with Confocal Laser Scanning Microscopy. H. Sugihara, K. Katsura, T. Takamatsu and S. Fujita Department of Pathology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokohji, Kamigyoku, Kyoto 602, Japan.

Using a confocal laser scanning microscope (LSM), we could count the centromeres stained with anti-centromere antibody in the nuclei of tissue sections. We fixed 2 5 - 30 ~m thick frozen sections of human cancer tissues with PLP (0.507o paraformaldehyde) at 4~ for 5 to 30 rain. The sections were incubated successively with 1:50 dilution of crude antiserum, obtained from a patient of CREST syndrome, biotinylated anti-human-Ig G and FITC-labelled avidin and mounted with anti fade solution (1070 phenylenediamine). We took microtomographs of interphase nuclei at 1/~m pitch with a confocal LSM (Olympus LSM GB). Stereo view of 3D images of intact nuclei in the sections, reconstructed from microtomographs, enabled us to count the number of centromeres even in each of interlacing nuclei in tissue sections. We successfully detected 4 4 - 4 6 centromeres in normal fibroblasts and could count as many as 70 centromeres easily in a cancerous nucleus, although the size of spots varied remarkably. We identified some near diploid aneuploid cells with around 50 centromeres, which had the shift of DNA index smaller than 0.1 that is difficult to detect by flow cytometry. With this technique, the distribution of chromosome number can be mapped in the tissue section of tumors even in the areas of cytometrically uniform ploidy mode,

P9.10 The Use of Sodium Hydroxide-Formaldehyde as a Fixative to Simplify the Staining of DNA by NAMAUr Technique. P. S. Testillano ~'2, C. J. Tandler 3, A. Olmedilla ~ and M. C. Risuefio JCentro Inv. Biol., CSIC, Veldzquez 144, 28006 Madrid, Spain. 2Dep. CC. Morfot. y Cirugi~t. Univ. Alcatd, Spain. 3Ito.Biol. Cet. Fac. Medicine, Conieet, Buenos Aires Argentina.

The NAMA-Ur is a specific method for staining DNA at the EM level (1). It is based on a weak alkaline hydrolysis to

Poster sessions remove RNA and phosphate groups of proteins, followed by Methylation-Acetylation (MA) to block the Ur stainable groups of proteins, which is performed on aldehyde fixed tissues. In this work the samples were directly fixed overnight in a 0.25N NaOH solution in 4% formaldehyde which turned out to be a good fixative for DNA. Subsequent M A treatment and uranyl staining provided a specific contrast to DNA, similar to that obtained after NAMA-Ur technique. The method was performed on mouse testis and liver as well as on onion root meristems. The chromatin patches and fibres appeared densely stained whereas the interchromatin structures and the nucleolus showed no contrast except for the intranucleolar chromatin masses. No staining was observed in the cytoplasm. The NaOH-formaldehyde solution, with such a high pH, allows complete RNA hydrolysis, being at the same time a suitable fixative for ultrastructural studies of DNA. ~Testillano et al. J Histochem. Cytochem. 39,10 (1991) 1427. Supported by CICYT PB87 0332-C02-01 and Joint Research Program CSIC/CONICET.

P9.11 Ultrastrnetural Detection of RNA within the Nucleolus by Molecular Immunocytochemistry. M. Thiry and G. Goessens Laboratory of Cell and Tissue Biology, University of LiOge, Belgium.

The precise distribution of RNA within the nucleolus has been investigated by means of the in situ polyadenylate nucleotidyl transferase method. This technique relies on the fact that during sectioning RNA ends appear at the surface of the sections. These RNA ends can be specifically labelled using exogeneous polyadenylate nucleotidyl transferase and biotinylated ATP. Subsequently, the labelled sites can be detected by a very sensitive immunogold labelling procedure. On the other hand, a postembedding immunogold labelling technique involving monoclonal anti-RNA antibodies has been used for studying the exact location of RNA within the nucleolus. Under these two conditions, label is found over the granular component and the dense fibrillar component of the nucleolus. In addition, an evident label is also consistently visualized over the fibrillar centres, both in their peripheral regions and in their central part. This result contradicts earlier data based on high-resolution autoradiography after uridine incorporation. Together with other recent immunocytochemical and in situ hybridization observations, these results support the view that transcriptionally active rRNA genes are located in the confines of the fibrillar centres.

533 P9.12 Transcription by RNA Polymerase II (RPII) is Confined to Discrete Nuclear Domains. D. G. Wansink, W. Schul, I. van der Kraan, R. van Driel and L. de Jong E. C. Slater Institute for Biochemical Research, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands.

We have developed a novel method to visualize transcription in the nucleus in order to study the spatial distribution of sites of pre-mRNA synthesis. This technique is based on the ability of RPII to use the UTP-analogue 5-BrUTP efficiently as a substrate. Both run-on transcription in permeabilized cells, and microinjection in living cells were used to allow incorporation of BrUTP in RNA. Labelled RNA was visualized by indirect immunofluorescence microscopy, using a Br(d)Urd-specific mAb. The immunofluorescence pattern consisted of 2 0 0 - 5 0 0 speckles of different intensity throughout the nucleoplasm in all animal cells tested. The intensity but not the number of speckles seemed to increase as a function of incubation time with BrUTP. The immunofluorescence signal was completely sensitive to a-amanitin (1 gg/ ml) and RNase, indicating that the pattern reflects transcription by RPII. Indirect immunofluorescence with a mAb against RPII resulted in a similar speckled pattern. BrUTP-labelled nucleoplasmic speckles may correspond to sites of RNA synthesis on single genes, or, more likely, on clusters of active genes. This BrUTP-labelling technique will enable us to study the spatial distribution of pre-mRNA transcription sites with respect to sites of other nuclear activities (e.g. RNA processing and replication) in double labelling experiments.

P9.13 Different DNA Topoisomerases are Localized in Specific Nuclear Domains. N. Zini, S. Santi ~ P. Sabatelli, F. Marinelli, L. M. Neri* and N. M. Maraldi Inst. Citomorfologia CNR, Bologna; ~ Biol. Cell. and M.E., IOR, Bologna; *Inst. Anatomia, University of Ferrara, Italy.

Monoclonal antibodies have been raised against Topoisomerase I and 170 and 180 Kd isoforms of Topoisomerase II (Negri, C. et al. Exptl. Cell Res. in press), which, by immunofluorescence, localize in different nuclear areas, being the 180 Kd isoform detected in the nucleolus by immunoelectron microscopy (Zini, N. et al. Exptl. Cell Res. in press). Here we further investigated the subnuclear distribution of the different Topoisomerases by confocal microscopy and E.M. immunocytochemistry. The immunolocalization has been performed in K562 and HeLa cells and in isolated nuclear matrices from K562. Topoisomerase I is present in the interchromatin as well as in the nucleolar domains; the 170 Kd Topoisomerase II is mainly present in the interchromatin and in the inner network of the nuclear matrix, while the 180 Kd isoform is exclusively localized in the nucleolus and in the nucleolar remnant of the nuclear matrix. These results indicate that the different Topoisomerases play specific roles in DNA replication and transcription and are integral part of some nucleoskeleton structures.

534 P10. HISTOCHEMISTRY

Poster sessions IN DIAGNOSTIC

PATHOLOGY

P10.1

Histochemicai Characterization of Specific Carbohydrate Binding Sites of Epithelial Neoplasms - - Their Variability Associated with Malignancy. H. Bahn, J. Knolle, D. Stiller, F. W. Rath and H. J. Gabius Martin-Luther-University Halle, Philipps-University Marburg, FRG. By applying neoglycoproteins (NGP) it is possible to decide whether the expression of carbohydrate-binding sites correlates with different grades of malignancy. A panel of biotinylated NGPs was used for the detection of endogenous sugar receptors in formalin-fixed, paraffin-embedded specimens of different type of carcinomas. The NGPs used consisted of conjugates of carbohydrate-free bovine serum albumin (BSA) and different sugars, labelled by biotinylation. The following biotinylated NGPs have been used in this study: fucose-, lactose-, maltose-, mannose-, a-N-acetyl-Dgalactosamine-, /3-N-acetyl-D-galactosamine-, and N-acetylgtucosamine-BSA-biotin. The binding to the tissue sections can be made visible by using the avidin-peroxidase technique and by staining with the H2Oz/diaminobenzidine method. The ability of different enzymes to improve the staining was tested, as well as the variation of conditions such as incubation time, temperature and concentration of NGP. Control reactions were performed in order to clearly recognize or exclude endogenous peroxidase, endogenous biotin, and unspecific interactions by using additional agents, e.g. carbohydrate-free BSA-biotin or biotin-free carbohydrate-BSA in special tests of competitive inhibition. In different stages of carcinogenesis of the cervical epithelium and urothelium the binding of the NGP varies depending on the dedifferentiation.

worth using in the obstetrics and gynaecologic field. Hopefully, the present study on malignant ovarian tumor imprinted smear may serve as parameter in distinguishing between benign and malignant tumor.

P10.3

Morphological Study on the Action of PGR Decoction in Prevention of the Development of Experimental Liver Cirrhosis. L. Bian, X. Shen, Z. Shu, H. Wang and H. Huang Nlngxia Medical College, Yinchuan, China. This paper presents the histochemical and ultrastructural study on liver cirrhosis of rats treated by PGR decoction (Paeonia rubra, Gardenia jasminoides, Rheum palmatum). Forty-eight wistar rats were divided into three groups. The liver cirrhosis group (14) was given CC14 and other compound factors, the treated group (20) PGR decoction through gastric intubation for forty-one days besides CCL14 and other compound factors, while the control group (14) normal foods and water only. The result of the treated group shows the rate of liver cirrhosis was obviously decreased as compared with the liver cirrhosis group (P 0.01). In hepatic lobule the necrotic area and the liver cellular fat droplets decreased. And RNA, glycoge increased, nucleate and SDH, G-6-Pase activity increased as well. The rough endoplasmic reticulum recovered almost to normal range, the collagenous fibre in perisinusoidal space decreased. This experiment PGR decoction available can decrease liver cirrhosis caused by CC14 and other compound factors. This paper discusses the possibility of the preventive organism on the liver cirrhosis.

P10.2

PI0.4

Cytodiagnosis of Malignant Ovarian Tumor.

Expression of Nuclear Lamins in Lung Cancer.

K. Ito and K. Noda

J. L. V. Broers, H. Kuijpers and F. C. S. Ramaekers University of Limburg, Dept. of Molecular Cell Biology & Genetics, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Lamins belong to the family of intermediate filament proteins and make up a fibrillary network that constitutes the nuclear lamina. It has been established that lamins A and C (A-type lamins), but not B-type lamins are differentially expressed during embryogenesis, being absent in undifferentiated cells and the earliest stages of development. We have examined small cell lung cancer (SCLC) and nonSCLC cell lines for their lamin expression patterns, using different monoclonal antibodies to A- and B-type lamins in immunocytochemistry and immunoblotting experiments. Non-SCLC cell lines were positive with both B-type and A-type lamin antibodies, while SCLC cell lines were in general positive with B-type lamin antibodies but negative for A-type lamins. These data were confirmed by Northern blotting experiments using a cDNA probe hybridizing to lamin A and lamin C mRNA. In freshly frozen tung tumor specimens of SCLC the absence of expression of A-type lamins as seen in SCLC cell lines was confirmed. Surprisingly, solid non-SCLC showed, next to the presence of A-type lamins in cell nuclei, also strong cytoplasmic staining with A-type lamin antibodies

Kinki Univ. Sch. Med. Obst. Gyne., Osaka, Japan.

To investigate the possibility of distinguishing benign tumor from malignant tumor and assuming histological types in imprinting cytodiagnosis of ovarian carcinoma, imprinted cells of tumor were compared with histological findings of operated tumor specimens. Imprinted smear was cytoanalyzed from a malignant group of 94 cases [serous cystadenocarcinoma (38), mucinous cystadenocarcinoma (17), clear cell adenocarcinoma (5), endometrioid carcinoma, (4), etc.] and a benign group of 25 cases serving as controls [serous cystadenoma (5) and mucinous cystadenoma (10)]. From the Papanicolaou, periodic acid, alcian blue and mucicarmine stains, and immunochemical stains of a-fetoprotein and protein S-100 of the imprinted material of tumor from each cases, cells were compared with those of operated tumor specimens. Cytodiagnosis showed that in many cases, the histological types of the malignant tumors were identifiable from their morphological and cytochemical characteristics. Imprinted smear examination seemed comparable in usefulness with histological examination using rapidly frozen sections. Then former (4 minutes) was superior to the latter timewise. It was demonstrated that imprinted smear would be

Poster sessions in a number of cases. B-type lamins were consistently expressed in all SCLC examined while in non-SCLC some cases showed no immunoreactivity with B-type lamin antibodies in a considerable number of tumor cells within otherwise positive tumors. Lamin antibodies may therefore become applicable in lung cancer diagnosis.

P10.5 Blood Cell Cytochemistry as an Indicator of Therapeutic Efficiency of MM-Range Electromagnetic Radiation. L. S. Bundyuk, G. S. Litvinov and L. I. Matseiko SRC "Vidguk'" Vladimirskaya 61-b, Kiev, 252033, Ukraine. The development of pathological processes may be judged from blood cell functional state estimated by enzyme cytochemical investigation because blood cell enzyme activity has been changing before clinical symptoms of disease manifest themselves. The activity of energy metabolism enzymes (SDG, LDG) of peripheral blood lymphocytes was studied in 70 white rats with Heren's carcinoma and in 60 mice C57B1/6 with Lewis's carcinoma of lungs. Animals were exposed to radiation frequency of 35,9-42,3 GHz (group 1), of 42,3-48,9 GHz (group 2) and of 48,9-55,1 GHz (group 3). It was established that with the tumor growth lymphocyte energy metabolism in animals was characterized by substantial shift towards glycolysis. On the 13th day after tumor inoculation SDG/ LDG in rats and mice amounted to 0.3, and in the intact animals - - t o 2.1 and 1.3, respectively. After 10 sessions of exposure for 20 min/day in rats and mice of groups 1 - 3 SDG/LDG amounted to 1.6, 0.9, 0.7 and 0.8, 0.6, 1.3, respectively. Restoration of lymphocyte energy metabolism correlated with the data of tumor growth rate, metastatic spread and with the rate of tumor complete regression. Thus blood cell cytochemistry may be used as indicator of ramrange EMR therapeutic efficiency in oncology.

P10.6 Glycoeonjugate Histochemistry of Gastric Carcinomas Using the Lectin, Griffonia simplicifolia Agglutinin-II. L. Chen and P. Liang 1Pathological Department, Fujian Medical College, Fuzhou, P.R. of China; and 2Laboratory of Electron Mircoscopy. The histochemical binding of biotin-conjugated Griffonia simplicifolia agglutinin-II (GSA-II) to paraffin sections of 107 gastric carcinomas is described. This lectin is specific for N-acetylglucosamine and a valuable marker for studying mucous neck cells. Binding of GSA-II to tumor cells appeared inconsistent, but its reactivity, binding patterns and frequency of positive cell had relations with differentiation and histotogie types. In well-differentiated tubular and papillary adenocarcinomas, GSA-II labelling was usually restricted to luminal surfaces and apical cytoplasm. Extracellular mucus pools in mucoid adenocarcinoma might be stained or unstained with GSA-II. GSA-II binding to signet ring cells was rare. Their cytoplasmic vacuoles were usually negative. In undifferentiated carcinoma GSA-II binding sites could be

535 found in entire cytoplasm, although PAS and HID/AB (pH 2.5) reactions were usually negative. No significant difference in GSA-II reactivity was discerned between the diffuse type carcinoma thought to have originated from mucous neck cells and the intestinal type carcinoma supposed not originated from them. The microscopic features of gastric cacinomas are complicated and heterogeneous, it is suggested not trying to establish the cellular origin simply from their phenotypic changes. In this paper, the factors influencing the expression of GSA-II binding sites were also discussed.

P10.7

Expression of PCNA Immunoreactivity in Sertoli Cells from Human Cryptorchid Testes. J. Codesal, L. Santamaria, I. Gonzalez-Mediero, R. Martin, A. Queizan and M. Nistal Dpt Morphology, Sch Medicine, Autonomous University (JC, LS, RM); Dpt Pathology, Hospital "Ni~o Jesus'. (IGM); Dpt Pediatrics (AQ) and Dpt Pathology (MN), Hospital "La Paz"; Madrid, Spain It is well known the impairment of germ cells in human cryptorchidism, nevertheless, the development of alterations of Sertoli cells (SC) in this congenital condition are not fully studied, particularly in pediatric stages. The present study was carried out in cryptorchid testicular biopsies from: 7 cases ( 1 - 4 y. old), 20 cases ( 4 - 9 y. old), 20 cases ( 9 - 15 y. old) and in testicular specimens from 15 adult patients ( 2 1 - 32 y. old) affected by unilateral cryptorchidism. Testicles from autopsies were used as a controls. PCNA (proliferating cell nuclear antigen) immunoreactivity (IR) was immunohistochemically tested in all cases. A SC nucleus labelling index (LISC) was used to quantify PCNA-IR expression. LISC was significantly increased in cryptorchid patients from 4 to 15 years in comparison to controls of same age. The cryptorchid testes with type IV lesions (with SC hyperplasia) show a LISC higher than both controls and the other cryptorchidism types without SC hyperplasia. Then there is a correlation between the morphologic finding of SC hyperplasia and the immunohistochemical expression of PCNA in cryptorchid cases from 4 years to pubertal stage. In adult cryptorchidism no PCNA-IR SC nuclei were detected.

P10.8 Detection of Human Immunodeficiency Virus Type-1 (HIV-1) In Tissue Sections: Problems and Pitfalls. R. de Weger, D. van Wichen, H. Kuipers, P. Joling, M. Bosch* and H.-J. Schuurman Department of Pathology, University Hospital, P.O. Box 85.500, 3508 GA Utrecht, and *Natl. Inst. Publ. Health and Environm. Protection, Bilthoven, The Netherlands. For in situ detection of HIV-1 in tissue, we used immunohistochemistry with monoclonal antibodies to structural and regulatory proteins, and in situ hybridisation with full-length and oligonucleotide probes. Immunohistochemistry in frozen tissue sections: in follicles of lymphoid tissue from HIV-l-infected patients, HIV-1 structural proteins gag p24 and env gp 41 localize on the protrusions of follicular dendritic cells (FDC). These antigens

536 presumably concentrate there in the form of immune complexes. Outside follicles, and in other tissues, HIV-1 proteins are observed in solitary ceils, mainly of monocytemacrophage origin including dendritic macrophages. However, the antibodies show binding to tissue of noninfected individuals, especially those to regulatory proteins tat, rev, and nef. In situ hybridization: using full-length probes and synthetic oligonucleotide to HIV-1 RNA, a specific signal is obtained in HIV-1 infected H9 cells and in frozen tissue sections. We adapted the method for the application on formalin-fixed substrates. This adaptation reduces the infectious risk in working with patient material. Also, signal detection is combined with a better cellular morphology. HIV-RNA positive cells occur scattered in tissue sections. In some lymph node cases, a faint signal is observed over the network of follicular dendritic cells. This indicates a state of infection of FDC, or the concentration of free virions in the FDC labyrinth. Interpretation: HIV-1 structural proteins and HIV-1 RNA are easily detectable in tissue components of HIV-1 infected patients. Combined with in situ hybridization, the main location is in germinal centres of lymphoid tissue. In immunohistochemistry for HIV-1 proteins one should be aware of apparent~\crossreactivity to uninfected tissue components. This phenomenon may be ascribed to the expression of unknown endogenous retroviral(-like) elements, or to molecular mimicry of epitopes shared by viral and host molecules.

PlO.9 Identification of Infections Agents in the Blood of Patients with the Immunoferritin Method. L. V. Didenko, N. D. Konstantinova, V. L. Popov and

U. K. Plotnikova The Gamaleja Institute of Epidemiology and Microbiology, Rus. Acad. Med. Sci., Russia.

We have used immunoferritin method for detection and identification of microorganisms and corresponding antigenes in the blood of patients. The blood cells were isolated by centrifugation from patient blood of an abdominal fever, dysenteria Flexner and Sonne, fixed by Ito and Karnovsky method [J. Cell Biol. v. 39, 168-169]. The ceils were incubated with antibodies against L-form of Salmonella tiphi and Shigella Flexner, dehydrated in Araldit (Fluka) and were studied by electron microscopy. Spheroplasts of Salmonella tiphi were revealed in blood preparations of patients suffering from abdominal fever. It has been interpreted as a result of L-transformation. Gramm-negative microorganisms were detected in blood of patients with dysenteria Flexner. In contrast in the case dysenteria Sonne microorganisms were not found, but we observed amorphous mass containing specific immunoferroglobulins. We found that specific labels were adsorbed on the perimeter of membranes of platelets, but other blood cells had labels in local sites of cell membrane. Owing to immunoferritin method we can describe and identify infection agents in different forms in blood of patients, suffering from abdominal fever, dysenteria Flexner and Sonne.

Poster sessions

P10.10 The Use of Peptidases in Differential Diagnosis Various Forms of Chronic Virus Hepatitis. E. V. Esaulenko RC St.-Petersburg, Pavlov's Medical Institute, Russia.

Activity hystochemetry was used to study the localization of depeptidyl peptidase (DPT IV), aminopeptidase A (APA), aminopeptidase N (APN), g-glutamyl transpeptidase (gGTP) in liver biopsies of patients with different forms of chronic virus hepatitis (CVH): persistent (CPH) and active (CAH). Our findings allow to suggest the next general propositions: 1) The most common sites of DPP IV, APN, g-GTP are biliary pole of hepatocytes; APA - - the capillary endothelium; 2) The localization of enzymes and the intensity of reaction in liver lobule has been dependent on activity of the chronic pathological process; 3) Histochemistry of membrane proteases may be used for diagnosis of CVH and differential diagnosis between the two types of chronic hepatitis: CPH and CAH.

P10.11 L D H Patterns as Indexes of Tumour Oxygenation. L Freitas, V. Bertone, P. Griffini and G. Gerzeli Dept. of Animal Biology and CNR Centre for Histochemistry, Univ. Pavia, Italy.

Tumour oxygenation is a key factor of therapy success. Hypoxic cells respond only to hyperthermia and nitroaromatic drugs. They manifest enhanced genetic instability, drug resistance and tendency to metastatize after reoxygenation. The assessment of local oxygenation levels would thus improve the therapeutic strategy. The histochemical visualization of LDH activity on biopsy sections, with PVA as tissue protectant, is proposed as a simple and economical way to achieve this purpose routinely. A trend from diffuse (Di), to diffuse plus granular (Di + Gr) and eventually granular (Gr) formazans were observed in ascites and solid tumours, in parallel with a temporal and/or spatial progression towards hypoxia and anoxia. Several mitotic and giant cells (Di + Gr) were often noticed in perinecrotic areas reached by infiltration of edema. These findings suggest that: (1) Di represents soluble isoenzymes which indicate good oxygenation; (2) Di + Gr: both soluble and bound enzyme forms, decreased 02 levels; (3) Gr: bound isoenzymes, active under hypoxiaanoxia; (4) the edema reoxygenates hypoxic cells. These assumptions are based on the following: (a) LDH is an ambiquitous enzyme; (b) H subunits, associated with good oxygenation, promote solubility; M subunits, hypoxiaassociated, and the anoxic shock protein LDHk promote binding to particulate structures; (c) mitochondiral-LDH may oxidize excess lactate under stress situations; (d) tissues with predominance of H subunits (ex. cardiac muscle) display Di pattern and those with predominance of M subunits (ex. scheletal muscle) show mostly the Gr product; (e) an inhibitor (pyruvate) and an activator (cyanide) of H subunits reflect on Di; a M inhibitor (urea) depresses Gr. Lipid degeneration (an hypoxia index) causes mild reduction of the tetrazolium salt and redistribution of the formazan. (Funds from MURST 60~

Poster sessions P10.12

Immunohistochemicai Demonstration of SP-IR Nerve Fibres in Multiple Glomus Turnout. C. Alessandrini, M. Guarna, A. M. Pucci ~ M. Fimiani, M. Garosi, and C. Fruschelli Histology and General Embryology Institute, and Department of Dermatology ~ University of Siena, Italy. An immunocytochemical study was carried out in a case of multiple glomus tumour of the skin, in order to investigate the presence and distribution of Substance P immunoreactive (SPIR) nerve fibres in the turnout parenchyma by using an immunofluorescence technique. Numerous SP-IR nerve fibres were observed near the glomus cells of the irregular, dilated, vascular channels which are present in the glomus tumour. The present findings are in agreement with the clinical picture of the tumour which is painful despite the fact that its histological and ultrastructural aspect is typical of asymptomafic tumours. In conclusion the numerous SP-IR nerve fibres, can mediate nociception or produce vasodilation in the glomus tumour.

PI0.13

Substance P Immunoreactive Nerve Fibers are Decreased in the Skin of Patients with Rett Syndrome. M. Guarna, Y. Hayek*, C. Alessandrini, A. M. Pucci and C. Fruschelli Histology and Gen. Embr. Inst., University Siena, *Children Neuropsychiatry, USL 30, Italy. We have studied the distribution of nerve fibers containing substance P(SP) in the skin of patients suffering of Rett syndrome. The study has been carried out on skin biopsies of (5) patients with "clinical" Rett Syndrome, according to the criteria of Hagberg and Witt-Engerstrom. Diagnostic skin biopsies of patients with autism (3) were employed as controls. For the localization of S P a rat monoclonal antibody against SP and an indirect immunofluorescence method were used. SP-IR nerve fibers in the skin of patients with Rett Syndrome were remarkable reduced and few fibers were only observable at the dermis-epidermis junction. Controls show numerous nerve fibers localized not only in dermis around blood vessels and glands, but also in epidermis. Our findings suggest that sensory nerve fibers of small diameter containing peptides such as SP are reduced in the skin of these patients and confirm the involvement of peripheral nervous system in this syndrome.

P10.14 Application of Immunocytochemistry and Different Classes of Reagents for Cancer Ceils Detection in Serous Effusions. D. F. Gluzman, I. V. Abramenko, L. M. Sklyarenko and A. 2. Evsev'eva institute for Oncology and Radiobiology Problems, Academy of Sciences, Kiev, Ukraine. Cancer cells in serous effusions of 175 oncological patients were detected using immunocytochemical methods, monoclonal antibodies (Moabs), peroxidase-conjugated lectins and

537 different neoglycoprotein probes. 65 noncancer patients consisted control group. In addition to well-known Moabs which were tested in our laboratory such as anti-CEA, B6.2, DAKO-Ber-EP4, anti-PEM, anti-NSE, anti-CD15 it was shown diagnostic significance of detection of lectin's (PNA, HPL, SBA, SJA, LAL) and cytochemical determination of alkaline phosphatase activity. Lectin's receptors were identified on cancer cell membranes without previous application of neuraminidase and in cytoplasm of mesothelial cells and histiocytes after neuraminidase pretreatment. The de- and hyposyalisation of surface antigens and vimentincytokeratins coexpression were common signs of different origin cancer cells in effusions (lung, mammary, stomach, ovary cancer). Other signs as expression of oncoassociated antigens and oligosaccharide-binding molecules varied. Among oligosaccharide containing probes TF-, Fucal->2Galand Gal/31-->3 GLcNAc- probes are most perspective in diagnostic plane.

P10.15 Value of Immunohistochemistry in Diagnosis of Malignant Lymphomas and Lymph Node Metastatis. O. V. Yurchenko, B. J. Goldshmid and D. F. Gluzman Institute for Oncology and Radiobiology Problems, Academy of Sciences, Kiev, Ukraine. Paraffin and cryostate sections of lymph nodes from 58 patients with malignant lymphomas and metastatic-lesions were observed by routinely morphological methods and using immunohistochemistry. The panel of monoclonal antibodies (Moabs) included direct against leucocyte common antigen (CD45), kappa and tambda light chains of immunoglobulins, CEA-1 and CEA-4, anticytokeratines (HI, H4), vimentin, desmin, against of epithelial membrane antigen (Ber-EP4), Ki-I and Ber-H2 antigens (Bio Tec Med, Denmark). Sections were stained by PAP-method and corresponding controls were performed. Introduction of listed panel of markers has brought the accurate identification of the majority of lymph node lesions. On immunostaining the neoplastic cells of highgrade non-Hodgkin's lymphomas (NHL) were positive for the CD45 and negative for H1, H4, CEA, Ber-EP4. The criterion for B-cell NHL was demonstration of a monoclonal lymphoid cell population expressing either lambda or kappa light chains, but not both. Moabs to cytokeratins showed high specificity for cancer cells. Expression of CEA epitopes, defined by Moabs CEA-1 and CEA-4 depended on the turnout cells origin.

P10.16 Oligosaccharide-Binding Molecules on the Surface of Human Hematopoietic Cells. I. V, Abramenko, D. F. Gluzman, N. V. Bovin and A. I. Evsev'eva Institute for Oncology and Radiobiology Problems, Academy of Sciences, Kiev, Ukraine. Using ABC-method and 22 probes (biotinylated PAA to which oligosaccharides are attached via alyphatic spacer, in particular blood-group related, Thomson-Friedenreich haptens and fragments of LECAM receptors) surface carbohydrate-binding abilities of blood mononuclears from 11

538 healthy persons and 46 patients with different forms of leukemias and non-Hodgkin's lymphomas (NHL) were studied. All cases were examined cytochemically and were labelled with a panel of monoclonal antibodies recognizing leukocyte surface antigens. Patients with common ALL and pre-B-ALL (12), pre-T-ALL (3), AML (4), AMonL (3), AMML (7), erythromyelosis (1), plasma cell leukemia (PC) (1), B-CLL (6), HCL (3), NHL in leukemization stage (7) were included in this study. Receptors to different oligosaccharides were detected on cell surface membranes, but only Lact-Nprobe (mature B lymphocytes and plasma cells) and GalNAcprobe (erythroblasts) showed speciflcities of binding to peculiar cell types. In general maximal carbohydrate-binding abilities were detected in NHL, PL, AMonL, erythromyelosis; middle - - in AMML, HCL, ALL; minimal - - in B-CLL, AML, healthy men. Detection of oligosaccharide receptors may be useful in differential diagnosis of B-CLL and NHL in leukemization stage.

P10.17

Interphase Cytogenetics in the Analysis of Chromosome Aberrations Correlated with Bladder Cancer Progression. A. H. N. Hopman, P. Poddighe, M. Vallinga, A. W. G. B.

Smeets ~ and F. C. S. Ramaekers Dept. of Molecular Cell Biology & Genetics, Univ. of Limburg, Maastricht and IStiehting Ziekenhuis en Klinisch Laboratorium, Venray, The Netherlands.

Transitional cell carcinomas (TCCs) of the urinary bladder were examined for numerical aberrations involving chromosomes 1, 7, 9, 11, 15 and 17 at different stages of clinical progression. Monosomy for chromosome 9 was detected in about 30~ of low grade, non-invasive TCCs. Invasive and non-invasive aneuploid/tetraploid TCCs showed a profound imbalance between the different chromosomes. An evident underrepresentation of chromosomes 9, 11 and 15 in comparison to chromosomes 1, 7 and 17 was detected. The underrepresentation of chromosome 9 in diploid, as well as aneuploid TCCs, may be explained by a process of tetraploidization. Therefore, loss of chromosome 9 may be one of the primary events, while tetraploidization and loss of chromosomes 11 and 15 can be correlated to tumor progression.

P10.18 Identification of the Primary Sites of Brain Metastases. An lmmnnohistochemical Study. J. Zhang Department of Pathology, Hubei Cancer Hospital, Wuhan, P.R. of China.

147 specimens of brain metastases were labelled by thirteen kinds of antibodies (McAb or PcAb), by means of AvidinBiofin-Peroxidase Complex (ABC) method. These antibodies are specific markers to various primary cancers such as lung, breast, stomach, colon, thyroid, prostata, chriocarcinoma and astrocytoma etc. We expected that this method could detect the unknown primary sites of brain metastases rapidly and accurately after operations. The results showed that most of the primary sites of brain metastases could be found by

Poster sessions immunohistochemistry. These antibodies such as HLC3-AB, BG6, MG7, MC5, Thyroglobulin, PSA and HCG are specific markers to the brain metastases originating from lung, breast, stomach, colon, thyroid, prostata and chriocarcinoma, respectively. The true positive rates of all those antibodies were above ninety percent. Since 1987, it has been applied in routine neuropathological diagnosis and good results achieved. In comparison with some other measures such as Xray, Radionuclide and CT, our study suggests that immunohistochemistry is an economical, rapid and accurate method in identifying the unknown primary sites of brain metastases. In addition, it is also a useful tool in differentiation of pathological diagnosis between primary and secondary brain tumors.

P10.19 Ontogenetic Changes of the Localization of TumorAssociated 27 kDa Protein in the Human Placenta as Revealed by the Immunomicroscopical Technique. K. Kami ~, N. Sato ~, M. Ishikawa 2 and M. Nakai 2 Department of Human Morphology and Physiology, Tokiwa University, Mit#, and Department of Obstetrics and Gynaecology, Kitasato University, Sagamihard, Japan.

A tumor-associated antigen that shares antigenicity with a pregnancy-associated protein has been detected in ascitic fluid of patients with advanced ovarian cancer. The protein, prepared from malignant ascitic fluid by the combination of polyethylene glycol 4000 fractionation, DEAE-Sepharose chromatography and preparative-PAGE, was subsequently separated on SDS-PAGE, as a single band corresponding to an approximate molecular mass of 27 kDa. Immunological analysis showed that this protein was a circulating species of an ectopic developmental antigen that markedly increases in biological fluids in both the second trimester of pregnancy and tumor-bearing statuses. The localization of the protein has also been revealed in the placenta by an immunomicroscopical technique. It was noticeable that there were marked differences in immunoperoxidase (ABC) stainings between the first trimester and the third trimester of gestation. In the first trimester, the antigenic site was appeared to be oriented predominantly towards fetal connective tissue elements. After the tenth week of gestation, in the second trimester, the protein-containing vesicles were mainly present in the cytoplasm of syncytial trophoblasts. In many cases, these were fused with cell membrane at the crypt of microvilli. In the third trimester, however, the immunopositive sites were exclusively seen in the fetal blood vessels. These results suggest that the protein may be mediated by the mechanism of morphogenesis and physiogenesis in the ontogenetic development of placenta. Further study is being made along these lines.

P10.20 The Morphological Investigation of the Microscopic Mole. K. lto Kinki Univ. Sch. Med. Obst. Gyne., Osaka, Japan.

Purpose: The case in which cyst-formation of the villi recognized microscopically is defined as microscopic mole,

Poster sessions but its diagnostic criteria and biological behavior are not always clear-cut. We examined diagnostic criteria and clinical management of this microscopic mole. Method: Villi obtained from 40 cases of artificial abortion, 60 cases of spontaneous abortion and 5 cases of complete mole were used. The shape, branching and shade of the terminal villi were observed and then the minor axis of villi was measured. Central liquefaction, disappearance of villous vessels and proliferation of villous cells were observed. Result: (1) Disappearance of villous vessels, central liquefaction and proliferation of villous cells were observed not only in cases of abortion but in normal villi, though less frequently. (2) When the villi were composed of below 1 mm in minor axis, none of the findings, such as central liquefaction, disappearance of villous vessels and proliferation of villous cells were outstanding. (3) We could not distinguish cases whose minor axis of villi is above 1 mm from complete mole even when cyst-formation was not observed, and found occurrence of 2 cases of secondary tumor out of them. (4) Cases which meet the criteria (3) are separated as microscopic moles meaning that a relationship with complete mole cannot be denied. The other cases are named hydropic degeneration.

P10.21 Morphometric Evaluation of the Elastic Fiber Damage in Pneumonitis. S. Battaglia*, A. M. Casali** and A. R. Botticelli*** Departments of Pathology, University of Genova* and Pavia***, and Department of Histology and Embryology, University of Genova** Italy.

The degradation of elastic fibers in pneumonitis is consequent to imbalance o f the alpha-l-anti-trypsin/leukocyte elastase system. The aim of the present research is to evaluate the detectable amount of the elastic framework in inflammatory areas. Human samples of focal pneumonitis, as well as controls, were analysed by morphometric methods. The study was carried out on specimens fixed with 10070 formalin solution, embedded in paraffin, cut at 5 ~m and stained with modified elastic tissue-Masson trichrome. Measurements were performed by means of the LEITZ ASM 68K semiautomatic image analysis system. The per cent area occupied by elastic fibres in the inflammatory foci was reduced to less than 15070 of the amount found in control lungs. Moreover, nearly the same figure was observed in the lung tissue closely surrounding the inflammatory foci. This feature suggests that the leakage of the leukocyte elastase affected also the surrounding free tissue to the same extent. We believe that such damage of the elastic bundles is too severe to allow a proper elastic remodelling. So far the future o f these inflammatory areas appears to be linked with the development of a focal panacinar emphysema.

P10.22 Morphological Analysis of Yolk Sac Tumor. K. Ito Kinki Univ. Sch. Med. Obst. Gyne., Osaka, Japan.

Morphological analysis of imprint cytology and histology was performed in 5 patients with yolk sac tumor (YST). The age of

539 the patients ranged from 14 to 37. The level of A F P ranged from 460 ng/mt to 13,464 ng/ml. Five patients had a primary lesion in the ovary. These five cases of YST were histologically evaluated and classified into 4 patterns. As a result, endodermal sinus pattern was most frequently identified, followed by polyvesicular vitelline pattern. As with imprint cytology findings of 4 cases YST, mostly cuboidal ceils were frequently observed; cuboidal cells and nuclei were both cudoidal in shape and 2 5 - 4 0 / ~ and 2 0 - 2 5 / ~ in diameter respectively. The chromatin showed fine to coarse granular patterns. The nuclear-cytoplasmic (N/C) ratio was 50 - 70~ and each nucleus contained one to four nucleoli. Next to cuboidal cells, spindle-shaped cells were most frequently noticed; 2 0 - 4 0 / ~ in size with spindle-shaped nuclei i 0 - 2 0 in diameter and fine to coarse granular chromatin pattern. Nuclei of 30-50~ of N / C ratio contained one to two nucleoli. Naked cells and bizarred cells were also noticed.

P10~

A Study on Significance of CA19-9 in Mature Cystic Teratoma, K. Ito Kinki Univ. Sch. Med. Obst. Gyne., Osaka, Japan.

We comparatively studied preoperative and postoperative serum CA19-9 levels in 170 mature cystic teratoma (MCT) cases. In MCT cases showing positive serum CA19-9 levels, immunohistochemistry using an enzyme immunoassay was performed to determine which tissues the high levels of serum CA19-9 had originated from. 1. Preoperative serum CA19-9 level was abnormally high, 83.8 U/mt, against postoperative low level, 32.9 U/rot. This suggested the possibility of CA19-9 being produced in the tumor of MCT. 2. There were 31 CA19-9 positive cases and CA19-9 was found in the bronchial gland tissue and surface epithelium in 9 and 4 cases, respectively, out of 23 cases showing serum levels of more than 101 U/ml. Out of 45 cases with normal CA19-9 levels, 4 had bronchial gland tissue and 3 had surface epithelium, both without CA19-9. Lewis system examination of those cases indicated Lewis~ - ) and L e w W ( - ) in all of them. 3. CA19-9 levels were higher in cystic fluid than in serum from MCT patients. This suggested two routes for the transfer into blood of tumor-tissue produced CA19-9: 1) to be directly excreted in serum via surface epithelium; 2) to be retained in the ovarian cyst, then leaking into surrounding capillary vessels via the interstitium. 4. Clinically, CA19-9 is a marker necessary for examining MCT recurrence.

P10.24 Clinical and Pathological Studies of Primary Mixed Mesodermal Tumor of the Ovary. K. Ito and K. Noda Kinki Univ. Seh. Med. Obst. Gyne., Osaka, Japan.

In two cases of mixed mesodermal tumor (MMT) primarily originating in the ovary, H-E stain, alcian blue stain, PAS stain, toluidine blue stain, and immunohistochemical stain of protein S-100 were performed. Both cases, aged 68 and 58

540 years, respectively, developed MMT after menopause. CA125 levels were 327 U / m l and 596 U/ml. High LDH levels, 1407 U / m l and 1092 U/ml, were noted. However, other tumor markers, CEA, CA19-9, etc., showed cut-off levels or less. Both cases were positive for the cytodiagnosis of abdominal ascites collected by pre-operative culdocentesis. Thus, it was judged that the cells had originated from an adenocarcinoma. According to clinical stage classification at laparotomy, the two cases were stages IV and III, respectively. Hence, 100 mg of CDDP was injected intraperitoneally at operation. Later, following intravenous administration of CAP (a total of 425 mg and 430 mg of CDDP to cases 1 and 2, respectively), the second look operation was performed. Histological examination showed that the cancer part was comprised of endometrioid carcinoma (case 1) and serous cystadenocarcinoma (case 2). Both cases contained chondrosarcoma as the sarcomatous element. Histopathologically, in both cases, this chondrosarcoma was positive for all of PAS stain, alcian bue stain, toluidine blue stain, and immunohistochemical stain of protein S100. However, the serous cystadenocarcinoma forming the cancer part was negative for all the stains. The endometrioid carcinoma also forming the cancer part was positive only for PAS stain. Both cases died from the primary disease although various kinds of potent chemotherapies were performed after the second look operation.

P10.25 Immunohistochemical Studies of Spleen in Patients with Idiopathic Thrombocytopenic Purpura (ITP). C.-Y. Li ~ and C.-S. Chang ~ JSection of Hematopathology, Mayo Clinic, Rochester, MN, U.S.A. and 2Division of Hemato-Oncology, Kaohsiung Medical College, Kaohsiung, Taiwan, China.

In our earlier study of 83 patients with the clinical diagnosis of ITP, the prominent secondary follicles and foamy macrophages were the characteristic features of the spleen in 90~ of the cases. Patients with prominent secondary follicles tended to be younger, female preponderance, higher antiplatelet antibody formation, higher post-splenectomy platelet elevation, and higher initial response to splenectomy. About 10~ of ITP patients showed neither lymphoid hyperplasia nor foamy macrophages. These patients had lower splenic weight and tended to not respond to splenectomy. Extensive immunohistochemical studies were done on 2 selected ITP spleens and 4 disease-free spleens from Hodgkin's disease staging or incidental removal during surgery. To study the macrophage-mediated platelet destruction, factor VIII antigen was stained on 4 cases with prominent foamy macrophages in paraffin sections, and glycoprotein I I b / I I I a complex was also stained using H P I - I D antibody on 2 splenic imprints. The conspicuous changes demonstrated by the immunohistochemical studies included: (1) expansion of marginal zone lymphocytes, especially in cases with prominent secondary follicles; (2) increased L26 (CD20)-positive cells in red pulp areas of ITP spleens; (3) positive factor VIII-related antigen and glycoprotein I I b / I I I a complex noted in the cytoplasm of foamy macrophages, confirming phagocytosis of platelets; (4) more extramedullary megakaryopoiesis in the case with prominent follicular hyperplasia; and (5) marked decrease of CD8 cells and HLA-DR expression of macrophages in the case

Poster sessions with prominent foamy macrophages. These immunohistochemical findings may offer direct evidence of macrophagemediated platelet destruction and suggests that there are different immunological responses and mechanisms involved in the immune destruction of platelets in patients with ITP.

P10.26 Polyamine Content Changes of Cells and Blood Plasma Under the Millimetre Wave Irradiation. N. Y. Gridina, G. S. Litvinov and L. I. Matseiko SRC "'Vidguk'; Vladimirskaya 61-b, Kiev, 252033, Ukraine. White rats with Heren's carcinoma were exposed to millimetre wave (MMW) irradiation at 3 5 - 55 GHz frequency and with power of 3 m W t / c m 2. The action of 3 5 - 4 2 GHz frequency resulted in decrease of tumor up to its complete elimination (group 1), and 4 8 - 5 5 GHz frequency action resulted in progress of tumor process (group 2). In group 1 a rapid decrease of spermidine and spermine content was observed. In group 2 polyamine content in blood cells had essentially increased on 1 0 - 13th day after tumor inoculation. For both groups in blood cells putrescine content was almost identical. By the end of the experiment polyamine content in blood plasma of group 1 was higher than of group 2. These data indicated the changes of erythrocyte membrane charge or alterations of their permeability and partial transfer of polyamines in plasma under MMW radiation. Evidently, MMW effect on cell and blood plasma transport function had an influence upon the polyamine content redistribution. In this case the decrease of polyamine concentration in blood plasma was observed only under positive MMW effect.

P10.27 Prospects of Interphase Silver-Stained Nucleolar Organizer Region Data in Practical Oncology. N. N. Mamaev, E. L. Neischtadt, A. B. Marcotchev, E. N. Grinyeva, S. E. Mamaeva, N. I. Komarova, N. V. Bebiya and V. A. Gutschin Cytogenetic Unit, Faculty Therapy Clinic, Pavlov Medical Institute; Toun Oncologic Dispensary; The Institute of Cytology, StPetersburg, Russia.

Nucleolar argentofilia (NA) of tumor cells from esophagial, gastric, colonic, thyroid, pulmonary, breast, prostatic and genital tract carcinomas were investigated at cytological and hystological levels by means of silver-stained technique. Unchanged cells from the mucosa of the esophagus, stomach, colon were used as controls. Silver-stained nucleoli of tumor cells were shown to be quite polymorphic and their NA is markedly higher than in controls. Furthermore, the content of Ag-grains per nucleus in most malignant tumors was significantly greater than that in cells from benign tumors and also from unchanged tissues. The latter seemed to be a very important feature for distinguishing malignant from nonmalignant cells at early malignisation stages, in vascular microinvasions especially. If the mean number of Ag-grains per nucleus fails to diagnose a tumor distribution of cells with various amounts of silver grains is to be studied. The amount of more than 10070 cells with 15 or more Ag-grains per nucleus is of a considerable diagnostic value. Finally, if the hystograms don't contain necessary diagnostic information

Poster sessions either, cytological as well as ultrastructural assessment of nucleolar polymorphism may help in case of doubt.

P10.28 Immunohistochemical Localization of Plasminogen Activators (PAs) in Human Laryngeal Squamous Cell Carcinoma. M. P. Molinari Tosatti, D. Rosa, S. Parolini, D. Flagiello and A. Di Nallo Istology, Dept. Biomedical Sciences and Biotechnology, University of Brescia, 25123 Brescia, Italy. PAs have been implicated in various aspects of malignancy, such as tumor spread, invasion and metastasis. Two types of P A can be distinguished, based on molecular weight, immunological reactivity, and immunohistochemical distribution. They are urokinase-type (u-PA), usually associated with cell migration and tissue destruction, and the tissue-type (t-PA) associated with trombolysis. The aim of our study was to investigate the immunohistochemical localization of the PAs in histological sections of neoplastic laryngeal tissue. We have analyzed surgical specimens from 60 patients with laryngeal squamous cell carcinoma of different histological grading. The avidin-biotin-peroxidase complex method was used for immunostaining. There was heterogeneity in the u-PA immunoreactivity of different parts of individual carcinoma: some neoplastic cells were strongly stained while others were devoid of immunoreactivity. Immunostaining was strong in well differentiated squamous cell carcinomas, whereas positivity progressively decreased in moderately and poorly differentiated tumors. Positivity for u-PA was observed in disseminated connective tissue cells with a fibroblast-like morphology and in the epithelial cells of the ducts, t-PA staining was evident in endothelial cells of blood vessels and in the peritumoral stroma as well as in areas where squamous cell carcinoma shows necrotic changes. Fibrinogen and Plasminogen were also present in the tumoral stroma and in tumor necrotic areas. PAs were also found in inflammatory areas as extracellular and intracellular immunostaining. These studies have shown that u-PA and t-PA have very different distribution, suggesting they may be involved in different biological processes.

P10.29 Systematic Registration in a Computer Database and Analysis of Routine Immunohistochemistry Data in the Identification of the Unknown Primary. Presented on behalf of the Institutes of Pathology from Dordrecht, Leeuwarden and Winschoten and the Dutch Centre for Healthcare Information by M. Nap.

The aim of the study was to analyze the use and outcome of routine immunohistochemistry (I.H.) in the identification of the unknown primary. In 394 cases with metastatic tumor localisations from three different institutes we analyzed the immunohistochemical results obtained from routine practice. Markers were grouped in epithelial, mesenchymal and lymphoid types. Tissue samples were arranged according to either the known origin of the primary tumour or as metastasis with unknown primary.

541 In 237 cases we had a known primary and in 157 the primary was not known. A first analysis of the use of one or more markers from each group led to the following results. Epithelial markers: 18 not used, 367 used. Known primary: 189/237 positive. Unknown primary: 123/157 positive. Mesenchymal markers were not used in 126 cases and used in 268 cases. In known primaries 55 positives and 109 negatives were found and in unknown primaries 18 positive and 86 negative. Lymphoid markers were used in 146 cases only and they were positive in 3 known and 3 unknown primaries. Among the epithelial markers, CEA was most often used, followed by the polyclonal keratin antibody Ker Z 622, OC 125 and the monoclonal keratin Cam 5.2. Whereas CEA and OC125 showed a strong organ bound positive score for respectively digestive tract and female genital tract, the two keratin reagents showed a non-organ bound, but carcinoma specific pattern. PSA and TG did not leave any doubt with regard to organ specificity. Isolated Vimentin positivity was found predominantly in metastases from the urinary tract (kidney), followed by female genital tract and respiratory tract. Only a low number of unknown primaries showed Vimentin positivity. Analysis of combined epithelial and mesenchymal marker profiles showed that the combination of positive reactions in both groups were found in the same sequential order. Although systematic collection and analysis of I.H. data from routine practice may give more insight in the diagnostic possibilities of this technique, it is not likely that I.H. alone will solve the problem of the unknown primary.

P10.30 Immunohistochemical Analysis of Tumor Angiogenesis in Human Mammary Tumors: an Image Analysis Study. K. Oda, J. Itoh, M. Kubota, Y. Tokuda, T. Tajima and R. Y. Osamura Dept. of Pathol., Cell Biol. Res. Lab. and Surg., Tokai Univ. Sch. of Med. Isehara, Kanagawa 259-11, Japan.

Although it has been suggested that vascularization is an important prognostic factor in mammary carcinomas, there are few data on morphological characteristics of the tumor vessels. The present report is an application of image analysis on immunohistochemically stained tumor vessels to elucidate the new morphological parameters far the analysis of vascularization in mammary tumors. Materials and Methods: Paraffin-embedded sections of 8 human mammary tumors (5 invasive ductal carcinomas(CA), 2 fibroadenomas (FA) and 1 phytlodes tumor) were stained by indirect peroxidase labelled methods using monoclonal antibody to factor VIII (DAKOPATTS). Using IBAS image analysis system (Carl Zeiss), we performed quantitative analysis on 112 images (2410 vessels), measuring: DMIN; the shortest diameter, FCIRCLE; circularity shape factor defined as (4 • n • area)/(perimeter2), FSHAPE; aspect ratio defined as D M I N / D M A X (maximum diameter). Results and Comments: In the tumor and in the surrounding breast tissue (BR) of 2 FAs, there was no significant difference in the average values (AV) of these three parameters. In all 5 cases of CA, AV: DMIN was lower in the tumor than in the surrounding BR. 4 of the 5 CAs revealed lower AV: F S H A P E and all 5 CAs demonstrate lower AV: FCIRCLE compared

542

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with those in the surrounding breast tissue. These findings may suggest the significance of these parameters in analyzing the vascularization of mammary tumors.

retain, modify or lose the functional characteristics of normal ceils, thus resulting in morphological and histochemical polymorphism in carcinomas.

P10.31 Prognostic Significance of Mucin Histochemistry for Patients with Recurrent Colorectal Cancer.

P10.33

A. G. Perevoschikov Cancer Research Centre of Russian Academy of Medical Sciences, Moscow, Russia.

Comparative and retrospective histochemical study of different types of mucins in 35 cancerous tumours of rectum and surrounding mucosa has been accomplished on patients with the local relapse occurred after radical operations and without it. The analysis of prognostic significance of histochemical data was obtained by specific programmable algorhythmical complex on the basis of personal computer. With relation to prognostic aspect the indication of favourable prognosis for patients is availability of moderate or minor amounts of sulphomucins in mucosa around tumour as well as complete absence of sulpho- and sialomucins in cancerous cells. Unfavourable factors indicating probable development of tumour relapse after operation were the following: availability of moderate amounts of sialomucins and/or minor amounts of sulphomucins in cancerous cells, as well as complete absence of sulphomucins in mucosa around tumour. An effective method for individual prognostication of disease consequences and its reliability which came to 80 per cent has been developed on the basis of results obtained.

P10.32

Ultracytochemical Study of Phosphohydrolases and Oxidoreductases in Human Gastric Carcinoma Cells. A. G. Perevoschikov Cancer Research Center of Russian Acad. of Med. Sci. Moscow, Russia.

Ultracytochemical techniques were used to study functional maturity of cancer cells. It was found that various organelles of cancer cells may possess the same functional characteristics as their respective normal cell analogues. The subcellular distribution of ATPase, IDPase and acid phosphatase activities in the cancer cells exhibiting ultrastructural features of chief, parietal, endocrine cells and border-enterocytes was similar to that in corresponding normal cells. The alkaline phosphatase activity in microvilli of the tumour cells reminding border-enterocytes as well as activity IDPase in granular endoplasmic reticulum, nuclear envelope and plasma membrane of cancer ceils reminding mucoid and parietal cells and oxidore-ductase activity (cytochrome oxidase, succinate dehydrogenase, NADH-dehydrogenase) in mitochondria of the parietal type cells may disappear. While a number of organospecific features in the cancer cells disappear or are retained within the normal range, certain properties might be increased (e.g. the level of acid phosphatase activity in the cancer cells reminding chief cells and border-enterocytes) or appear anew (glucose-6-phosphatase activity in the granular endoplasmic reticulum cavities of the cancer cells). Our findings show that the tumour cells of gastric carcinomas may

Expression of Cytokeratin Polypeptides, CEA in Cancer of the Cervix Uteri Metaplastic and Ectocervical Origin: an Immnnohistochemical Study. S. V. Petrov* and N. T. Raikhlin** *Kazan Medical Institute, Kazan, 420012, Russia; **Cancer Research Centre, Moscow, 115478, Russia.

Immunohistochemical study of squamous cell carcinomas (SCC) of the cervix uteri was carried out with help of panel monoclonal antibodies to cytokeratins (Dia-M-Russia, Biosoft, Dakopatts) and polyclonal antibodies to CEA by indirect immunoperoxidase and PAP methods. SCC ectocervical type (I) and reserve-cell type (II) could be distinguished by cytokeratin and CEA expression. Cytokeratins N 1, 2, 10, 5, 14, 15 were revealed in I in a small amount, while in II cytokeratins N 8, 18, 17 were found in a large amount and also CEA were expressed by cancer cells. Among 87 investigated SCC about 90% tumors contained immunophenotypic properties of the reserve cells carcinomas. We consider these data have important significance for the diagnosis and treatment of the cervical SCC. But these markers cannot be employed in immunohistochemical differentiation of the cervical dysplasia from SCC.

P10.34 OVTL12/30 (Keratin 7 Antibody) as Diagnostic Marker in Paraffin Embedded Lung Carcinoma Tissues. F. J. J. M. van de Molengraft', P. H. K. Jap 2, C. C. van Niekerk 2 and L. G. Poels2 1Department of Pathology, Medical Center Alkmaar; 2Department of Cell Biology and Histology, Medical School University of Nijmegen, The Netherlands.

Histological typing of lung carcinomas, particularly of bronchus carcinomas, can be hampered in small biopts due to its cellular heterogeneity. In order to improve the diagnostics of lung carcinomas monoclonal antibodies against keratins have been investigated. In this study we report the immunoreactivity of the anti-keratin-7 monoclonal antibody OVTL12/30 on paraffin embedded tissue sections of lung carcinomas from 61 patients. A modified AECimmunoperoxidase method was applied after mild predigestion with pepsin. Our results showed that all squamous cell carcinomas of the lung (n = 24) were negative regardless of the grade of differentiation. All adenocarcinomas, including bronchioalveolar carcinomas (n = 20), were positive, regardless of their grade of differentiation. Since the large cell anaplastic carcinomas (n = 6) did not react with keratin 7 antibody, these carcinomas are thought to be of planocellular origin. Small cell anaplastic carcinomas were negative in 10 out of 11 cases. Our data show that OVTL12/30 keratin 7 antibody contributes to the differentiation between squamous cell - - and adenocarcinomas of the lung in routine clinical pathology with outcome for therapy and prognosis.

Poster sessions P10.35

A New Murine Monoclonal Antibody (COTL1) for Colon-Specific Mucin in Paraffin Embedded Colon Tumours. L. G. Poels ~, F. van de Molengraft 2, P. H. K. Jap 1, R. J. E. Pullens' and S. Roberts ~ tDepartment of Cell Biology & Histology, Medical School University of Nijmegen; 2Department of Pathology, Medical Centre Alkmaar, The Netherlands. A murine hybridoma cell line COTL1 was generated by fusion of spleen cells from immunized Balb/c mice with myeloma cells Sp2/0-Agl4. Mice were immunized with an extract prepared from a well differentiated human colon carcinoma. Hybridoma supernatants were screened initially by immunofluorescence assays on unfixed cryostat sections from normal colon biopts, benign as well as malignant colon turnouts. The IgG1 monoclonal antibody COTL1 did bind exclusively to colon epithelial cells and not to any other normal human cell type. Within the colon epithelium mainly mucus producing cells were stained intensively. Within the group of colon polyps (n = 20) as well as colon carcinomas (n = 25) all samples were stained positively. The staining rate, however, was correlated to the differentiation grade, i.e. the mucus production by turnout cells. Other digestive tract tumours, i.e. small intestine Peutz Jeghers tumour, were all negative with this monoclonal antibody. The antibody reacted equally well with formalin fixed, paraffin embedded colon tissues. From these and previously published results we conclude that colon tumours can be discriminated from other digestive tract tumours by its negative reaction with OVTL 12/ 30 (a paraffin resistant monoclonal antibody to keratin 7) and a positive reaction with the paraffin resistant COTL1 monoclonal antibody.

P10.36

Evaluation of Morphological and Cytoehemieal Markers in some Variants of Acute NonLymphoblastie Leukemia (FAB). E. RlMulescu Dept. of Hematology, Oncologie Inst. Cluj, Romania. Some variants of acute non-lymphoblastic leukemia (ANLL) sometimes present diagnostic difficulties (M3, Ms, MT). According to morphology and cytochemistry the leukemic cells of 262 cases were identified. The myeloperoxidase (MPO), Sudan Black B(SBB) and chloroacetate esterase reactions revealed granulocytic differentiation in the various acute myeloid leukemia subtypes: acute myetoblastic leukemia without maturation (AML-M1), acute myeloblastic leukemia with maturation (AML-M2), acute promyetocytic leukemia (AML-M3) and acute myelomonocytic leukemia (AML-M4). The nonspecific esterases: naphthol AS-D acetate (NASDA) and alpha-naphthyl acetate esterase (ANAE), the acid phosphatase and lysozyme reactions demonstrated monocytic differentiation. The aim of the present study was to illustrate the diagnostic, prognostic and therapeutic value of cytochemical markers playing a determining role for the choice of therapy. The results obtained showed that AML-M3 variant presented much more diagnostic problems generated by morphological and cytochemical heterogeneity of the leukemic promyelocytes. The immunological and ultrastructural methods contributed to the recognition of relatively

543 rare types of AML (MO, M6, M7), which cannot be classified by conventional techniques. In conclusion, some cytochemical markers are useful in the rapid identification of AML subtypes with different prognosis and therapy.

P10.37

Characterization of the Immuuophenotypes in Leukaemia Using Monoclonai Antibodies. E. R~dulescu Dept. of Hematology, Oncologic Inst. Cluj, Romania. The aim of this paper was to demonstrate the contribution of immunoenzymatic techniques: indirect immunoperoxidase (iP), alkaline phosphatase anti alkaline phosphatase (APAAP) in the immunophenotyping of circulating blastic leukemic cells of 65 patients (56 with different types of leukemia and 9 with non-hodgkin lymphoma in leukemic phase) and the clinical significance of surface membrane markers. For the diagnosis monoclonal antibodies were used: HLA-DR, CD19, CD20, CD22, CD23, TDT, CD5, CD7, CH2, MPO, CDI3, CD33, CD14, Gero, AN51, J15. The results obtained showed that 90% of the cases of acute myeloid leukemia (AML) reacted with one or more of the panmyeloid monoclonal antibodies (MPO, CD13, CD33). CD13 was reactive in the cases with mixed cell proliferation. Its preferential activity was obvious in AML-MO with minimal differentiation; in M1-M4 and chronic granulocytic leukemia in blastic crisis. The anti-MPO antibody appeared to be the most sensitive and also the most specific immunological reagent for staining AML cells. The reactivity of the myeloid panel of monoclonal antibodies showed a certain correlation with the FAB classification of AML. A good correlation was found between M4, M5 subtypes and CD14, between M6 and Gero, M7 and AN51, J15. In conclusion, both these modern immunocytochemical techniques are very precise, very sensitive and specific in the establishment of the immunophenotype.

P10.38 Immunohistochemical Detection of Gonadotropin Producing Pituitary Adenomas. N. Sanno, K.-I. Inada, R. Y. Osamura and A. Teramoto Dept of Neurosurgery, Ushioda General Hosp. 4-138 Shitanoya, Tsurumi-ku, Yokohama 230, Japan and Dept. of Pathol., Tokai Univ. Sch. of Med. Isehara Kanagawa 259-11 Dept. of Neurosurgery, Univ. of Tokyo 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, Japan. 83 cases with clinically non-functioning adenoma were investigated by immunohistochemistry. Expression of one more gonadotropin subunits (a subunit, follicle-stimulating hormone (FSH/3), luteinizing hormone (LH/3) was found in 38(45.8%) of all ademonas studied, a subunit and FSH(/were positively stained in 28 (33.7%) and 27 (32.5%) cases respectively, whereas LH/3 was detected in seven (8.4%) adenomas. The presence of both a subunit and FSH/3 was found in 17 cases, while of only a subunit was positive in 8 cases and only FSH/3, in 10 cases, LH/3 was not detected was always accompanied by a subunit or FSH/L By the double staining method, a subunit and FSH/3 were not always colocalized in the same cells. These results suggested that

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P10.39 Modulation of Cell Surface Molecules During HIV-1 Infection of H9 Cells.

Biochemical analysis of the antigen occurring in NCI-H82 has shown that the antigen is entrapped in a larger aggregate of at least 500 kDa. Cell fractionating by differential centrifugation showed that most of the antigen remains present in the 4400X g pellet of cell homogenates of NCI-H82, but that it can be solubilized by Triton X-100. Immunoaffinity assays are now underway to isolate the large (>500 kD) protein complex for production of second generation antibodies.

H.-J. Schuurman ~, T. Meerlo ~, P. Joling ~, A. D. M. E. Osterhaus 2 and J. Goudsmit 3 ~Department of Pathology, University Hospital, P.O. Box 85.500, 3508 GA Utrecht, The Netherlands; 2Natl. Inst. Publ. Health and Environm. Hygiene, Bilthoven; 3Academic Medical Center, Amsterdam, The Netherlands.

P10.41 Carbonic Anhydrase III: a Marker of Cells with Myoepithelial Differentiation in Polymorphic Adenoma.

immunohistochemistry is a useful tool to disclose high gonadotropin producing pituitary adenomas among clinically nonfunctioning adenomas.

We performed immunogold electron microscopy of H9 T-cells in a chronic state of infection and cells 2 days after infection with HIV-I IIIB, both for cell surface molecules and HIV-1 proteins. Infected cells manifested HIV-1 proteins gag p24, pol, env gp41 and gpl20, in clusters in the extracellular area and in budding particles on the cell surface. Cell surface molecules including CD3, CD4, CD5, CD1 la, CD25, CD30, and CD54 antigen and HLA-DR are present in a random distribution along the cell surface. The CD63 antigen, a lysosomal membrane glycoprotein, was mainly located in the cytoplasm of uninfected cells. Cells 2 days after infection showed CD4 labelling on sites were virions were budding from or attached to the cell surface and on free virions. This was confirmed in double immunogold labelling for HIV-1 gag p24 and CD4. Virions showed also labelling by CD3, CD5, CD1 la, CD25, CD30, CD54 and CD63 antibodies and antiHLA-DR. In quantitative assessment, the cell membranes of infected H9 cells showed a significantly lower density of CD4, CD5, and anti-HLA-DR than the membrane of uninfected cells. The labelling density by CD63 was significantly higher. Conclusion: during the first phase of infection, host cell molecules including cell adhesion molecules concentrate on budding structures and newly generated HIV-1 virions. This phenomenon contributes to the disappearance of these molecules (like the CD4 molecule) from the cell membrane after infection.

PI0.40 A New Antigen Characteristic of Neuroendocrine Malignancies. N. H. M. Senden, J. L. V. Broers and F. C. S. Ramaekers University of Limburg, Dept. of Molecular Cell Biology & Genetics, Maastrict, The Netherlands. Two new monoclonal antibodies RNL2 and RNL3 were generated using the small cell lung cancer (SCLC) cell line NCI-H82. The antibodies reacted with a subset of neuroendocrine tissues and neuroendocrine neoplasms. In lung cancer both antibodies reacted only with some SCLC and carcinoids and not with non-neuroendocrine lung carcinomas. Western blot analysis revealed that RNL2 and RNL3 recognize a protein doublet with a molecular weight between 4 3 - 4 5 kd as well as a 25 kDa compound. Competition radioimmunoassays revealed that the two antibodies recognize different epitopes in the same protein. Preliminary Northern blot analysis has indicated that the gene encoding these proteins is mainly expressed in neuroendocrine tissues.

N. S. Shamsutdinov Kazan Medical Institute, Kazan, Russia.

Identification of the cells with myoepithelial differentiation is connected with some difficulties because the classic morphologic research methods are laborious and not always reliable. In a 1987 report (Vfifin/~nen, H. K., Harmainen, H. A., 1987) about the possibility of using antibodies to carbonic anhydrase III for revealing of smooth muscle cells of uterus, myoepithelial cells of mammary gland and prostate appeared. In this work the results of the immunohistochemical study of parenchymal cells of 20 polymorphic adenomas of parotid salivary gland using indirect Coons' method are reported. The paraffin sections were stained with hematoxylin and eosin for microscopic diagnosis. Studies of cryostate tumor specimens, stained with antibodies helped to reveal a positive immunoreactivity in the majority of "solid" fields of parenchyma and some separate cells of chondromixoid structures. The cells that line the glandular structures have shown a negative immunoreactivity. It was settled, that most of parenchymal cells of polymorphic adenoma have myoepithelial differentiation.

P10.42 Immunohistochemical Evaluation of Cell Proliferation in Breast Carcinomas by Three AntiPCNA Antibodies. S. M. Siitonen, J. J. Isola, I. S. Rantala and H. J. Helin Pathology Unit, Tampere University Hospital, P.O. Box 2000, SF-33521 Tampere, Finland. We have studied the proliferative activity of breast carcinoma using PCNA-immunohistochemistry (streptavidin-biotin peroxidase technique) with monoclonai antibodies 19F4 (frozen sections), 19A2 and PC 10 (archival sections). With the 19A2 immunostaining we used a new antigen retrieval method which significantly augmented the PCNA immunostaining. Immunostaining was evaluated conventionally or with the CAS 200 image analysis system. The proportion of total PCNA~9r4-positive nuclear area (PCNA~F4 score) correlated significantly with flow cytometric S-phase fraction (SPF; linear regression, r = 0.469, p<0.001, n = 85) in frozen sections as did the proportion of PCNA~9A2-positive cells (PCNAj9A2 score; r = 0.593, p<0.001, n = 73) in paraffin sections. The PCNAvc~o score did not correlate with SPF or with the PCNA~9A2 score of the same tumor. There was a strong positive relation between the PCNA~9A2 score and histological tumor grade (ANOVA, p = 0.007). The patients

P o s t e r sessions with high PCNA19A2 score (PCNA19A2 score 8% was used as a cut-off) had significantly (p = 0.003, n = 87) worse prognosis than those with low PCNA~gAz score. We conclude that anti-PCNA antibodies 19F4 and 19A2 can be used as a marker of tumor proliferation in breast carcinoma. The PCNA,gA2-immunostaining with the antigen retrieval method can be done on archival, paraffin embedded tissue sections. The relation of the PCNA,gA2 score with patient outcome favours its use in the clinical setting.

P10.43 The Usefulness of the Lactate Dehydrogenase Macroreaction in Autopsy Practice. F. Smedts*, K. Kubat** and P. Vooijs** *Department of Pathology Diagnostic Center SSDZ Delft; **Department of Pathology University Hospital Nijmegen, The Netherlands. The detection of recent myocardial damage by means of the macroscopic LDH reaction (LDHr) linked to NADHd, conducted in liquid medium is greatly insensitive to postmortal autolysis. In 10 heart cones stored at 6~ up to 114 hours after death no autolytic artefacts appeared on freshly cut surfaces of myocardial slices. In 10 cones kept at room temperature up to 95 hours after death no artefacts appeared in 8 cases, in 2 cases the LDHr was impaired by postmortal bacterial spread and decomposition of the myocardium. Intravital perfusion of an injured myocardial area increases the sensitivity of the test. Postmortal cessation of circulation is decisive in preserving the dehydrogenase activity in undamaged myocardium. Artificial decrease of enzymic activity always appears in nonrecent cut surfaces of myocardial slices exposed to air and oozing fluids, even if slices are kept at 6~ and wrapped in plastic. In normal practice, when bodies are stored in a refrigerating unit, the LDHr may still indicate myocardial damage many hours after death.

P10.44 Detection of Keratin 7 in Papanicoulaou Stained Slides Provides a Strong Tool in the Differential Diagnosis of Effusions. J. Baars*, J. de Ruijter*, F. Smedts*, C. van Niekerk**, L. Poels**, C. Seldenrijk* and F. Ramaekers***, Diagnostisch Centrum SSDZ, Delft* Katholieke Universiteit Nijmegen**, Universiteit van Limburg***, The Netherlands. Reinier de Graafweg 7, 2625 AD Delft, The Netherlands. Keratin 7 shows a tissue specific distribution in epithelia. It is not found in epithelia lining the colon and in colonic cancers, but is however usually present in the epithelia lining the female genital tract and in tumours arising from these epithelia. Monoclonal antibodies directed against this keratin subtype have been found useful in the differential diagnosis of these tumour types. OVTL 12/30 is a recently developed monoclonal antibody directed against keratin 7. It has been shown to be reactive with formalin fixed paraffin embedded tissues, and has been proven useful in the aforementioned differential diagnosis. The aim of this study was to evaluate whether OVTL 12/30

545 would react with prestained cytologic slides and if it could be useful as a marker in indicating the origin of the primary tumour in effusions. For this purpose the antibody was tested in a series of Papanicolaou (PAP) and May Grfinwald-Giemsa stained slides from effusions of patients presenting with histologically confirmed cancers of colon (8), ovary (12), mesothelium (11), breast (I0), lung (6), esophagus (1), pancreas (2), urinary bladder (5), stomach (6), kidney (2) and prostate (1). The PAP prestained and embedded cells were reincubated with the antiserum in an immunohistochemical detection assay. The malignant ceils from ovarian adenocarcinomas displayed strong to moderate staining intensity in all cases. Expression in adenocarcinomas of gastric, pancreatic, renal, esophageal and mammary origin was less and variable, with also negative malignant cells, this expression pattern was also observed in transitional cell carcinomas originating in the urinary bladder. Squamous cell and undifferentiated small cell lung carcinomas were not reactive with OVTL 12/30, while a bronchioalveolar cell carcinoma was strongly positive. Malignant cells from adenocarcinomas of the colon and malignant mesotheliomas were negative. We feel that the application of this antibody may be a powerful tool in cytology, allowing the differentiation between adenocarcinomas originating in the ovary and colon as well as mesotheliomas. Moreover OVTL 12/30 is reactive with PAP stained cells.

P10.45 Cytokeratin, Chromogranin A, PsAP, PSA and CEA Expression in BPH, Atypia, and Well Differentiated Prostate Cancer. S. Frkovid-Grazio, I. Kraljid, M. Belicza and M. Tarle Clinical Hospital Sestre Milosrdnice, Zagreb, Republic of Croatia. In ongoing studies we found a high percentage of prostatic atypia (>20%) that becomes manifest cancer within 24 months after TURP. We have investigated 5 cases of glandular BPH, 5 cases of atypical glandular hyperplasia (AGH), 10 cases of well differentiated prostate cancer (WDPC). In 7 of these latter cases A G H was found within the gland adjacent to carcinoma. Immunohistochemical staining was performed by applying polyclonal Ab to PsAP, PSA, CEA, and monoclonal Ab to pan-cytokeratin (56-64 kD) and chromogranin A, purchased from Dakkopats. Significant differences in expression of PsAP, PSA and CEA were detected between WDPC and both BPH and A G H . Positive staining for CEA in BPH and A G H was found only in presence of inflammation. The expression of CEA in A G H adjacent to WDPC (0.06) was found in secretory cells regardless of inflammatory component and was higher than in tumor (0.03). In BPH basal cell layer was strongly cytokeratin positive. In A G H basal cell layer showed structural changes associated with weaker respective staining. Secretory cells in BPH were negative or weakly cytokeratin positive (0.6) while in secretory cells of A G H as well as in WDPC cells the intensity of reaction was moderate (1.2 and 1.0, respectively). Surprisingly, chromogranin A positive cells were more numerous in A G H than in both BPH and WDPC specimens. We advocate the use of these tissue markers as a tool for the recognition of potential A G H aggressiveness.

546

P10.46 Detection of Chromosome Changes in Archival Endocrine Tumors by Interphase In Sit~ Hybridization. H. van Dekken, R. Teygeman, T. A. Tersteeg*, G. P. Vooijs*, F. T. Bosman and A. Verhofstad* Depts. of Pathology, Erasmus University Rotterdam, and *University Hospital Nijmegen, The Netherlands.

We have applied in situ hybridization (ISH) for the cytogenetic and histologic analysis of familial and sporadic phaeochromocytomas within 4 ~ m deparaffinized tissue sections. In an earlier study 8 phaeochromocytomas were evaluated for the occurrence of numerical abnormalities with a defined chromosome specific repetitive DNA probe set. Extra copies of chromosome 1 or 7 (P<0.01) were seen in two primary tumors, whereas an underrepresentation of chromosome 15 (P<0.01) was distinguished in a metastasis. We are currently extending this series. Also the presence of chromosomal deletions will be investigated, since RFLP studies on phaeoehromocytomas have indicated the loss of genetic material on chromosome arms lp, 3p, 17p, and 22q. For this purpose ISH with the appropriate chromosome region specific DNA probes (YAC's and overlapping sets of cosmids) will be applied. In preliminary experiments with such probes (lp36, 6p21) the feasibility of this approach was demonstrated. We conclude that interphase ISH can be used for the cytogenetic inspection of archival (previously nonkaryotyped) solid tumor specimens.

P10.47 The Use of Monoclonal Antibodies in Ovarian Tumor Pathology. C. C. van Niekerk*, F. C. S. Ramaekers***, A. G. J. M. Hanselaar**, J. Aldeweireldt** and L. G. Poels* Departments of Cell Biology & Histology* and Pathology**, University of Nijmegen, Nijmegen. Molecular Cell Biology & Genetics *** R UL Maastricht, The Netherlands. A comparative histopathological study was performed on normal, benign and malignant cells of the human ovary. Our data show that Keratin expression is not limited to the group of epithelial tumors, but is also found in some gonadal as well as in germ cell tumors. Vimentin expression does not discriminate between epithelial and mesenchymal derived tumors. The pan-epithelial marker BW 495/36 marked the onset of tumorigenesis from mesothelial cells to ovarian cancer cells by increasing its expression. Tag-72 and OV632 in general discriminate between negatively staining mesothelial cells, cystomas, serous adenomas and positively reacting ovarian mucinous adenomas and ovarian carcinomas. Due to its lower sensitivity in the group of endometrioid, clear cell and adenocarcinomas, the antibody OV632 was useful as a marker for progression within the serous group. Gonadal tumors could be distinguished from germ cell tumors by the absence of TAG-72 and HMFG-2, while germ cell tumors usually expressed TAG-72 and to a lesser extent also HMFG2. Ovarian tumor markers (OC 125, OV-TL 3, OV-TL 16 and MOv 18) reacted with almost all samples of the epithelial ovarian tumors with an increasing expression from benign cases to carcinomas. In conclusion: a combination of BW

Poster sessions 495/36, TAG-72, OV 632 and one of the ovarian tumor Mabs (OC 125, OV-TL 3, OV-TL 16 or MOv 18) are useful markers in the discrimination of normal mesothelial cells, ovarian cystomas, -sereus, -mucinous adenomas and carcinomas, while ovarian gonadal and germ cell tumors can be distinguished by the Mabs TAG-72 and HMFG-2.

P10.48 Quantitative Cytochemical Detection of Malignant and Potentially Malignant Cells in the Colon. C. J. F. van Noorde#, A. J. Best ~, I. M. C. Vogels ~ and P. K. Das 2 ZLaboratory of Cell Biology and Histology, and 2Department of Pathology, Academic Medical Centre, University of Amsterdam, The Netherlands. Activities of certain enzymes such as glucose-6-phosphate dehydrogenase are increased at early stages of malignancy and are detectable before any morphological changes. However, it is not sufficient simply to demonstrate its activity in tissue sections marking the areas of high activity as malignant. High activity may be present in any proliferating cell, malignant, benign or normal, but it was found possible to distinguish malignant cells from normal cells by using an oxygen sensitive tetrazolium salt (neotetrazolium) for the histochemical demonstration of glucose-6-phosphate dehydrogenase activity in cryostat sections of human colon. We have studied 12 cases of established adenocarcinoma of the colon in addition to 4 of ulcerative colitis and 5 of adenomatous polyposis (polyposis coli). In a nitrogen atmosphere the activities of malignant and normal cells were similar. However, after incubation in an atmosphere of pure oxygen, only malignant cells gave a positive reaction after 5 min. Three of the five cases of adenomatous polyposis showed areas of "potential malignancy". These areas gave a positive reaction for glucose6-phosphate dehydrogenase activity in oxygen in a manner similar to malignant cells, but could not be classified as malignant by morphological criteria. The areas were considered to be either in the premalignant stages or predisposed to the development of malignancy. All other cases of adenomatous polyposis and ulcerative colitis gave a reaction in oxygen, compatible with normal cells. An explanation for the difference in oxygen sensitivity between normal and cancer cells appeared to be a decreased amount of lipid peroxidation products present in proliferating tissue. We suggest this test may be exploited for the early diagnosis of malignancy in conditions known to be (pre)malignant.

P10.49 Immunohistochemicai Study of Brain Tumours for Prognostic Aims. S. Y. Kasumova, Y. Y. Vyaltseva, R. G. Vasilov and L B. Buchwalow Research Institute for Biotechnology, Moscow, Russia. Cryostat and paraffin sections of 94 human brain turnouts of various histogenesis have been immunostained using monoclonal antibodies to glial fibrillar acid protein (GFAP), neurofilaments (NF), vimentin (V), laminin (L) and proteoglican (PG). G F A P was detected in all turnouts of astrocyte genesis, in 50070 of oligodendrogliomas and in 50~

Poster sessions of medulloblastomas while the intensity of GFAP staining markedly decreased with an increasing malignancy. NF were detected in 50% of medulloblastomas. V was detected in 50% of neuroepithelial and in 70% of mesenchymal tumours while its manifestation was enhanced with an increasing malignancy. L and PG were detected in the stroma of large blood vessels of various turnouts irrespective of their genesis. Immunostaining with a monoclonal antibody to NF permitted to differentiate neurocytomas and primitive neuroepithelial tumours within histologically diagnosed oligodendrogliomas and glioblastomas respectively, in cases when part of the cells were of neuronal differentiation.

P10.50

Monoclonal Antibody for Diagnosis of Tumours of Epithelial Origin Tumors. G. V. Vikha, R. I. Yakubovskaya, T. N. Karmakova, A. A. Suchno and A. U. Barishnikov Scientific-Production Association "Biotechnotogia'; Moscow, Russia.

We have developed a strategy for production of highly purified monoclonal antibody ICO-25 against a component of human milk fat globule membranes. The aims of this study were to determine and localize efficiency of the monoclonal antibody with tumor. Various types of tumor were examined including tumors of mammary gland, lung, ovary, uterus, colon, kidney, thyroid, salivary gland, urinary bladder, nasopharyngeal region, blood cells and normal human tissues. Ethanol fixed deparaffinized and fresh frozen section of tumors taken were stained by an indirect two stage immunofluorescent assay and also immunoperoxidase procedure and counterstained with hematoxylin. ICO-25 specifically reacts with unilayer epithelium having secretory activity. Antigen recognized by ICO-25 is resistant to formalin fixation and paraffin embedding. Our results show clearly that the antigen detected by the ICO-25 antibody is localized to both the membrane and cytoplasm of malignant cells. It is found with the cells of mammary gland, lung, stomach, uterus. There are cells with only membrane staining on the tumor kidney, ovary, lung, urinary bladder, thyroid, colon. The antibody reacts very strongly with breast (98/98), lung (14/19), ovary (16/17) tumors. There are specific reactions with adenocarcinoma and squamate tumor cells. The concentration of antibody used in immunochemistry was 5 ~g/ml. The antibody reacts very weakly or not at all with normal tissue. Between malignant and normal epithelium considerable variation exists at the distribution of the antigen. The antibody does not react with blood cells.

547 embedded in paraffin or Epon-812 according to a standard technique. For ultracytochemical investigation skin samples were incubated in standard media for evaluation of ATPase and adenylate eyclase (AC) activities. In psoriatic epidermis we found a moderate ATPase activity on plasma membranes, desmosomes, nuclei, endoplasmic reticulum and on keratohyaline granules of rare granular cells. ATPase activity in derma and in lower layers of epidermis was higher than in granular cells and corneous layer. There was practically no ATPase activity in the spinous layer of epidermis. AC activity was found in all layers of epidermis mainly at the inner side of the plasma membrane, desmosomes, and also in endoplasmic reticulum and nuclear membranes. After combined EsPUVA-therapy an increase of ATPase and AC activities was observed. An enhancement of AC activity detected previously by biochemists can be accounted for by an appearance of melanosomes in all layers of epidermis and by a restoration of the granular layer in which there are cells with keratohyaline granules that have a higher enzymatic activity.

P10.52

Immunohistochemical Study of Bone Morphogenetic Protein in 48 Cases of Mesenchymal Tumors. S.-X. Zhang, D.-F Li and J. Liu The Fourth Military Medical University, Xian, P.R. China.

Paraffin sections from 36 cases of bone tumors (including fibrous dysplasia (2), ossifying fibroma (2), chondroblastoma (1), benign osteoblastoma (5), chondrosarcoma (3), mesenchymal chondrosarcoma (3), osteosarcoma (19) and malignant fibrous histiocytoma (1)) and 12 cases of tumor of the soft tissue (including fibroma(2), rhabdomyosarcoma (3), leiomyosarcoma (7)) were immunostained by ABC method using monoclonal antibody against bone morphogenetic protein (BMP) for investigating the distribution and diagnostic role of BMP in human bone tumors. The result showed that 32 of 36 cases of bone tumors (excepting chondrosarcoma (1), mesenchymal chondrosarcoma (2), and malignant fibrous histiocytoma (1)) were positive. BMPMcAb may be used to differentiate the tumors which derived from bone-forming cell lines, from the others. A lot of bone tumors contain BMP, but only osteosarcomas, chondrosarcomas and osteoid osteoma have been found to induce bone formation by human tumor transplants in athymic nude mouse. It seems possible that inducing bone formation by transplants relates not only to existence of BMP but also the quantity of BMP which the tumor contains and has released. Immunostaining is better than transplants to supplement the morphological criteria which pathologists rely on exclusively for diagnosis of bone tumors.

P10.51

Enzymes of the Nucleotide Turnover in Psoriatic Skin. L B. Buchwalow, E. I. Dunaevskaya, L. E. Zavalishina,

P10.53

Immunohistochemicai Study of Bone Morphogenetic Protein in 19 Cases of Osteosarcoma.

E. Bocharova, G. A. Drnitriev, M. Gomberg and A. L. Mashkilleison

S.oX. Zhang, D.-F. Li and J. Liu The Fourth ?Jilitary Medical University, Xian. P.R. China.

Research Institute for Biotechnology, Moscow, Russia.

Paraffin sections from 19 cases of osteosarcoma were immunostained by ABC method using monoclonal antibody against bone morphogenetic protein (BMP) for investigating the distribution of BMP in human osteosarcoma. The result

Adult patients with psoriasis received Essentiale-forte (orally and intravenously) in combination with PUVA-therapy. Skin bioptates from the lesions were fixed in formaldehyde and

548 showed that all osteosarcoma, no matter which type they histopathologically or roentgenographically belong to, express BMP and the majority of the osteosarcomas with perpendicular spicules were more intensive. We think BMPMcAb may help us decide the histogenesis of tumor.

P10.54 Ewing's Sarcoma can Express Bone Morphogenetic Protein. S.-X. Zhang, D.-F Li and J. Liu The Fourth Military Medical University, Xian, P.R. China. Paraffin sections from 6 cases of Ewing's sarcoma were immunostained by ABC method using BMP-McAb for investigating the existence of BMP in Ewing's Sarcoma. All cases were positive. The result coincided with human tumor transplants in athymic nude mouse by Bauer. It is shown that Ewing's sarcoma can express BMP. So we support the hypothesis that Ewing's sarcoma arises from primitive multipotential cells and can pluripotentially differentiate.

P10.55 An Ultrastructural and Immunohistochemical Study of Human Pregnant "Transitional Syncytiotrophoblasts". R. Zhang Laboratory of Electron Microscopy, Cheng-de Medical College, Cheng-de 067000 Hebei Province, P.R. China.

The ultrastructure of trophoblasts of terminal villi (67 cases of different weeks' age, 5 - 41 weeks), normal placental beds (10 cases), hydatidiformole (10 cases), malignant mole (10 cases) and choriocarcinoma (10 cases) were studied under TEM, and also with anti-HCG Ab, anti-HPL Ab, and anti-SP-1 Ab; immunohistochemical study was also performed on the trophoblasts of the last 4 kinds of specimens. The results showed, under light microscope, the "transitional syncytiotrophoblasts" may be mono-, di-, or multiplenucleated. Under TEM, there were trophoblasts with nuclei similar to those of cytotrophoblasts, while the cytoplasm similar to that of syncytiotrophoblasts, were called "transitional syncytiotrophoblasts" (some called "intermediate") which probably were from fusion of cytotrophoblasts or syncytiotrophoblasts and cytotrophoblasts. In the trophoblasts of trophoblastic tumors, we found there were different-sized microcysts with microvilli and different-sized secretion granules in their cytoplasm. Immunohistochemical study of "transitional syncytiotrophoblasts" revealed that they contained different amounts of HCG, HPL and SP-1, HPL ranging first. This kind of cell gradually turned into syncytiotrophoblasts. Then, the immunohistochemical study showed some of them containing HCG, HPL, SP-1 showing most strongly positive.

P o s t e r sessions

P10.56 Enzymatic Changes in the Oesophageal Diseases. D. Hopwood ~, J. Jankowski 2, G. CoghilP, L. McLellan 2 and K. G. Wormsley2 Departments of Pathologj and Medicine2, Ninewells Hospital and Medical School, Dundee, UK.

Oesophageal squamous carcinoma accounts for many deaths each year. There are no reliable histological markers for premalignant change, including dysplasia. Abnormal expression of specific enzymes is of functional importance to the pathogenesis of oesophagitis and in carcinogenesis. In an attempt to assess further their expression in oesophageal diseases, we assessed 50 patients; 17 normal, 18 with oesophagitis and 15 with oesophageal squamous carcinoma. We assessed the presence of enzymes, by histochemistry, known to be implicated in altered cellular metabolism of the oesophageal mucosa (non-specific esterases, N A D H and 4 gtutathione transferases. We found that inflamed oesophageal mucosa expressed a greater amount of NADH and microsomal glutathione transferases compared with normal mucosa (p<0.01 and 0.05 respectively). In addition, squamous cell carcinomas expressed NADH and cytosolic (Pi-isoenzyme) glutathione transferases to a greater extent compared with adjacent benign mucosa (p<0.01). Nonspecific esterases and microsomal glutathione transferases were, however, decreased in squamous cell carcinomas (p<0.01). We conclude that these findings may suggest differential adaptation of enzyme expression in benign compared with malignant oesophageal disease.

P10.57

Mechanisms Involved in the Release of Superficial Squames from Oesophageal Squamous Mucosa. D. Hopwood ~, H. Dobson t, J. Jankowski 2, K. Howar G. Milne ~ and K. G. Wormsley~ Departments of Pathologj and Medicine~, Ninewells Hospital and Medical School, Dundee, UK.

The oesophageal mucosal function is to withstand the physical and chemical trauma from its luminal contents. The paradox is for it to remain strong and intact and yet to shed effete cells from its surface. Changes in adhesive mechanisms between basal and surface cells were sought. Histometrically, a significant change in desmosome diameter was found, those on the basal cells being much bigger than those on superficial cells. There were most desmosomes per micrometer of cell membrane on the superficial cells. Desmosomes were sensitive to an endogenous serine proteinase. Cell adhesion molecules were also investigated by immunohistochemistry and in vitro techniques. CD15, a member of the immunoglobulin superfamily, was found in the suprabasal cells, distributed in a chicken wire pattern. Cadherins and some integrins showed a similar pattern. They were also demonstrated in vitro using various cell adhesion techniques. Shed superficial cells were washed from endoscopes and counted. Cell adhesion in the oesophagus involves a variety of mechanisms that change qualitatively and quantitatively from basal cell to surface, finally allowing the shedding of effete superficial cells.

Poster sessions

549

P l l . IMMUNOHISTOCHEMICAL METHODS Pll.1 Cell Rounding Studied by Orientated Horizontal and Vertical Sections of Monolayer Cultures Under Transmission Electron Microscopy. K. H. Sit, H. L. Chart and B. t l . Bay Department of Anatomy, Faculty of Medicine, National University of Singapore, Singapore 0511. situ non-homogeneous embedding using polystyrene, solvent resistant permanox or Melinex as culture supports presents a shearing cell-substratal interface resulting in uplifted cell processes and varied intervals between opposing surfaces of the substratum. To circumvent these problems, we have previously described a technique of using araldite for substratal coating and embedding in a Nunc cover glass slide (Sit et aL J. Electron Micros. Tech. 19:271 - 2 7 2 (1991)). The araldite is dropped into the chamber slide to give a coating of 1.5-2 mm thickness and put in a 60~ oven with cap loosened and allowed to polymerize overnight. The surface is rinsed once with Dulbecco's modified Eagle's medium and trypsinized cells suspended in 0.2 ml of foetal bovine serum are seeded onto each well for 1 hour before the addition of 1.8 ml of DMEM. In preparation for TEM, the cells are fixed in 5~ glutaraldehyde in 0.1 M sodium cacodylate buffer, osmicated in 1070 osmium tetroxide before infiltration with araldite. The polymerized specimen is dipped in liquid nitrogen to remove the cover glass. In the present ultrastructural study, the focal attachment sites of monolayer Chang liver cells before and after Na+/H + antiporter mediated cell rounding with Na + bicarbonate saline buffers (Sit and Wong Tissue & Cell 21:388 - 4 0 0 (1989); Sit et al. J. Tissue Cult. Meth. 1 3 : 2 5 7 - 2 6 0 (1991)) are demonstrated together with the huge endocytic channels that characterize antiporter mediated rounding (Sit et al. Tissue & Cell 22: 785 - 802).

In

PI 1.2 Paraffin Embedding and Immunohistochemistry. P. Bolhuis Working-Committee on Immunohisto- and Cytochemistry, Region 2; The Netherlands'.

The influence of eight different, commercially available paraffin-based embedding media, on immunohistochemical staining was investigated. Eight Dutch departments of pathology participated in this comparative experiment. All paraffin samples were code numbered and distributed among the participating laboratories, Fixation, dehydration, clearing and paraffin embedding, as well as immunohistochemical procedures, were performed according to standard (institutional) procedures. Paraffin embedding temperatures ranged from 58~ to 65~ Five monoclonal antibodies (directed against keratin, vimentin, desmin, L C A and EMA) and one polyclonal antiserum (S-100) were used to evaluate the effect on immunohistochemical staining. The result of this comparative investigation revealed only marginal (negligible) differences in immunohistochemical reactivity for the eight tested paraffinbased embedding media.

Pll.3

Immunolocalization of Isoprenoid Enzymes in Plastids: Chemical Fixation versus Cryofixation. C. Cheniclef, F. Rafia ~, A. Verna 2 and J. P. Carde ~ ~Physiologie Cellulaire Vdgdtale, URA CNRS 568, Universit~ Bordeaux 1, F-33405 Talence, 2Neurocytochimie-Cytologie, URA CNRS 339, Universitd Bordeaux 2, F-33405 Talence, France.

The enzyme geranylgeranylpyrophosphate synthase (GGPPS), which controls the formation of diterpenes and carotenoids in plastids, was localized in chloroplasts and chromoplasts from green and red Capsicum fruits, respectively, by immunogold EM cytochemistry of Lowicryl embedded material after chemical aldehydic fixation or cryofixation by slam-freezing on a nitrogen cooled copper block (1) followed by freezesubstitution with pure acetone. I n green fruits, where the GGPPS amount is low, the enzyme was nearly undetectable in chemically fixed material, whereas a precise labelling was observed in cryofixed chloroplasts, mainly in the plastid stroma and plastoglobuli. In chromoplasts of red fruits, which contain large amounts of GGPPS, a high density of labelling was observed in both chemically fixed and cryofixed materials. Cryofixation followed by freeze-substitution seems therefore the more reliable method to localize some antigens present in small amounts in the tissues and/or highly modified or lost during chemical fixation and dehydration. ~Verna, A., Biol. Cell 49, 9 5 - 9 8 (1983).

Pll.5 Stabilization in Ethylene Glycol at Low Temperature. Polar Resin Embedding for Light Microscopy, Avoiding Chemical Fixation. B. Dote, A. Paraninfo and P. Usai Dipartimento di Biologia Animale, Facotta "di Scienze MFN, Universita "di Torino, v. Accademia Albertina 17, 1 10123 Torino, Italy.

In the course of several years we revealed enzymatic activities in Vertebrate and Invertebrate tissues (sections 2 micron or less thick) embedded in polar resin (Glycol Methacrylate: Techovit 7100, Kulzer; Historesin, LKB), at low temperature. A brief ( 3 0 - 6 0 rain) and mild fixation in 1~ paraformaldehyde (in cacodylate buffer 0.2 M, pH 7.3), a mild and incomplete dehydration in acetone (at 4~ temperature), followed by infiltration and polymerization of the monomer at a maximum of 6~ always working in ice bath, let a good preservation of morphology and, moreover, of several enzymes. Enzyme activity may be revealed also in tissue embedded some years before the histochemical reactions. Furthermore, similar results are obtained from sections of not fixed embedded tissues. These tissues were previously quenched in liquid Nitrogen or simply cooled ( - 2 0 ~ or lower temperatures) in Ethylene Glycol (EG) solution, dehydrated in EG/water mixtures at - 2 0 ~ (or lower) then embedded. In a good many cases, tissue sections show a considerable morphological preservation and enzymatic activities are more easily demonstrable than in the briefly fixed tissues.

550 Moreover, small Invertebrates (Rotifera, Anellida) can be stabilized in EG solutions (at - 20~ then several enzymatic and histochemical reactions can be performed in toto, as well. Work supported by MURST grants (60%, 40%).

Pll,6 Substitution of Xylene with a Non-Toxic Fatty Acid Ester in the Paraffin Technique. L L. Holm ~ and H. O. Lyon 2 ~School of Hospital Laboratory Technicians, Copenhagen, 2Dept. of Pathology, Hvidovre Hospital, Hvidovre and the Substitution Group, Denmark.

The aromatic hydrocarbon, xylene, used in the paraffin technique for clearing and for deparaffinizing tissue sections, is neurotoxic. Substitution of this solvent with a non-toxic agent is therefore desirable. We investigated tentative substitutes and compared their clearing properties to xylene. We made an evaluation of toxicity and price of agents showing good clearing properties. We chose the commercial fatty acid ester Estiplast 3124 for a further multicentre testing. The test included 230 tissue specimens. The only change in the routine of the departments was to clear one half of the specimens in Estiplast 312 R. The other half served as controls. Staining methods were Aluminium-haematein and Eosin and Oil Red O. A systematic registration of criteria for assessing section quality and morphology took place. In general, we got resuks with Estiplast 312 R comparable to those with xylene. We did, however, meet some difficulties with fatty tissue in a few cases. We further tested whether Estiplast 312 R at temperatures ranging from 25 - 60 ~ could substitute xylene for deparaffinizing tissue sections. This was the case with Estiplast 3124 heated to 50~ Our conclusion is that the non-toxic ester, Estiplast 3124 can replace xylene and toluene both for clearing and deparaffinizing.

Pll.7

A New Application of Confocal Laser Scanning Microscopy (C-LSM) to Observe Subcellular Organelles Utilizing Non-fluorescent Probe (Osmium Black). J. Itoh, H. Utsunomiya, N. Komatsu, S. Takekoshi,

R. Y. Osamura and K. Watanabe Cell Biol. Res. Lab. and Pathol., Tokai Univ. Sch. of Med. Isehara Kanagawa Japan 259-11.

Recently, the confocal laser scanning microscopy (C-LSM) was introduced into the fields of biology and medicine and its application has surprisingly been increased in both frequency and variety. In fact, the C-LSM enabled us to observe subcellular organelles with light microscopic specimens by presenting fine optical tomography and to reconstruct them three-dimensionally. In these observations, however, fluorescent signal is solely available to be detected by the C-LSM. Since fluorescent signal is easily and quickly deteriorated, it is impossible to get permanently preservable specimens which make retrospective and objective observations possible.

Poster sessions In the present report, we employed the combination of horseradish peroxidase-DAB-osmium (final product is osmium-black) as the detection probe instead of the fluorescent one in C-LSM (the combination of LSM-IO and IBAS Carl Zeiss was used) and successfully observed fine three-dimensional configurations of subcellular organelles such as microtubules, endoplasmic reticula and mitochondria.

Pll.8 The Effect of Proteolytic Enzymes Pretreatment and that of ARS (Antigen Retrieval Solution) on Immunohistochemistry. B. Klein Brink Working-Committee on Immunohisto- and Cytochemistry, Region 1; The Netherlands.

Twelve laboratories have participated in testing three commercially available proteolytic enzymes (protease type XXIV, pepsin and trypsin) and the effect of ARS on immunohistochemistry, in relation to non-pretreated tissue. Five monoclonal antibodies (directed against keratin, vimentin, desmin, LCA and EMA) and three polyclonal antisera (S-100, CD3 and IgA) were tested on buffered formaldehyde fixed colonic tissue. The results of this investigation showed a different antigen expression in immunohistochemistry for some antibodies and antisera after pretreatment with proteolytic enzymes or with ARS.

Pll.9

Repeated PAP or Repeated PAP and ABC Methods. Y. Liu and S. Yang Department of Pathology, Institute of Basic Medical Sciences, Chang Le Xi Road, Xi'an, 710032, P.R. China.

In demonstration of the viral antigen of epidemic hemorrhagic fever (EHF) in formalin fixed and paraffin embedded autopsy tissues, more sensitive immunohistochemical methods are needed. Because the antigen is so weak that traditional double PAP and ABC methods usually failed to take effect in long preserved (more than 10 year) paraffin sections. The repeated PAP method was characterized by repeated use of PAP. In our laboratory, after double PAP, bridge antibody and PAP were added and the same procedures were repeated for 8 times (8 PAP). The repeated PAP and ABC method was performed first by repeated PAP in which biotinylated bridge antibody should be used and then the procedure was finished by application of vector ABC complex for once. Therefore, PAP and ABC were combined together, the number of ABC complex were combined with the same number of the bridge antibody molecules. Two kinds of first monoclonal antibodies against EHFV and insulin were used in this procedure. The results showed that the larger the number of PAP or ABC used, the more sensitive the positive result reaction. The increased sensitivity was shown not only by the larger percentage of positive rate but also by the higher OD value obtained by the more repeated PAP or more PAP + ABC under Leitz Microspectrophotometer.

Poster sessions Pll.10

lmmunohistochemieal Study with Monoclonal Antibody to 140 kd Human Kidney Brush Border Protein. M. Shiozawa, M. Ohyama, T. Ezaki, K. Yasuda and S. Aiso Department of Anatomy, Keio University, Tokyo, Japan.

We prepared monoclonal antibodies of the IgG1 type, which reacted specifically with the 140 kd human brush border protein, estimated by SDS PAGE. The reaction of the MAb was shown

551 to be definitely specific to the antigen by ELISA, banding analysis on blots from SDS-PAGE, and the immunohistochemical reaction. The immunohistochemicat staining of human tissues, using this MAb, revealed that the antigen was localized on the brush border in kidney, luminal membrane of the epithelial cells in gall bladder and prostate, and along the bile canaliculi of normal liver as well as in hepatoma and foetal liver. The intensity of immunostaining in hepatoma was stronger than that in normal and foetal liver. We also studied threedimensional structure of bile duct system by immunostaining with this MAb using Confocal Laser Scanning Microscope System (Molecular Dynamics Japan, Inc.).

P12. QUANTITATIVE HISTOCHEMISTRY P12.1 Quantitative Enzyme Histochemistry on FreezeSubstituted Glycol Methacrylate-Embedded Rat Liver.

P12.2 Histochemical and Stereological Methods for Diagnosis of Thyroid Tumours.

K. S. Bosch and W. M. Frederiks Laboratory of Cell Biology and Histology, University of Amsterdam, The Netherlands.

A. Co'r and Z. Pajer Institute of Histology and Embryology, Medical Faculty in Ljubljana, Slovenia.

During the last decade many reliable procedures have been developed for the demonstration of enzyme activities in situ. Unfixed cyrostat sections are recommended to detect optimum enzyme activities. A disadvantage of the use of unfixed cryostat sections is the rather poor morphology of the tissue, especially under pathological conditions. As an alternative, freeze-substituted tissues embedded in glycol methacrylate can be applied. In the present study we have compared the activities of several enzymes as demonstrated in cryostat sections and GMA-sections of normal liver. The following enzymes could be detected in GMA-sections: 5'-nucleotidase (lead salt method), alkaline phosphatase (indoxyl-tetrazolium salt method), catalase (diaminobenzidine method), acid phosphatase (diazonium salt method), lactate dehydrogenase (tetrazolium salt method) and glutamate dehydrogenase (tetrazolium salt method). The activities of all these enzymes were dramatically decreased in GMA-sections in comparison with the activities demonstrated in unfixed cryostat sections. The activities of the following enzymes could not be detected in GMA-sections: glucose-6-phosphate dehydrogenase (tetrazolium salt method), D-amino acid oxidase (ceriumdiaminobenzidine-cobalt-hydrogen peroxide technique) and glucose-6-phosphatase (cerium-diaminobenzidine technique). We assume that this may be caused by inactivation of these enzymes or extraction of compounds, e.g. lipids, necessary for the enzyme activities. The role of limited penetration of substrates and/or dyes into the resin was studied by comparing the enzyme activities in GMA-sections of 3 and 6 ~m thickness, whereas a possible retarded penetration of these compounds into the resin was investigated by measuring the amount of final reaction product with a cytophotometer during incubation. From the cytophotometric results it was concluded that thermal and/or chemical denaturation of proteins during embedding in the resin monomer was the main cause of the decrease in enzyme activities.

According to the histologic typing system of thyroid tumours set up by the WHO, follicular neoplasms are classified as follicular carcinomas. The most important diagnostic parameter for cancer is the evidence of vascular and/or capsular invasion. The aim of this work was to find the method which would lead to the improvement of thyroid tumour diagnosis. The thyroids from the experiment, where mice have drunk thyrostatic (1.2% NaC104) and have been irradiated with 0.8 Gy gamma rays in five continuous days, were used. 10 histologically normal thyroids, 10 with hypertrophy and hyperplasia, 8 follicular adenomas and 10 follicular carcinomas were analysed. The average number of Ag-NOR/ nucleus and the average nuclear volume of epithelial cells (Vn) with nucleator were determined. Both methods were applicable to distinguish between adenomas and carcinomas. The results showed significant differences in Ag-NOR/nucleus (P 0.005) and V, (P 0.05 between follicular adenomas and follicular carcinomas. The method of counting Ag-NOR/nucleus was more valid, more accurate and more efficient than the method of measuring nuclear volume.

P12.3 A Novel Technique for In Situ Measurement of Substrate Concentrations. W. J. C. Geerts*, J. M. Koopdonk-KooF, A. Jonker**, W. H. Lamers* and C. J. F. van Noorden t *Department of Anatomy and Embryology; tDepartment of Cell Biology and Histology, University of Amsterdam, Meibergdreef 15, 1105 A Z Amsterdam, The Netherlands.

Metabolic activity of cells, i.e. flux through metabolic pathways, not only depends on cellular enzyme concentrations but also on the cellular concentration of substrates and cofactors. For the determination of the cellular concentration of the substrate glucose-6-phosphate (G6P), gelatin model sections containing varying amounts of substrate, were made.

552 A second gelatin model section, containing the enzyme (glucose-6-phosphate dehydrogenase), activators, cofactors and a tetrazolium salt, were sandwiched on the substrate model section. The formazan produced can be measured, with either a scanning and integrating cytophotometer or an image analysis system, and related to the substrate concentration. Good correlations were found between the substrate concentration and the amount of formazan produced. The method was reproducible and 85 _+ 4~ of G6P could be demonstrated. The sensitivity of this method appeared to be at least 100 ~M and enabled the measurement and localization of physiological substrate concentrations in sections of liver where G6P concentrations are in the same order of magnitude.

P12.4 Distribution of Alkaline Phosphatase Activity in the Periodontium of the Rat Incisor. M. Groeneveld, V. Everts and W. Beertsen Dept. Periodontol., Acad. Centre for Dentistry Amsterdam (A CTA), The Netherlands.

Alkaline phosphatases (ALPs) are membrane-bound glycoproteins which have the capacity to hydrolyze monophosphate esters. The L/B/K-isoenzyme is considered to play a role in the process of mineralization and/or transport of ions or nutrients across cell membranes. This isoenzyme, which is present in liver, bone and kidney, is also very abundant in the periodontal ligament. In order to obtain information on its spatial distribution in the latter tissue, enzyme activity was quantitatively assessed in the periodontal tissues associated with the continuously growing rat incisor. The lower jaws of five Wistar rats were dissected out immediately after killing of the animals, embedded in gelatin and frozen in liquid nitrogen. Undecalcified cryosections of 6/am thickness were cut parallel to the longitudinal axis of the incisor. The indoxyl-tetrazolium salt method was used for the histochemical demonstration of ALP-activity and the amount of reaction product quantified using a Vickers M85a microdensitometer at a wavelength of 585 nm. As a parameter for cell density Feulgen-DNA was assessed in adjoining tissue sections using the same instrument at a wavelength of 560 nm. At eight predetermined sites along the incisor measurements were carried out across the width of the periodontal ligament from the cementum to the wall of the alveolar bone. Measurements did also include the supracrestal extension of the periodontal ligament and the lamina propria of the gingiva. The data clearly demonstrated a heterogeneous distribution of ALP-activity in the rat incisor periodontium. The highest activity was found in the bone half of the ligament. In all other regions of the ligament (including its supracrestal extension) the activity was significantly lower (ANOVA; p<0.05), but still much higher than in the lamina propria of the free gingiva. This heterogeneous distribution is taken to suggest that considerable inter- and intratissue variations may occur in the periodontium as to the capacity to hydrolyze phosphate esters.

P12.5 Comparison of Sensitivity Among Multi-Layered Peroxidase Labelled Antibody (MLP) Method, Avidin-Biotin-Complex (ABC) Method and Peroxidase-anti-Peroxidase (PAP) Method.

Poster sessions S.-L lzurni, M. Shin and P. K. Nakane Department of Anatomy, Nagasaki University School of Medicine, Nagasaki, 852, Japan.

Sensitivities of immuno-peroxidase methods vary, however objective quantitative comparative information is scarce. Hence, we compared the sensitivities. Rabbit aT-T-DNA immune-complexed with T-T dimerized salmon sperm DNA in plastic wells and that complexed with UV induced T-T dimers in paraffin-embedded rat liver sections were used as antigen for ELISA and immuno-peroxidase methods, respectively. These plastic plates and sections were reacted HRP-goat antirabbit IgG (HRP-GaRIgG) (MBL, Japan) [I~ I~ and HRP-rabbit anti-goat IgG (HRP-RaGIgG) (Cappel, USA) [2~ 2~ and HRP-GaRIgG [3~ 3~ and HRP-RaGIgG [4~ biotinylated GaRIgG and HRP-avidin from Histofine SAB-PO KIT (Nichirei, Japan) and from Vectastain (Vector Lab. USA) [ABC], aRIgG and rabbitaHRP included from DAKO PAP KIT and RABBIT PAP IMMUNOSTAINING KIT (Zymed Lab. USA) [PAP]. The reagents from the kits were diluted as specified. The generated signal and background were determined spectrophotometrically for ELISA and by a computer assisted image analyzer for immuno-peroxidase method. Both by ELISA and immuno-peroxidase method, the sensitivity (signal-back ground) was found to be [4~ >[3~176 = [ABC] = [I~ >= [PAP].

P12.6 Quantitative Histochemical Monitoring of the Response of Tibialis Anterior Muscle in Paraplegic Subjects to Electrical Stimulation. L. Rochester ~, M. Barron 2, C. Chandler 3, R. Sutton ~, S. Miller s and M. Johnson 2 tRegional Spinal Injuries Unit, Hexham General Hospital; 2School of Neurosciences, University of Newcastle upon Tyne; JHealth and Behavioural Sciences Department, Newcastle upon Tyne Polytechnic, UK.

The primary aim of this study was to determine whether regimes of electrical stimulation could improve fatigue resistance and muscle strength in subjects with spinal injury. Seven paraplegic subjects aged 1 9 - 4 3 years with complete thoracic spinal trans-section of 2 - 1 4 years' duration were included in the investigation. Biopsies of left and right tibialis anterior (TA) were taken before and after intermittent percutaneous stimulation of one TA muscle, the unstimulated/pre-stimulated muscles acting as controls. The proportion of Type 1 (slow-twitch oxidative), Type 2A (fasttwitch oxidative/glycolytic) and Type 2B (fast-twitch glycolytic) muscle fibres were monitored in tissue sections in which the activity of myofibrillar ATPase was demonstrated. Almost all pre-stimulation biopsies showed an abnormally low proportion of Type l fibres and this did not alter in response to stimulation. The activity of succinate dehydrogenase (SDH) was measured in individual muscle fibres by microphotometric enzyme assay (MEA) using Nitro BT. In all 7 subjects, SDH activity in TA was significantly increased following stimulation as compared with pre-stimulation values in the same muscle. This correlated with enhanced fatigue-resistance demonstrated using electrophysiological techniques. Capillary density was monitored in tissue sections using Ulex europaeus agglutinin I (UEA-I) as a marker of vascular endothelial cells

Poster sessions but only one patient showed an increased capillary supply in TA muscle following electrical stimulation.

P12.7 Two Dimensional Quantification of Histochemical Reactions by Image Analysis. A. Jonker *t, W. J. C. Geerts*, W. H. Lamers* and C. J. F. van Noorden + *Department of Anatomy and Embryology; tDepartment of Cell Biology and Histology, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Image analysis in metabolic research can be used for quantitative analysis of enzyme activity in situ in tissues or cells, Local variations in enzyme activity and its distribution can be detected two dimensionally by time-lapse measurements, and images of precipitated coloured final reaction product can be stored. This enables analysis of kinetic behaviour of enzymes in various loci within tissues. With a monochrome Sony CCD camera and a Sun VideoPix frame grabber, time-lapse measurements of histochemical reactions were made. All measurements were stored on hard disk. Absorption values were computed and an intensity map showed differences in enzyme activity related to tissue morphology. Both test and control reactions were recorded in serial sections. Afterward, the specific reaction velocity was calculated for different areas by subtracting the control images from the test images. It appeared that image analysis is a very attractive tool for enzyme reactions in situ.

P12.8 Two Simple Methods for Estimating the Initial Reaction Velocity of a Soluble Dehydrogenase In Sit~. Y. Nakae ~ and P. J. Stoward z IDepartment of Oral Anatomy, Tokushima University School of Dentistry, Japan and 2Department of Anatomy and Physiology, University of Dundee, UK.

Gel films containing 0.8% low gelling temperature agarose, 80 mM Tris-HC1 buffer (pH 7.5), 5 mM EDTA, 10 mM NAN,, 1.2 mM Nitro BT, 0.26 mM PMS, 1.5 mM NAD and 0 - 7 0 mM lithium lactate were placed on 4/am - - thick unfixed sections of various mouse tissues. The sections plus films were incubated on a thermostatically-controlled microscope stage at 37~ Images of the sections illuminated with monochromatic light (584 nm) were captured at 10 sec intervals with an ARGUS-100 image analyser system. The images were processed at leisure and the absorbances of selected cells were plotted as a function of incubation time. The initial reaction rates (vJ were found to fit the equations v~ = a~~ and v~ = v + a2~ where v and ~ are respectively the gradients and the intercepts on the absorbance-axis of the linear regression lines of the absorbance on incubation time plots for times betwen 1 and 3 rain, and a~ and as are constants. The values of a, and as were determined from linear plots of ~ against v~ and (v~-v) against ~ respectively. They were in the ranges 2 . 6 - 3.3 and 1 . 2 - 2 . 0 respectively for hepatocytes, cardiac muscle fibres, skeletal muscle fibres (gastrocnemius), gastric parietal cells,

553 oocytes, and glandular and ductal epithelia of the parotid gland.

P12.9 Quantitative Histochemistry and Image Analysis in Cancer Prognosis. R. A. Prochuhanov ~, L. S. Agroskin, V. B. Makulov 2 ~St. Firm Bioinf., St. Petersburg, Russia; 2S. L Vavilov State Optical Institute, St. Petersburg, Russia.

The main objective of the work is early diagnostics and disease prognosis estimation for prophylaxis and radical treatment in oncology. The following ideas form the basis of the work: - - characteristics of the histohematic barriers (epithelium, endothelium and stromal cells) components are taken as indexes of tissue and organs state; - - quantitative methods of the morphometry and citochemistry of the biopsy and operational material are used to estimate these characteristics; - - the main instrument is based on methods of automatic digital image processing. The main advantage of this approach is the possibility of diagnostics based on background processes. That implies the diagnostics in the absence of evident (conventional) symptoms. The result of work is an automatic system for cancer diagnostics. In the present time the cancer of the mammary glands diagnostic method has been tested with the model system. The estimate of classification quality was done using the methods of discriminate analysis and it demonstrated 90 percent of trustworthiness. There are theoretical and technological reasons for this system to be extended for use on other localization of cancer and different diseases connected with the immune system.

P12.10 Quantification and Reconstruction of the Surface Lectin/Immunogold Labelling in Cell Membranes. D. A. Rusakov ~ and G. G. Skibo 2 ~State University, Dniepropetrovsk 320625, 2Bogomoletz Institute of Physiology, Kiev 252601, Ukraine.

Lectin/immunogold labelling of membrane molecules at the electron microscope level is known to be a powerful tool to study properties of cell membranes. Currently used methods to quantify the labelling usually neglect its stochastic nature as well as the fact that micrographs represent random membrane profiles. In nerve cells developing in monolayer culture for 5 - 2 0 days, we studied the topography of immunogold labelled neural cell adhesion molecules (hippocampus of new born rats) and wheat germ agglutinin gold labelled membrane glycoconjugates (spinal cells from 12-day-old mouse embryo). Electron micrographs were collected in statistical samples according to the cell types, membrane profile parts and developmental terms which they represent. The pictures were considered to be random plane sections of infinite membranes. Observed label distributions along membrane profiles were analysed like stochastic processes where events are a label or label aggregate. Standard statistical analysis of the processes allowed a proper description of the corresponding label arrangement. A developed stereological approach based on these data was used to parameterize and

554 reconstruct by means of Monte Carlo simulation the typical label lateral topography on the 'unfolded' surface of studied membranes, The results obtained allow the stochastic geometry of the surface arrangement of labelled molecules to be described as well as providing insights into the lateral organization of studied membranes under different experimental conditions.

P12.11 Quantitative Histoenzymology and its Potentialities in System(ic) Analysis of Lung Pathology. V. v. Tomson RC St. Petersburg Parlor's Medical Institute, Russia.

Quantitation by histochemical methods of some enzymes reflecting cellular or tissue key metabolic activities serves to elucidate a functional heterogeneity of structural elements which cannot be reached by conventional morphological techniques. In this study, systemic analysis of a few marker enzyme activities in human lung inflammatory pathologies has been performed for the examination of essential differences in structural and metabolic remodelling as to the elements of aerohaematic and bronchovascular barriers beyond the area of the lesions. Since early post-mortem sampling, within one hour after the patient's death, was used throughout the study, the analytical approach applied has turned out to be sensitive enough for evaluation of tissue alterations which accompany both acid-alkaline disbalance and progression of acute as well as chronic respiratory failure in lung tissue.

P12.12 Determination of Local Metabolite Concentrations in Organs of the Rat using Bioluminescence. M. Ademes, S. Walenta and W. Mueller-Klieser Institute of Physiology and Pathophysiology, University of Mainz, Duesbergweg 6, D-6500 Mainz, FRG. The local concentrations of ATP, lactate and glucose were determined with high spatial resolution in various organs of

Poster sessions the rat using a bioluminescence technique (Mueller-Klieser et al., J. Natl. Cancer Inst. 80: 842, 1988). The technique allows for the correlation of the substrate distribution obtained and the histological structure of the tissue. Organs such as kidney, heart, spleen, liver, and skeletal muscle were taken from Sprague Dawley rats (weight: 200-300 g). Under anesthesia and continuous control of temperature, blood-gas, and bloodpressure each organ was rapidly covered with liquid nitrogen in situ. Frozen sections from the organs were used for the bioluminescence reaction. Metabolite distributions were rather homogeneous, yet regional differences were found that were often correlated with the histological structure of the organ. For example, maximum values in the kidney for ATP (1.6 mM), and glucose (4.5 raM) were located in the cortex, whereas lactate (1.8 mM) seemed to be accumulated in the transitional region between cortex and medulla. Thus, imaging bioluminescence may be used to assess information on the metabolic state of tissue in relation to the histological architecture of various organs. (Supported by the BMFT 01 ZO 8801).

P12.13

The use of Quantitative Histochemistry for Analysis of Compensatory Changes in the Lungs. V. V. Tomson, L. A. Zlotnikova, L. N. Danilov and Y. A. Kirillov RC St. Petersburg Pavlov's Medical Institute, Russia.

The development of fibrosis provoked by bleomycetin administration is accompanied by an accumulation of plasmocytes in the alveolar wall, its fibrosive thickening and nucleus degeneration in second type alveolocytes. All of these changes are revealed by electron microscopy. The quantitative histoenzymological analysis of second type alveolocytes, alveolar macrophages and endothelium of capillaries in the alveolar wall was carried out in the early stage of the experiment. This analysis has shown morphofunctional compensatory changes of the bioenergetics in the cells which are connected with surfactant metabolism. The changes cannot be defined by ordinary histological methods.

P 1 3 , I N S I T U HYBRIDIZATION P13.1

Optimization of Biotinylated Probe Detection. G. A l i a # and S. McQuaid 2 ~Veterinary Sciences Division, 2Royal Victoria Hospital, Belfast, Ireland.

Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus specific RNA probe on formalin-fixed, paraffinembedded central nervous system tissue infected with measles virus. We report that maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their

failure to detect viral nucleic acid in infected white matter ceils. Only by increasing the number of steps in the detection protocols were these infected cells demon.strable. Unless prehybridization, hybridization and detection protocols are optimized the results obtained in pathogenicity studies using ISH could be misinterpreted leading to false conclusions about nucleic acid distribution. This also applies to the ever increasing use of ISH for diagnostic purposes. P13.2

Marker Chromosome Analysis Using Reverse Painting. J. Bayer, C. Tan, A. Martens and J. Bauman TNO Institute of Applied Radiobiology and Immunology, Lange Kleiweg 151, 2288 GJ Rijswijk, The Netherlands.

A new method for generating chromosome specific painting

Poster sessions probes starting from minimal numbers of flow-sorted chromosomes is described. This method is of special use in the cytogenetic analysis of unidentified marker chromosomes in human neoplasia. After MboI digestion of the purified chromosomal DNA, synthetic oligonucleotide adaptor molecules were ligated onto the sticky ends of the digested chromosomal DNA. The resulting DNA fragments can be amplified irrespective of their chromosomal DNA composition, using adaptor-specific primers in a Polymerase Chain Reaction (PCR). Like this, DNA from as few as 1,000 copies of a specific chromosome was amplified in a sequence independent fashion. Chromosomal In Situ Suppression hybridization with these PCR products after labelling with biotin or digoxigenin was shown to very evenly "paint" the correct chromosomes in human metaphase spreads. Until now we have applied the procedure to generate painting probes for normal human chromosomes as well as marker chromosomes. Application of the painting probe for the CML-specific Philadelphia chromosome to normal human metaphase spreads allowed unequivocal identification of the regions that make up this marker. The study of other cytogenetically undefined marker chromosomes using this "reverse painting" approach is in progress. "Reverse painting" provides information on the exact bands of normal chromosomes that contribute to marker chromosomes. It is thus ideally fit for identifying translocation breakpoints. Furthermore it produces clonable marker specific DNA that can be used for further molecular biological characterization of marker chromosomes.

P13.3

Combination of Immunohistochemistry with both Radioactive and Non-Radioactive In Situ Hybridization: Application to the Simultaneous Detection of one Antigen and two mRNAs in the Same Brain Tissue Section. A. Trembleau, D. Roche and A . Calas Ddpartement de Cytologic, Institut des Neurosciences CNRS URA 1488, Universitd P. & M. Curie, 7 quai Saint Bernard, 75252 Paris cedex 05, France. We present a simple method for combining non-radioactive and radioactive in situ hybridization, and immunohistochemistry on the same brain tissue section. This approach was developed using the well-characterized rat hypothalamoposthypophyseal system, which allowed the optimization of the triple-labelling procedure and verification of the labelling specificity. We report here a few examples of triple labellings in 12 gm cryostat sections of hypothalamus of dehydrated rats, including the simultaneous detection of vasopressin mRNA, oxytocin mRNA and oxytocin peptide, as well as the simultaneous detection of vasopressin mRNA, oxytocin mRNA and tyrosine hydroxylase (TH). This was performed on floating sections as follows: first, the two probes were hybridized simultaneously, second the peptide (or TH) was detected with an immunoperoxidase-DAB procedure, third the digoxigenin-labelled probe was detected with an alkaline phosphatase-NBT/BCIP technique, and finally the ~S-labelled probe was detected by histological autoradiography. We demonstrated that the combination of the three techniques did not significantly decrease their specificity nor their sensitivity. Moreover, we showed that the three markers

555 used in our protocol were sufficiently distinguishable to allow intracellular colocalization studies. In conclusion, this new method allowing the simultaneous detection of three different products of gene expression in the same section could be useful for further analysis of the phenotypic organization and of its plasticity in heterocellular systems such as endocrine or nervous tissues.

P13.4 DO Tumour Clones Unrelated to Hodgkin- and ReedSternberg Cells Exist in Hodgkin's Disease? J. Deerberg ~, K. Weber-Matthiesen, B. Schlegelberger and

W. Grote Dept. o f Human Genetics, University o f Kiel, Germany.

Hodgkin- and Reed-Sternberg cells (HRS-cells) are generally regarded as the malignant cells in Hodgkin's disease. Nevertheless it cannot be excluded that additional cell populations coexist in Hodgkin's disease. Coexistence of additional clones is for example indicated by the presence of chromosomally aberrant single cells, which are frequently revealed by standard cytogenetic analyses. Because only few mitosis are usually available for investigation, clonatity of the additional cells still remains obscure. Moreover lineage assignment of these cells is impossible due to destruction of cell membranes and cytoplasm. Therefore we have recently developed a technique to combine fluorescence immunophenotyping and in situ hybridization on the single cell level. This technique is referred to as "Fluorescence-immunophenotyping and Interphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)". Using this approach we were able to show in a case of Hodgkin's disease that a chromosomally aberrant T-cell clone was existent in addition to the CD30 + HRS-cells. q. D. was supported by grants of the FriedrichNaumann-Sfiftung.

P13.5

Testicular Angiotensin-Converting Enzyme Localization with Isoform Specific Antibodies and Nucleic Acid Probes. J.-M. Gasc, M. Sibony, D. Segretain, S. Shanmugam and

P. Corvol I N S E R M U36 - - Collkge de France 3, rue d'Ulm, 75005 Paris - France.

A short isoform of the Angiotensin-Converting Enzyme (ACE) is expressed in the sexually mature testis. This truncated germinal form and the endothelial (somatic) form differ by the length of their peptidic sequence (in the mouse 732 and 1278 amino-acids, respectively) and N-terminal amino-acid sequences. These differences result from two alternate transcription initiation sites. Using antibodies to synthetic peptides corresponding to the specific N-terminal amino-acid sequence of each of the two isoforms, and antibodies to other parts common to both isoforms, it was shown that: - the endothelial and testicular isoforms are exclusively restricted to somatic and germinal cells, respectively; - the germinal isoform appears after the pachytene stage and is particularly expressed in spermatids; -

556 in germ cells ACE is located in the cytoplasm, at the plasma membrane (particularly over the flagella) and in the acrosome. Using in situ hybridization, with [35S]-labelled riboprobes specific for the germinal isoform, it was shown that germinal ACE is exclusively expressed in germ cells (and not in Sertoli or Leydig cells); transcription of ACE in germ cells occurs only after completion of meiosis and stops when germ cells are mature and ready to slough off into the lumen. The specificity of the antibodies and nucleic acid probes, and the complementarity of the two morphological techniques used (immunohistochemistry and In Situ hybridization) will allow a study of the regulation mechanisms of the germinal ACE expression, whose function still remains unknown.

P13.6 In Situ Hybridization (ISH) of K-, H-, N-ras mRNA in Gastric Carcinoma Using Thymine-Thymine (T-T) Dimerized Synthetic Oligo-DNA Probes. T. Hashimoto*, K. Motojima*, T. Kanematsu*, M. Shin**, S.-I. Izumi**, M. Nozawa** and P. K. Nakane** *2nd Dept. of Surgery and **3rd Dept. of Anatomy, Nagasaki Univ. School of Medicine, Nagasaki 852, Japan. K-, H-, N-ras oncogenes are highly homologous and encode proteins which may be involved in growth signal transduction and are often activated organ specific manner in malignancies. Because of the homology between the ras, we utilize ISH using T-T dimerized synthetic oligo-DNA probes shown to be unique to each ras in order to define the ras transcribed in gastric carcinoma, in addition to performing immunohistochemistry (IHC) using monoclonal antibody to K-ras p21. Surgically resected gastric carcinoma were formalin fixed and were either sectioned frozen or embedded in paraffin and sectioned for ISH and IHC. Among 25 cases examined, K-ras mRNAs alone were detected in 10, H-ras alone in 2, N-ras alone in 2 and both K-ras and H-ras in 1. There was no obvious correlation between the histological types or the degrees of differentiation of tumors and kind of ras oncogenes expressed. The expression of K-ras mRNAs was not always consistent with the level of K-ras p21. Deeply invaded tumor cell clusters tended to express higher levels of K-ras mRNA, whereas the level of K-ras p21 tended to be similar between the superficial and deeply invaded tumors. In this study with the T-T dimerized oligo-DNA probes, we could distinguish the three ras mRNAs and showed that K-ras mRNAs were most frequently expressed in gastric carcinoma.

P13.7

Chromosome 7 Aberrations in Adenomas of the Colon. J. Herbergs, A. de Bruine, M. Vallinga, F. C. S. Ramaekers, A. F. P. M, de Goeij, J. ten Kate and A. H. N. Hopman Department of Pathology and Department of Molecular Cell Biology & Genetics, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Colonic adenomas (n = 38) were analysed by fluorescent in situ hybridization using centromere probes specific for chromosomes 1, 7, 17, X and Y. Fresh adenomas were divided in tissue blocks, which were: a) fixed in formaldehyde and

P o s t e r sessions embedded in paraffin, b) snap frozen or c) minced. Single cell suspensions were prepared by treating the minced tissue blocks in phosphate buffered saline containing 1 mM EDTA, 1 mM EGTA, 0.5 mM DTT. The cells were fixed in 70% ethanol at - 20oc. A numerical aberration for at least one of the chromosomes was detected in 15 out of 38 adenomas. 5 out of these 15 cases were aberrant for almost all investigated chromosomes and therefore considered to be aneuploid. A trisomy for chromosome 7 was detected in 8 out of 33 diploid adenomas. In 5 out of 8 cases a trisomy for chromosome 7 was the only numerical aberration found, In one case it was shown by combining immunocytochemistry and in situ hybridization in smears of the same suspension, that the cells aberrant for chromosome 7 were epithelial cells and not inflammatory cells.

P13.8 Interphase Cytogenetics in the Analysis of Chromosome Aberrations in Paraffin Sections of Solid Tumours. A. 11. N. Hopman, M. Vallinga and F. C. S. Ramaekers Dept. of Molecular Cell Biology & Genetics, Univ. of Limburg, P.O. Box 616, 6200 MD, Maastricht, The Netherlands. In situ hybridization (ISH) using repetitive probes can be performed in routinely processed, paraffin-embedded tissue sections from solid tumors. This enabled us to study chromosomal aberrations in archival pathological material. An important parameter to obtain strong ISH signals and reproducible results in sections appeared to be the proteolytic treatment of the sections prior the ISH reaction. Different parameters were studied involving influence of nuclear truncation in the detection of numerical chromosome aberrations, the detection of tumor cells among stromal and inflammatory cells, flow cytometric DNA index in relation to chromosome copy number, and chromosome heterogeneity. Paraffin sections of several routinely processed tumors were studied. In bladder cancer we compared the detection of trisomy up to nonasomy in cells isolated from tumors with the results obtained in paraffin sections. In mesotheliomas we compared the results of classical karyoptyping with the detection of aberrations in the paraffin section. In hydatiform moles and hydropic abortions we compared sex chromosome patterns, chromosome aneuploidy and chromosome composition in different subpopulations. I n case of a melanoma we compared human melanoma cell lines with tissue sections of xenografted lesions obtained from these cell lines in nude mice. The results of these studies in different types of cancer will be discussed.

P13.9 Expression of some Oncogenes Correlates with Distinct Differentiation States of Neuroendocrine Carcinomas of the Lung. J.-P. Da I, W. Li2 and J.-J. Bao 3 ~Department of Pathology, General Hospital of the Air Force. 2Department of Pathology, Great Wall Hospital. 3Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. P.R. of China. Expression of p53, c-H-ras and v-myb oncogenes was studied in neuroendocrine carcinoma of the lung including typical

P o s t e r sessions carcinoid (4 cases), atypical carcinoid (4 cases) and small cell lung carcinoma (4 cases) by in situ hybridization. All materials were obtained from surgical specimens. They were immediately fixed in 4o7o paraformaldehyde and then embedded in paraffin. The major steps of DNA-mRNA in situ hybridization consisted of (1) labelling p53, c-H-ras and v-myb with non-radioactive digoxigenin-11-dUTP by random primer extension; (2) pretreatment of slides; (3) prehybridizafion and hybridization; (4)immunological detection, p53 labelled with 35S was used as a control. The results showed that expression of p53 and c-H-ras was high in 6 out of 12 cases and 8 out of 12, respectively. For v-myb high expression was only in 2 out of 12. There was no expression of p53 in typical carcinoid. Expression of both p53 and c-H-ras in atypical carcinoid and small cell lung carcinoma seemed to play a cooperative role in malignant transformation process of neuroendocrine carcinomas. So, digoxigenin labelling is as sensitive as that of isotope, and it is more convenient to operate and gives no contamination.

PI3.10 A new Option for Chemical Labelling of Oligonucleotide Probes with Biotin Moities for In Situ and Dot Hybridization. V. V. Samoshin, A. A. Manykin and S. M. Klimenko The D. L Ivanovski Institute of Virology, Rus. Acad. Med. Sci., Moscow, Russia. We propose a new convenient method for introducing single or multiple biotin residues into hybridization probes. The main principle of the method is a combination of two chemical reactions allowing to form internucleotide bonds of different stability during a solid phase H-phosphonate oligonucleotide synthesis [Samoshin, V. V. et al. (1991) Bioorg, Khimia (USSR). V. 17. Pp. 241 - 245]. In particular, pltosphoramidate and phosphoralkoxy bonds were utilized. The former to introduce alkylamino groups and the latter to protect P-H bonds from undesirable amidation. This novel approach (patent pending) avoids the main problem of a "direct" solid phase biatinylation of oligonucleotide probes, namely, a tedious and time consuming preparation and purification of biotin-containing synthones. No special syntheses of additional building blocks are required. Biotin can now be incorporated at any position in oligonucleotide during the machine synthesis. A series of oligonucleotide probes labelled with one, two or four biotin residues were synthesized and used in light and electron microscopy in situ hybridization when investigating virus Sindbis infection. Sensitivity of the probes slightly depends on a number of biotin residues (data not shown).

P13.11

Y.Chromosome Painting by In Situ Suppression Hybridization Using Microdissected Probe in Pregnant Women.

557 probe sets such as complete DNA library derived from sorted human chromosome or microdissected probe. Recently there have been some trials where fetal cells are separated from mother's blood by noninvasive technique for some prenatal diagnosis. But there are variabilities about the proportion of the fetal cells depending on the methods and techniques. We have used fluorescence in situ hybridization to determine the proportion of foetal cells in pregnant women bearing the male fetus. Positive cells were found in all 15 women who had the male fetus. The proportion was one in 20000 to 160000 cells. There are trend to increase of fetal cells as the weeks of pregnancy increase. The 5 women bearing the female fetus showed no positive cells. Normal male also disclosed no positive cells. Furthermore it might be useful as a noninvasive technique of cytogenetic analysis of fetus in addition to sex chromosomal distinction.

P13.12 The Ultrastructnrai In Situ Hybridization: Comparison of Three Methods Using Non Radioactive Probe. D. Le Guellec 1, A. Trembleau 2 and G. MoreP ICNRS UPR 412, Univ. Lyon L 69622 Villeurbanne, 2CNRS U 1199, Univ. Curie, 75252 Paris and 3CNRS U 1454 Univ. Lyon sud, 69600 Oullins, France.

The aim of this work is to compare GH mRNA localization at the ultrastructural level using three different methods: (1) on vibratome sections before epoxy resin embedding, (2) on ultrathin frozen sections and (3) on ultrathin sections of tissue embedded in Lowicryl resin. We have compared the sensitivity, the resolution and the ultrastructural tissue preservation of the three methods. The probe was a 30 mer oligoprobe labelled at the 3' end using biotin-21-dUTP. All these methods gave similar results, mRNA were located on lamellar endoplasmic reticulum of Somatotrophs. Several signal detection systems have been tested (enzyme and gold particles). The best sensitivity was obtained when biotin was detected by a rabbit anti-biotin serum revealed by anti-rabbit IgG conjugated with gold particles (5 to 15 nm) that gave the highest resolution. The pre-embedding method gave the best ultrastructural preservation with a low resolution with the enzymatic detection system (gold particles cannot be used without pretreatment that impair the ultrastructure preservation) and an intermediate sensitivity. The frozen sections method gave the best sensitivity but the lowest ultrastructural preservation. On Lowicryl ultrathin sections, ultrastructural preservation was intermediate and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.

P13.13 I n S~tu Hybridization and Electron Microscopy.

S. H. Koo, C. B. Lim and J. W. Park Dept. of Clinical Pathology, Chungnam Univ., Daejon, Korea.

M. V. E. Macvilte, A. G. M. van Dorp, J. A. M. Fransen and A. K. Raap Dpts. Cytoehemistry & Cytometry, and Electron Microscopy, Leiden University, The Netherlands.

Recently chromosome in situ suppression hybridization methods have been developed that allow the use of complex

In EM-ISH a workable balance between EM morphology and ISH signals must be found, because measures to increase

558 penetration of probe and antibody are contra-indicated for preservation of morphology. In various cell lines we have used rRNA and specific mRNAs as targets. DNA probes were labelled with digoxigenin and hybrids were detected by antidig-HRP/DAB reaction or anti-dig-1 nm gold/silver staining. In chess-board experiments we used fixatives based on formaldehyde and/or glutaraldehyde and various pepsin concentrations and incubation times for permeabilization. In pre-sectioning EM-ISH, DAB stained cells are monitored by reflection-contrast microscopy (RCM) before embedding for rapid screening on the effects of fixative and pepsin. Ultrathin cross-sections showed full penetration of DAB ISH-signals in RCM when hybridized according to a LM protocol, whereas an EM protocol showed only poor penetration, however EM examining showed unacceptable morphology. Although in post-sectioning EM-ISH, intracellular targets are easier to access, mild pepsin treatment increased ISH-signals for rRNA, and was even crucial for mRNA. Thus, EM-ISH is feasible in a post-sectioning approach under narrowly defined conditions.

Poster sessions techniques. Monoclonal antibodies were used to detect the receptor protein while specific oligonucleotide probes were used to detect the RNA of these receptor types. The probes were labelled with alkaline phosphatase and the signals, on the histological sections, were detected by using Vector Red or Vector Blue. The stained sections were scored from 0 - 3 for intensity and distribution of staining. The receptor proteins for ER, PgR and EGFR were detected on 66%, 49% and 36% of specimens respectively. In situ hybridization detected the RNA for these receptors in 51%, 68% and 72%. The failure to detect RNA for ER when the protein product was demonstrated may be due to significant loss of RNA in the paraffin embedded tumour samples. In the other cases, the finding of increased levels of RNA when compared to the protein product may be due to a reduced translation of the messages. The use of two different techniques to detect receptor proteins and nucleic acids will further our understanding of the interrelationship between the different receptors and will give valuable information concerning the control of growth of breast cancer cells.

P13.14 In $itu Hybridization of Human Chromosomes

Centrifuged on Glass.Slides. G. Mazzotti, G. Lattanzi, M. Falconi*, S. Santi#, E. Rizzi and N. M. Maraldi* Istituto di Anatomia Umana Normale Universita "di Bologna; *Istituto di Citomorfologia Normale e Patologica del C.N.R. c/o IOR Bologna; #Laboratorio Biologia Cellulare e M.E. fOR Bologna, Italy.

Human metaphase chromosomes obtained by citric acid isolation method and centrifuged on glass-slides were used for in situ hybridization. For this purpose HeLa cells were harvested by mitotic shake off after 2 hrs exposure to colcemid. Several fixatives and DNA denaturation methods were tested. Biotinylated human centromeric DNA probes were used for in situ hybridization. The reaction was visualized by immunofluorescence using fluoresceinisothiocyanate (FITC)-labelled avidin and DNA was counterstained with propidium iodide. The morphology of the chromosomes, as judged by observation under both light and confocal microscope, is better preserved after 2% paraformaldehyde fixation followed by denaturation at 70~ with formamide. A high chromosome yield in a very small area together with a good resolution of fluorescent spots has been obtained. We suggest that this procedure may have useful applications for high resolution mapping of in situ hybridization probes by field emission scanning electron microscopy. P13.15

Detection of Epidermal Growth Factor Receptor, Estrogen Receptor and Progesterone Receptor in Breast Cancers by Immunocytochemistry and In Situ Hybridization. J. S. Milliken, J.-M. Mathijs* and A. M. Bilous Department of Anatomical Pathology and Virology*, Westmead Hospital, Westmead N S W 2145, Australia.

47 frozen and paraffin embedded samples of breast carcinoma were processed to detect the presence of EGFR, ER and PgR using immunocytochemistry and in situ hybridization

P13.16 Use of Silver Enhancement in the Immunoperoxidase Detection of DNA and RNA Sequences In Situ and its Applications in Double Immuno/Hybridocytoehemistry. H. Mullink ~, N. M. Jiwa t, W. Vos ~, A. Horstman ~, J. M.

M. Walboomers ~and C. J. L. M. Meijer ~ Institute of Pathology, Free University Hospital, De Boelelaan 1117, 1081 HV, Amsterdam, The Netherlands.

In our search for sensitive, yet simple methods for the lightmicroscopical detection of viral antigens and/or nucleic acid sequences in situ, we found silver intensification of the DAB/ Nickel endproduct of the peroxidation reaction according to the Merchentaler variant of the Gallyas method (J. Histoch. Cytoch. 37: 1563; 1989) to be optimal (Mullink et al. J. Histoch. Cytoch. 40: 495; 1992). An adaption of this method was found to be very satisfactory in the hybridocytochemical detection of low abundant DNA sequences in routinely processed pathological tissue (e.g. HPV and EBV in resp. CIN lesions and (non)hodgkin lymphomas). A very low noise-tosignal ratio could be obtained. Another application was found for the demonstration of specific RNA sequences using RNA in situ hybridization (RISH) with biotinylated and digoxigenin-labelled RNA probes. Comparison with immunogold/silver methods generally turned out to be in favour of the DAB/Ni/Ag staining. Subsequently these DNA and RNA detection methods were tested for their applicability for double staining in combination with immunohistochemistry (IH). For the combination of DISH (using DAB/Ni/Ag detection) with immunohistochemistry best results were obtained when IH was performed first, using standard DAB/H202 immunostaining followed by DISH. Combination of RISH with IH, on the contrary, proved to be optimal when RISH preceded IH. Best results were obtained when the silver intensification step of the RNA detection was performed after the standard DAB/H2Oz staining for IH. In this way, cells expressing viral genes in Hodgkin lymphomas could be characterized immuno-

P o s t e r sessions phenotypically very well. Further details of the technique will be given.

P13.17

Resistance to Methotrexate and Dihydrofolate Reductase Gene Amplification in Patients with Acute Leukemia. R. Nano, G. Gerzeli, R. Invernizzi* and C. Morandi** Dept. of Animal Biology, University of Pavia and Center for the Study of Histochemistry, C.N.R., Pavia; *Dept. of Internal Medicine, Section o f Second Clinical Medicine, University of Pavia; **Institute of Biological Sciences, University of Verona, Italy. Methotrexate is the most widely studied drug used in cancer chemotherapy. The primary site of action of MTX is dihydrofolate reductase (DHFR; EC 1.5.1.3) enzyme involved in nucleic acid metabolism and thus in proliferation and differentiation processes. With regards to leukemia, cells can develop resistance to MTX. The cytochemical distribution of DHFR in peripheral and bone marrow blasts from cases of acute leukemia, part at onset, part in relapse was studied. In addition the number of copies of the DHFR gene were analysed by in situ hybridization in the same cases compared to normal granulocytes and MTX-resistant HeLa cells (carrying DHFR gene amplification) used as negative and positive controls respectively. The results indicate heterogeneous hybridization patterns at the single cell level A M L showed an increased DHFR activity due to gene amplification, while some cases of ALL at onset showed amplification without overexpression of the enzyme; on the contrary in relapse. Therefore it can be supposed that the amplification is not the only mechanism of resistance to MTX.

P13.18 Digoxigenin as a Probe Label for In Situ Hybridization on Oral Verruca Vulgaris G. Premoli-De-Percoco, I. Galindo, J. L. Ramirez and M. Perrone Universidad Centrai de Venezuela, Apartado 51513, Caracas 1050, Venezuela. A sensitive in situ hybridization test under low stringency condition (LSC) with a mixture of digoxigenin labelled human papillomavirus mixed probes (D-L HPV MP), revealed a positive reaction in 8 of 10 cases of oral verruca vulgaris (OVV). Ages ranged from 5 to 37 years with a mean of 14.5 of these patients. 4007o of all cases were located intraorally on hard palate followed by the commissures. These preliminary findings provide further evidence of the role of HPV in OVV in the Venezuelan population. We show that in situ hybridization conducted under LSC is useful in HPV detection (regardless of the type) and the digoxigenin-labelling system is a rapid, relatively easy and specific method. Also, this technique permits the retrospective evaluation of routinely processed material thus widening the investigative spectrum for HPV.

559

P13.19 Structural Chromosome 1 Aberrations in Transitional Cell Carcinoma of the Bladder. Interphase Cytogenetics Combining a Centromeric, Telomeric and Library DNA Probe. P. J. Poddighe, F. C. S. Ramaekers, A. W. G. B. Smeets, G. P. Vooijs and A. H. N. Hopman Dept. Molecular Cell Biology & Genetics, Maastricht; Dept. Pathology, University Hospital Nijmegen, The Netherlands. Fluorescence in situ hybridization (FISH) was used to study numerical and structural chromosome 1 aberrations in interphase nuclei of transitional cell carcinomas (TCCs) of the urinary bladder. One of the characteristic numerical aberrations as detected previously in low-grade noninvasive TCCs, included trisomy for chromosome 1 (Hopman et al., Cancer Res. 51, 644; 1991). We examined in more detail 22 cases with a centromeric- (lq12) and a telomeric-associated (lp36) DNA probe, and with a library DNA probe from sorted human chromosome I in single- and double-target FISH procedures. All flow cytometrically determined DNA diploid TCCs (13 cases), which showed three spots for lq12 (six cases), had two spots for lp36. Since the library DNA probe showed three separate domains in the nuclei of these cases, the additional copy for lq12 could be explained as an extra chromosome lp-, containing the l q t 2 target. This means that in case of a trisomy for chromosome 1, which occurred at a frequency of about 20~ in low grade, noninvasive TCCs, 1p36 was specifically deleted. In the flow cytometrically DNA tetraploid/aneuploid tumors the results were more complex. In 6 out of 9 cases we observed an overrepresentation of lq12 as compared to lp36, also suggesting the presence of extra copies of lp- chromosomes. The present study demonstrates the utility of the FISH method to assess structural chromosome aberrations in interphase nuclei of solid tumors.

P13.20

A Method to Determine the Origin of Hemopoietic Progenitor Cells by Interphase Cytogenetics. P. J. Poddighe, N. van der Lely, G. P. Vooijs, T. De Witte, F. C. S. Ramaekers and A. H. N. Hopman Dept. Molecular Cell Biology & Genetics, Maastricht; Depts. Pathology & Hematology, University Hospitat Nijmegen, The Netherlands. We developed a method to determine the origin of progenitor cells in agar cultures by in situ hybridization (ISH). For this purpose bone marrow samples of three patients suffering from acute myeloid leukemia (AML) were hybridized with centromeric-associated DNA probes specific for chromosomes 1 and 8. A numerical chromosome aberration, for one or both target chromosomes, as detected by karyotyping and ISH in the three bone marrow samples, was used as a marker. After applying the ISH technique on "the cells of the same samples growing in agar, the method enabled us to discriminate between the clonogenic and non-clonogenic cells in culture. Using this approach, it is possible to detect numerical chromosome aberrations in interphase nuclei in order to characterize progenitor cells in case of hemopoietic malignancies.

560

P13.21 Comparison of Interphase Cytogenetics on Frozen Tissue Sections and Southern Blot Analysis of Transitional Carcinoma of the Bladder. P. J. Poddighe, P. P. Bringuler, J. A. Schalken, F. C. S. Ramaekers and A. H. N. Hopman Dept. Molecular Cell Biology & Genetics, Maastricht; Depts. Pathology & Urology, University Hospital Nijmegen, The Netherlands. The purpose of this study was to assess the correlation between RFLP analysis and Interphase cytogenetics on transitional cell carcinomas of the urinary bladder. The results of RFLP analysis were compared to chromosome aneuploidy, numerical chromosome aberrations, imbalance between chromosomes, and heterogeneity of those aberrations within the tumor. We performed Southern blot analysis on DNA which was isolated from frozen tissue samples, containing more than 80% tumor ceils. We screened for loss of heterozygosity for chromosome loci 9q34, llp15, 16q22-24, 17p13 and 18q21. The data from the informative cases were compared with the interphase cytogenetics data on both frozen tissue sections and isolated nuclei of the same tumor, using centromericassociated DNA probes for the same chromosomes. We will discuss the usefulness of combining RFLP and interphase cytogenetic analysis for the detection of genetic markers associated with tumor progression.

P13.22

Rapid and Sensitive Detection of Human Papilloma Virus DNA in Oral Mucosal Lesions by In Sitn CytoHybridization with Digoxigenin-Labelled Probes. G. Premoli-De-Percoco, I. Galindo and J. L. Ramirez Universidad Central de Venezuela, Apartado 51513, Caracas 1050, Venezuela. The presence of human papilloma virus (HPV) nucleotide sequences in paraffin sections of oral biopsies were examined by in situ cyto-hybridization using non-isotopic, digoxigeninlabelled DNA probes representing HPV types 6, 11, 16 and 18. The hybrids were detected by using anti-digoxigenin alkaline phosphatase conjugate and visualized with an enzymecatalyzed color reaction. The in situ cyto-hybridization protocol was adapted in such a manner that it could be accomplished in 24 hours (or less). Also the modification introduced in the protocol enabled a non-specially trained technician to handle easily 40 specimen slides. The results indicated that this method was specific, simple and sensitive enough to be used for retrospective and routine screenings of HPV-suspect oral lesions, without radioactive hazard or the assistance of highly trained personnel. In addition, the test allows the study of the tissue morphology, along with the precise location of the viral particle, without background staining. Also, the modification introduced in the protocol enabled a non-specially trained technician to handle easily 40 specimen slides.

Poster sessions P13.23 In $itn Hybridization Based on Reflection of

Different Enzyme Cytochemical Reaction Products. E. J. M. Speet, M. Kamps, J. Bonnet*, F. C. S. Ramaekers and A. H. N. Hopman Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands and *Department of Cytochemistry and Cytometry, University of Leiden, Leiden, The Netherlands. So far only the polymerized DAB reaction product of the cytochemical peroxidase reaction has been used to detect in situ hybridized DNA probes with high sensitivity by means of reflection contrast microscopy (RCM). For clear analysis of the biological specimens oil (with a refractive index equal to glass) has to be applied between the lens and the slide to prevent reflection at the glass-air interphase. The use of oil however hampered the detection of several other enzyme cytochemical reaction products by RCM, due to their solubility in organic solutions. By fixation of these enzyme precipitates in an artificial matrix we were able to prevent the reaction products from being solved in oil. Therefore these enzyme precipitates could be analyzed by RCM, resulting in the detection of different reflection colors. This finding has been applied to single as well as double-target in situ hybridization (ISH) on both interphase and metaphase preparations to detect: a. a repetitive DNA probe for the centromere of chromosome I with several enzyme precipitates that reflect in different colors, b. two different centromerespecific DNA probes with two different reflecting enzyme reaction products, c. a cosmid DNA probe to demonstrate the sensitivity of the presented procedure. One may conclude that these results make RCM an attractive method to use for the visualization of different enzyme cytochemical reaction products in biological specimens, as demonstrated by the ISH experiments.

P13.24 In Situ Localization of Pepsinogen mRNA in Normal

Guinea Pig's Gastric Mucosa Using Thymine. Thymine Dimerized Probes. S. Tsukada ~, M. Ichinose ~, K. Miki ~, S. Ishihama ~, M. Matsushima ~, N. Kakei ~, N. Yahagi j, K. Kurokawa ~, H. Fukamachi 2, M. Nozawa 3, S. Izumi 3 and P. K. Nakane 3 ~lst Department o f Internal Medicine, Faculty of Medicine, University of Tokyo; 2Laboratory of Molecular Embryology, Zoological Institute Faculty of Science, University o f Tokyo; JDepartment of Anatomy, Nagasaki University School of Medicine. (Purpose) Pepsinogens are the precursor forms of Pepsins, typical aspartic proteinases normally present in the gastric mucosa of vertebrates. They are secreted into the gastric lumen and activated autocatlytically to Pepsins under acidic conditions. In order to understand the Pepsinogen gene regulation at the cellular level, we analysed the distribution of its mRNA in the stomach mucosa using in situ hybridization technique. (Method) The stomach from guinea pig was used for the analysis. In situ localization of Pepsinogen mRNA was analysed by T-T dimer method (P. K. Nakane et al.) Three synthetic oligo-DNA's complementary to the different regions of the guinea pig Pepsinogen mRNA were used as probes. The

Poster sessions serial sections of the same samples were analysed immunohistochemically using the anti-guinea pig Pepsinogen antibody. (Results) In situ localization of Pepsinogen mRNA were detected in the gastric mucosa. The specific staining of Pepsinogen mRNA was observed only in the immunohistochemically Pepsinogen-positive cells, especially in chief cells. These results strongly suggest that Pepsinogen production is regulated at the level of transcription and cell-specific manner.

561 rhodamine derivates). Chromosome-detection kits have been successfully used in practice of cytogenetic laboratories in Russia for diagnosis of common chromosomal syndromes (Down, Edvards, Klinefelter, Terrier, tryplo-X syndromes, polysomy X and Y, addition marker chromosomes) as well as for routine chromosome identification in post and prenatal diagnosis.

P13.27 P13.25

"Fluorescence-Immunopbenotyping and Interphase Cytogenetics as a T o o l for Investigation of Neoplasms ( F I C T I O N ) " . K. Weber-Matthiesen, M. Winkemann, J. Deerberg,

B. Schlegelberger and W. Grote Dept. of Human Genetics, University of Kiel, Germany.

In immunocytochemical studies the phenotypical evaluation of tumor cells is often complicated by accompanying normal cells, representing the original tissue or infiltrating leukocytes. This holds particularly true for tissues with a great morphological and immunophenotypicai variability, such as bone marrow. Similar difficulties are given in cases with very low percentages of tumor cells. In addition, cytogenetically distinct tumor clones with a similar immunophenotype, as described in peripheral T cell lymphomas, cannot be differentiated within immunohistochemical studies. We demonstrate three color "Fluorescence-immunophenotying and Interphase Cytogenetics as a Tool for Investigation of Neoplasms (FICTION)". The method combines fluorescence immunophenotyping and in situ hybridization with two different centromere specific probes. Using FICTION, chromosomally aberrant tumor cells can be identified by interphase cytogenetics and simultaneously characterized immunophenotypically. Since every individual interphase cell can be analysed, we are not limited by number and banding quality of mitotic cells.

Expression of Angiogenic Factors in Xenografts of Human Melanoma Cell Lines. M. Denijn, R. van 't Hullenaar, G. N. van Muijen,

R. M. de Waal, P. C. de Wilde and D. J. Ruiter Dept. of Pathology, University Hospital Nijmegen, The Netherlands.

Angiogenesis is crucial for tumor growth and progression. In primary cutaneous melanoma and in xenografts of human melanoma cell lines in nude mice, the degree of vascularization is related to the occurrence of metastasis. This finding suggests that tumors with metastatic potential induce a high degree and a particular type of vascularization. In this study, the expression of several angiogenic factors in xenografts of 6 human melanoma cell lines with a different degree of metastatic potential is investigated. The expression of basic FGF, TNF alpha, TGR alpha and beta, and angiogenin is studied by means of RNA in situ hybridization, and the expression of the angiogenic factors is compared to the quality and the quantity of the microvasculature and the variable degree of metastatic potential of the xenografts.

P13.28 DNA-In Situ Hybridization and DNA-Cytometry of the Same Nuclei. P. E. J. de Wit, H. M. J. Kerstens, A. G. J. M.

Hanselaar, P. C. M. de Wilde, G. P. Vooijs and D. J. Ruiter Dept. of Pathology, University Hospital, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

P13.26 Elaboration of Chromosome-Detection Kits Based on Non-Isotopic In Situ Hybridization with Chromosome-Specific DNA Probes. Y. Yurov ~, E. Rogaev ~'3, I. Ovchinnikov 2'3, S. Vorsanova ~ and I. Lebedeva 4 tNational Research Centre of Mental Health, Moscow, Russia," 2VNII Genetika, Moscow, Russia; JResearch Diagnostic Centre "Genotype S'" (Kristina Group Ltd.), Moscow, Russia; ~Moscow State University, Russia. Non-isotopic in situ hybridization provides a modern

approach to identification of human chromosomes in many areas of biomedicine, including genetics, cytology, oneology, obstetrics, hematology. We had developed the chromosome detection kits, based on original collection of alpha- and "classical" DNA probes for many human chromosomes, including 1, 3, 4, 5 and 19, 6, 7, 8, 9, 10, 11, 13 and 21, 14 and 22, 15, 16, 17, 18, X, Y (Yurov et al,, 1989, Cytogen Cell Genet. 46, 1 - 4 , 1114). Visualization of biotinylated probes after in situ hybridization was realized by enzymatic or fluorescent methods (including two colors fluorescein and

We investigated whether it is possible to apply the DNA in situ hybridization (ISH) technique and DNA cytometry to the same nuclei. This in order to be able to study specific DNA targets in a nucleus in correlation with its total DNA content. We used lymphocyte nuclei from disaggregated formalin fixed paraffin embedded blocks. The ISH procedure (chromosome 1 centromere regions visualized as DAB spots) preceded the Feulgen staining with Schiffs reagens. Positive Feulgen staining in combination wth DAB-ISH spots was observed in nearly all lymphocyte nuclei, which were well recognizable. Measurements showed that the intensity of the Feulgen staining decreased when it was preceded by the ISH procedure. Measurement was possible in nearly all nuclei. The countability and the actual number of DAt3kISH spots in the lymphocyte nuclei was not influenced by the subsequent Feulgen procedure, although the quality was less. DAB-ISH spots could be counted in the same nucleus of which the total DNA content was measured, and the data stored per nucleus. We conclude that, compromising between the two techniques, it seems possible to study specific DNA targets in a nucleus in correlation with data informative for its total DNA content.

562 P13.29

A New Option for Chemical Labelling of Oligonucleotide Probes with Biotin Moities for In Situ and Dot Hybridization. V. V. Samoshin, A. A. Manykin and S. M. Klimenko The D. L Ivanovski Institute of Virology, Rus. Acad. Med. Sci., Russia. We propose a new convenient method for introducing a single or multiple biotin residues into hybridization probes. The main principle of the method is a combination of two chemical reactions allowing to form an internucleotide bonds of different stability during a solid phase H-phosphonate oligonucleotide synthesis [Samoshin, V. V. et al. (1991). Bioorg. Khimia (USSR). V. 17. Pp. 241 -245]. In particular, phosphoramidate and phosphoralkoxy bonds were utilized. The former to introduce an alkylamino groups and the latter to protect a P-H bonds from undesirable amidation. This novel approach (patent pending) avoids the main problem of a "direct" solid phase biotinylation of oligonucleotide probes, namely, a tedious and time consuming preparation and purification of biotin-containing synthones. No special syntheses of additional building blocks are required. Biotin can now be incorporated at any position in oligonucteotide during the machine synthesis. A series of oligonucleotide probes labelled with one, two or four biotin residues were synthesized and used in light and electron microscopy in situ hybridization when investigating virus Sindbis infection. Sensitivity of the probes slightly depends on a number of biotin residues (data not shown).

P13.30

Application of the Alkaline Phosphatase-Fast Red Reaction to Fluorescence In Situ Hybridization: High Sensitivity and Suitable Bleaching Properties. E. J. M. Speef, B. Schutte ~, J. Wiegant 2, F. C. S. Ramaeker# and A. H. N. Hopman' 1Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands, and 2Departrnent of Cytochemistry and Cytometry, University of Leiden, Leiden, The Netherlands.

In spite of the high sensitivity that can be achieved with the fluorescence in situ hybridization (ISH) method, fading of the signals hampers multiple analyses of biological specimens. We used Naphtol-ASMX-phosphate and Fast Red TR in combination with alkaline phosphatase (APase) to produce fluorescent precipitated reaction products in ISH. For optimal and discrete localization of the strongly red fluorescent signals we optimized the enzyme precipitation reaction conditions, such as reaction time, the concentrations of substrate and

Poster sessions capture agent as welt as the viscosity of the reaction mixture. Our results show that the APase-Fast Red TR detection method has at least the same sensitivity as currently used conventional immunofluorescent detection systems. Single copy DNA sequences could be localized with high efficiency in metaphase spreads as well as in interphase nuclei. Multilabelling procedures, combining FITC and azo-dye fluorescence, become feasible with this technique. The red fluorescent ISH signals showed minimal fading as compared to FITC fluorescence upon exposure to either mercury arc or laser light. The permanent character of the signals allows a better analysis and three-dimensional localization of cell components, detected after ISH or immunocytochemistry by means of confocal scanning laser microscopy.

P13.31

Analysis of Dislocated, Kinetochore-lacking Chromosomes in Tumor Cells by In Situ Hybridization with Centromeric Probes. D. Schiffmann, S. Kirchner and B. K. Vig t Institute of Toxicology, W-8700 Wfirzburg, Germany; ~Dept. of Biology, University of Nevada Reno, USA. Some chromosomes in transformed rat cells and somatic cell hybrids fail to display kinetochores as detected by antikinetochore antibody (anti-CENP-A, -B, -C, -D). Such chromosomes (K--chromosomes) lack connection to the spindle apparatus and therefore fail to migrate normally in mitosis. Thus, K--chromosomes may constitute a novel mechanism for the genesis of aneuploidy and the loss of tumor suppressor genes. We have studied diploid primary Syrian hamster embryo (SHE) flbroblasts in comparison with malignant human (HeLa) and mouse (MBT) tumor cells. K-chromosomes were observed in 2.5070 of SHE cells, in 5070 of HeLa and in 10% of MBT cells along with our previous data (Vig et al., Mutagenesis 6, 361-367, 1991) these results suggest a correlation between the frequency of K-chromosomes and known variability in chromosome number in these tumor cells. In order to analyse whether the loss of kinetochores was due to a loss of centromeric sequences, we carried out in situ hybridization with minor satellite DNA for MBT cells and alpha sequences for HeLa cells (the minor satellite sequence is located at the lateral surface of the primary constriction of the chromosome; the used alpha sequences were of pericentric orign). We observed hybridization signals in the absence of kinetochores. Furthermore the frequency of these signals exceeded the frequency of kinetochore-lacking chromosomes. These results show that toss of kinetochores is not necessarily due to a loss of the centromeric DNA.

P o s t e r sessions

563

P14. H I S T O C H E M I S T R Y OF T H E E X T R A C E L L U L A R M A T R I X P14.1

P14.3

Phenotypic Modulation and Elastin Synthesis of Cultured Aortic Smooth Muscle Cells (SMCs) Grown on Collagen Gels.

Phospholipids in Mineralized Tissues Visualized with MGA and/or PLAz-Gold Complexes.

M. Akita, E. Murata, K. Fujita and K. Kaneko Dep. of Anatomy, Saitama Medical School, Saitama 350-04, Japan. Collagen gels provide a more physiological, isotropic environment that has been shown to promote the organization of different cell types into three-dimensional, tissue-like structures. The substrate upon which the cells rest can influence the shape of cells and extracellular matrix-synthesis. In the present study, we examined the phenotypic modulation and elastin synthesis of SMCs grown on collagen gels. The cells grown on collagen gels showed a multilayered growth with nodules formation and exhibited a contractile state having numerous microfilament bundles with dense bodies. In contrast, the cells grown within collagen gels did not form the nodules observed on the collagen gels, and the phenotypic property showed a synthetic state having welldeveloped rough ER and Golgi complex, but few microfilament bundles. Floating gels accelerated elastin synthesis. These findings suggest that the physical properties of the substrate relate to the cell differentiation and synthesis of extracellular matrix components.

P14.2

Immunolocalization of an Amyloid Precursor (SAA) in Aids Brain Cortex. M. P. Beltrano ~ F. M. Albedi ~ D. Taruscio ~ G. Costanzi*, L. Vago* and G. Donelli ~ ~ Superiore di Sanitd', Roma e *V Cattedra di Anatomia Patologica dell" Universit~t, Milano, Italia.

In AIDS patients increased levels of circulating A amyloid precursor (SAA) have been observed. To verify if this protein is present also in tissues, we studied the immunocytochemical localization of SAA in brain cortical autoptic samples from 16 AIDS patients, both at light and electron microscope level, by the P A P technique. G F A P for astrocytes and collagen VI as an extracellular matrix component were also evaluated in the same samples on paraffin sections. In nervous tissue the presence of SAA were diffusely detected. Ultrastructural immunohistochemistry showed a positivity for SAA in the vasal lumen and endothelial cells, inside intracytoplasmic vesicles. The basal membrane was negative; on its outer side small peroxidase-labelled areas, which correspond to astrocyte foot processes, were observed. When compared to control tissue, collagen VI showed an anomalous positivity both in the basal membrane and in the perivasal nervous tissue. These results c o n f r m that brain capillary wall is permeated by SAA without contemporaneous occurrence of perivasal amyloid fibrillogenesis. The abnormal deposition of collagen VI may represent a tissue response to the presence of amyloid. These observations emphasize that the capillary wall and, more in general, the blood brain barrier is significantly affected in neuro-AIDS as corresponding to one of the targets in the pathogenetic mechanism of CNS damage by HIV.

M. Goldberg ~, S. Lecolle ~, D. Septier 1, J. R. Nefussi 2 and H. Chardin ~ ~Fac Chir Dent, Paris V; 2Paris VlI-France.

Most lipids are lost during conventional processing for electron microscopy. Fixation with aldehyde in presence of malachite green (MGA) reduces the loss of lipids and allows proper visualization of phospholipids after OsO4 postfixation. Using this procedure, electron-dense rod-like structures, radiating filaments a n d / o r dots were observed in the predentine and osteoide, located in the spaces between the collagen fibres. In dentine or in bone, alignments of particles or neddle-shaped structures were seen along individual or bundles of collagen fibres, in the form of crystal ghosts. In the forming enamel, visualization of M G A electron-dense material was more questionable. The same distribution was observed when PLA2- gold complexes were used. Gold particles were seen along ribbonlike structures in forming enamel. Comparison was carried out between (1) aldehyde fixed- Lowicryl embedded sections, (2) glutaraldehyde/paraformaldehyde -OsO4 fixed-Epon embedded sections and (3) MGA-OsO4 fixed-Epon embedded sections evidenced. The highest number of gold particles was scored in group 1. One third less particles was scored in group 2. In both cases, pre-treatment of the sections with free PLA2 firmly reduced the labelling. Group 3 gave poor and uncertain results. These investigations i) confirmed the existence of phospholipids in mineralized tissues, ii) indicated substantial changes related to phospholipid distribution between the unmineralized and mineralized tissues. In conclusion, these investigations support that phospholipids play role in mineralization. P14.4

Analysis of Tiger Stripe Pattern in Germ Cell Tumors of the Human Testis. J. S. Groenewoud, B. Timmer, L. H. J. Looijenga and W. J. Oosterhuis Dr. Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.

A remarkably consistent morphologic feature of imprints of seminomas is a striated background of amorphous, eosinophilic material, known as "tiger stripe pattern". We have investigated the specificity of this tiger stripe pattern for seminomas and carcinoma in situ (CIS) in the context of testicular germ cell tumors. In addition we have tentatively characterized the material using immunohistochemistry. Material: 10 seminomas, 2 combined tumors and 10 nonseminomas (imprints, frozen sections, paraffin sections). Reagents: poly- and monoclonal antibodies against vitronectin, fibronectin, collagen I and IV, laminin, fibrogen, myosin and desmin. Results: In keeping with the literature tiger stripe pattern is consistently found in acetone fixed imprints of seminomas. It is rarely present in imprints of embryonal carcinoma. In other nonseminomatous tumor types it was not demonstrated.

564 Imprints of extensive CIS, both when adjacent to seminoma and nonseminoma, show characteristic tiger stripe pattern. Imprints of normal testis are negative. In ethanol (70~ v/v) fixed imprints the tiger stripe pattern is blurred. Frozen section when mounted on glass slides coated with tissue adhesive showed similar results. Tissue slits and artifactual cracks in the sections were filled with the tiger stripe pattern. Paraffin sections were always negative. Immunohistochemically the tiger stripe pattern was most often positive for vitronectin, both in imprints and in frozen sections. Largely negative was the staining with the other reagents. Regularly we found staining for vitronectin within seminiferous tubules with CIS. We conclude that a tiger stripe pattern is not only highly characteristic and specific for imprints of seminoma, but also for CIS. Vitronectin seems to be an important component of the material. The intriguing observation that seminiferous tubules with CIS contain vitronectin needs further investigation.

P14.5 Expression Patterns of Osteopontin in Developing Jaw Tissues of the Neonatal Rat. M. N. Helder, A. L. J. J. Bronckers and J. H. M. W61tgens Vrije Universiteit, Amsterdam, The Netherlands.

Molar tooth germs are used in our laboratory as a model to examine the role of organic matrix molecules in epitheliomesenchymal interactions. In the present study we focused on expression patterns of both type I collagen and osteopontin (OPN), which is a highly phosphorylated glycoprotein that is found in bone and possibly in (pre)dentin. Tissues studied were molars, alveolar bone and adhering soft tissues in upper jaws of 1 - 3 day old rats. Calvarial RNA extracts were used as a positive control. The expression patterns were determined by means of 1) immunohistochemistry, 2) Northern Blotting and 3) in situ hybridization. Type I collagen expression was found in osteoblasts adjacent to alveolar bone trabeculae and in odontoblasts and first subodontoblastic layers of pulpal cells, but not in the epithelial ameloblastic cells. In the case of OPN we were not able to detect any positive reaction in either the mesenchymal odontoblasts and pulpal cells or the epithelial ameloblastic layers. In contrast, osteoblasts of surrounding alveolar bone trabeculae showed strong positive signals for OPN with all three techniques, confirming the sensitivity and specificity of the detection methods. These results indicate that, although osteoblasts and molar tooth germ odontoblasts are both of mesenchymai origin and secrete a mineralizing connective tissue matrix, they differ in at least their OPN expression.

P14.6 Localization and Quantification by Image Analysis of Type II Collagen mRNA in the Rat Epiphyseai Cartilage. N. Balmain ~, D. Le Guellec2, D. Schoevaert 3 and D. Herbage 2 IInserm U120, Hdp. R. Debr(, 75019 Paris; 2Cytologie Mol(culaire, CNRS UPR 412, Univ. Lyon I, 69622 Villeurbanne; 3Mieroscopie quantitative, CHU Bic~tre, 94270 Kremlin-Bic#tre, France. Type II collagen mRNA was studied by in situ hybridization

P o s t e r sessions using 32p-labelled cDNA and synthetic oligonucleotide probes, on sections of rat epiphyseal cartilage. The Image Analysis System (Samba 2005 TITN Alcatel) was used to quantify the autoradiographs and to determine silver grain counts and the area analysed. Silver grains were localized on the cytoplasm of chondrocytes. All the chondrocytes of the entire epiphyseal cartilage had relatively high levels of type II collagen transcripts. The good correlation between silver grain counts and grain pixel areas in the chondrocytes of each zone showed that pixel area is a valid assay parameter. The transcripts varies throughout the epiphyseal cartilage, with the highest concentration in proliferative chondrocytes decreasing through the mature and hypertrophic chondrocytes until the calcifying zone. Chondrocytes of the calcified zone still containing significant amounts of type II mRNA. The results obtained with both the cDNA and the oligonucleotide probes were similar. We conclude that the distribution reflects the developmental stages of chondrocytes cells and that welldefined image analysis programs are rapid, precise tool for quantitative in situ hybridization studies in the epiphyseal cartilage. P14.7

Heterogeneous Expression of Dipeptidyl Peptidase IV on Human Breast Fibroblasts. A. J. Atherton, P. Monaghan, J. A. Kenny* and B. A. Gusterson Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, England; *Department of Biochemistry, University of Leeds, Leeds, LS2 9JT, UK. Dipeptidyl peptidase IV (DPP IV) is an integral membrane glycoprotein which acts to release dipeptides from the N-terminus of susceptible peptides. DPP IV may also interact with the extracellular matrix through binding to collagen or fibronectin. Immunohistochemical localisation of DPP IV in the normal human breast, revealed its presence on a subpopulation of fibroblasts. Positive staining was present on fibroblasts in the interlobular stroma but fibroblasts within intralobular stroma were negative. The venous capillary endothelial cells also expressed DPP IV. Ultrastructural localisation of DPP IV on both resin and cryo-sections demonstrated the presence of the enzyme on the surface membrane on interlobular fibroblasts. Within the endothelial cells there was positive staining both on the plasma membrane and within the cytoplasm. DPP IV positive fibroblasts were found both circumscribing and also within fibroadenomas where they were separated from the breast epithelium by DPP IV negative fibroblasts. In cytosarcoma phyUodes the stromal cells were uniformly negative. These results indicate that there are functional sub-populations of fibroblasts in the human breast and that future studies should be directed to additional phenotyping of these cells in the normal and diseased states.

P14.8 Expression of 3B3 Epitope in Loaded Articular Cartilage In Vitro. R. H. O'stendorf, G. P. J. VanKampen, R. J. VanDeStadt and J. K. VanDerKorst Jan van Breemen Instituut, 1056 AB Amsterdam, The Netherlands. The monoclonal antibody 3B3 recognizes unsaturated glucuronate residues adjacent to 6-sulphated Gal-NAc that are

Poster sessions left after digestion of chondroitin sulphate proteoglycans (PGs) with chondroitinase ABC. Without prior enzyme digestion 3B3 is also expressed in tissues undergoing rapid growth and development. In experimental osteoarthritis (OA) in dogs, induced by altered loading of the stifle joint, an increased expression of this antibody was reported. We studied the effect of loading on 3B3 expression in intact articular cartilage in vitro. Cartilage on sesamoid bones of 5 year-old cows, was loaded intermittently for 7 days. Controls were cultured without loading. During the last 17 hours of culture [35S]sulphate was added to the culture medium. Some sesamoid bones were labelled immediately after removal from the joint. After the experiment a slice of cartilage with underlying bone was sawn, fixed, decalcified and embedded in paraffin. Sections were used for autoradiography or immunolocalization with 3B3. Our results suggest that culturing increases the amount of 3B3-positive nonsynthesizing cells in the calcified layer. Loading of bovine sesamoid bones in vitro leads to expression of the 3B3-epitope around PG-synthesizing chondrocytes in the upper layers of the cartilage. A comparable distribution of the 3B3 epitope in the articular cartilage was recently reported in early experimental OA in dogs.

P14.9 Demonstration of Platelet-lnhibiting Activity within the Extracellular Matrix (ECM) in Glomeruli of the Rat Kidney. K. Poelstra, M. J. Hardonk, J. F. W. Bailer and

W. W. Bakker Department of Pathology, University of Groningen, The Netherlands.

Endothelial cells protect vessel walls from proaggregatory activity of subendothelial matrix constituents. However, within glomeruli the fenestrated endothelium may allow a more close contact between matrix and blood constituents. Previous ultra-structural studies suggest that ADPase activity is present in collagenous structures of the glomerular basement membrane of the rat kidney suggesting that the ECM can also inhibit platelets. To test this hypothesis, we studied the antithrombotic activity of glomerular ECM components using aggregometer assays. Suspensions of isolated rat glomeruli were incubated with 1000U/ml collagenase IV, to release ECM associated constituents. Supernatants were subsequently tested for platelet modulating activity with ADP (2.5/~M) as agonist. Histochemical experiments were performed to study the origin of the platelet modulating activity. Results show that supernatants of collagenase-treated glomeruli strongly inhibit ADP- induced aggregation. ADP-degrading activity can be detected in supernatants. Furthermore, cytochemical studies demonstrate that V205 is an inhibitor of glomerular ADPase and the antithrombotic activity is completely inhibited by VzOs. Therefore, the antithrombotic activity is mediated by ADPase. In vitro tests exclude the cytosol as source of this ADPase. Histochemical studies also exclude the possibility that ADPase activity in supernatants is released from plasma membranes. In line with these results is the histochemical demonstration of extracellular deposits of ADPase in cultures of glomerular epithelial and mesangial cells in which the enzyme is co-localized with collagen IV. It is concluded that

565 the ECM of rat glomeruli contains ADPase with strong potency to inhibit platelet aggregation. Therefore, the ECM not only activates platelets, but may also participate in the inhibition of thrombus formation.

P14.10 Age-Related Deposition of Amyioid P Component along Glomerular Basement Membranes of Human Kidney. I. S. Rantala Tampere University Hospital, Department of Pathology, POB 2000, SF-33521 Tampere, Finland.

Amyloid P Component (APC) is a normal matrix glycoprotein of adult human glomerular basement membranes (GBM). To study the age-related deposition of APC in kidney glomeruli, 25 renal biopsy specimens and 11 renal autopsy specimens were studied by irnmunofluorescence (IF) microscopy. Biopsy specimens included 7 cases with normal glomerutar morphology, ranging in age from 2 to 38 years, and 18 patients with various glomerulonephritides, ranging in age from 4 to 56 years. Autopsy specimens, all with normal glomerular morphology, ranged in age from 2 months to 28 years. In these specimens, APC was localized using cryostat sections and FITC conjugated anti-human APC antibodies. APC was not detected in glomeruli below 5 years of age. From 6 to 8 years, variability of IF intensity was seen. From the age of 9 years and upwards, clear IF along GBMs was invariably detected. By the age of 14 years, IF intensity reached that of the adults. Independently of glomerulonephritis, APC was present even if with varying intensity. Results indicate that the deposition of APC in kidney is age or maturity related as also described in skin and testis. In glomerulonephritis, abnormal patterns of deposition have been described. Possible pathogenetic significance remains, however, to be elucidated by future studies.

P14.11 Production of Single Domain Antibodies to N-CAM for Immunotargeting of Human Small Cell Lung Cancer Xenografts. R. Slobbe, O. Boerman, J. Broers, E. Mijnheere,

F. Ramaekers and G. van Eys Department of Molecular Cell Biology & Genetics, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands.

RNL-1, a mouse monoclonal antibody raised against the variant small cell lung cancer (SCLC) cell line NCI-H82 was characterized as a cluster-1 antibody recognizing an epitope of the neural cell adhesion molecule (N-CAM). Like other cluster-1 antibodies, RNL-1 reacts with SCLC tissue sections and lung carcinoids as well as with SCLC cell lines. In addition, reactivity was observed with normal neural tissue. As a first step towards application in immunodiagnosis and/or immunotherapy of small cell lung cancer, RNL-1 was labelled either with rain o r 125I and its biodistribution was examined in athymic mice carrying NCI-H82 xenografts. The maximum tumour accumulation was 11.8% injected dose per gram (% ID/g) for mIn-RNL-1 and 6.5% ID/g for ~25I-RNL-1. No accumulation of label was observed in

566 neuronal tissue of these mice, nor in neuronal tissue of rabbits injected with ~2SI-RNL-1. Single domain antibodies of RNL-1 have been produced with the objective to reduce the immunogenecity of the antibody, to increase tumour penetrating capacity and to improve the affinity of RNL-1 by further molecular manipulation. The variable regions of heavy and light chains of RNL-1 have been cloned, sequenced and expressed in a bacterial system. A rapid and specific purification of these proteins was achieved by using an N-terminal hexahistidine tag engineered into the expression vector. The ability of these single domain antibodies to recognize the antigen is currently under investigation. P14.12

Immunoelectron Microscopical Observation of Tracheobronchiai Amyloidosis. M . Toyoda, Y. Ebihara and S. Kita* Dep. Surg. Path. Tokyo Med. Colle. *Div. E. M. Tokyo Women's Med. Colle. 6 - 7 - 1 , Nishishinjuku, * 8 - 1 Kwadacho, Tokyo, Japan.

Since Lesser (1877) first described primary respiratory tract amytoidosis, many cases have been reported. This study was to identify the precursor protein of amyloidosis with immunogold electronmicroscopy (IEM). Biopsied specimens from tracheobronchiai amyloidosis (three cases) were studied. Its nature was confirmed by Congo red stain. No increase in plasma cells was seen in bone marrow or proximal to the amyloid deposition. Immunohistochemical examination was performed with anti-human amyloid A, amyloid P, IgG, IgA, IgM, kappa and lambda light chain (LC). Among these, tissues were fixed in 2~ glutaraldehyde 107~ osmium tetroxide and embedded in epon 812. These sections were pretreated with 4070 sodium metaperiodate and reacted with primary antibody, for which the prior antisera were reused. IEM demonstrated that amyloid fibril immunogold labelling density for either kappa or lambda (LC) significantly exceeded that of other primary antisera. Specificitical system. From these results, these cases were classified as AL-kappa (two cases) or AL-lambda (one case). P14.13

Invasion and Adhesion in Colonic Neoplasms. A . A . M . van der W u r f f , J. ten Kate, E. P. M. van der

Linden, W. N. M. Dinjens*, J.-W. Arends and

F. T. Bosman* Dept. of Pathology, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands; *Dept. of Pathology, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands.

L-CAM is a cell adhesion molecule, which is expressed at the

P o s t e r sessions intercellular borders of most epithelial cells. In carcinoma cell lines L-CAM has been demonstrated to act as an invasion suppressor. To determine whether or not L-CAM expression might distinguish between invasive and non-invasive or metastatic and non-metastatic colon neoplasms, we studied L-CAM expression in normal colon mucosa, colon adenomas with various degrees of dysplasia, colon carcinomas and their metastases in lymph node and liver by immunohistochemistry, using the 6F9 monoclonal anti L-CAM antibody. Normal mucosa showed evenly distributed distinct L-CAM immunoreactivity along intercellular borders. In adenomas as well as carcinomas a similar though weaker expression was observed. This pattern showed a trend to decrease in parallel with decreasing differentiation. No correlation, however, was found with Dukes stage or area within the turnout. In some carcinomas L-CAM was expressed at the luminal surface of the cells. In others invading cells were noticed which showed L-CAM expression. Some carcinomas did not have L-CAM immunoreactivity. Preliminary data suggest that L-CAM expression in metastases is similar to that in primary carcinomas. These results suggest that L-CAM expression is disregulated or lost as an early event in the development of colon neoplasia and indicate that L-CAM expression does not correlate with invasive or metastatic potential.

P14.14

Amyloidosis of the Ocular Fundic Tissues in Senile Maculodystrophy. V. V. Yermilov Volgograd Medical Institute, Chair o f Pathology, Russia.

Senile alteration of the ocular fundic tissues including senile maculodystrophies are widely prevalent diseases leading to a significant vision loss in both eyes and for that reason presenting a most important problem in modern ophthalmology. The role of senile amyloidosis in the pathogenesis of senile maculodystrophy has not been investigated yet. The aim of the present study was morphologic investigation of the ocular fundic tissues in senile maculodystrophy. 148 enucleated eyes from people deceased in homes for the elderly and psychiatric clinics, with in vivo ophthalmologic examination, were investigated. Histological, histochemical and selective tests for amyloid as well as tests with guanidine hydrochloride and potassium permanganate were used. Positive staining of several structural elements of the ocular fundic tissues for amyloid with alkaline Congo red, methyl violet and in a luminescent histochemical test with thioflavine S and T may indicate an amyloid nature of the observed alterations and suggests a certain role of the senile amyloid in the pathogenesis of senile maculodystrophy.

Poster sessions

567

P15. CYTOCHEMISTRY OF TIlE CYTOSKELETON P15.1 A Monocional Antibody that Reacts with Cilia and Muscular Cells Cytoskeleton. G. Bautista-Harris I, M. C. Lain6 and M. C. Marry ~Dept. Morfologia, Univ. Las Palmas de Gran Canaria, Spain; ~Centre de Biol. Cellulaire, CNRS, Ivry-sur-Seine, France. CC-248 monoclonal antibody is an IgM that reacts with ciliated cells and smooth muscle cells of quail oviduct sections. Cilia and cytoplasmic cytoskeleton of ciliated cells are recognized by immunocytochemistry, being the former labelled helicoidally at their distal region over the bend, as evidenced with colloidal gold labelling (Biol. Cell. 71: 191, 91). Cytoskeleton of smooth muscle cells from oviduct, vessels and gizzard, are also recognized. In addition, a banded pattern is observed on cross-striated, skeletal and cardiac, muscle cells. Labelled bands, corresponding to M line and I bands, differ on contracted and relaxed myofibrils. Whereas immunoblot results have been negative on ciliated cells and isolated cils samples, a 55 Kd band corresponding to desmin has been obtained on smooth muscle samples and a band at the same level on myofibrils. In regard to immunocytochemical and immunoblot results, it is thought that this antibody reacts with an epitope that is common to different cytoskeletal antigens related to mechanical movement,

P15.2 Interaction of Extraceilular Matrix with Cytoskeleton in NIH-3T3 Mouse Fibroblasts Transfected with a Human bFGF eDNA. M. Presta*, D. Flagiello, A. Di Nallo, A. Gualandris*, R. Cece*, S. Parolini, D. Rosa and M. P. Molinari-Tosatti Histology and *General Pathology, Dept. Biomedical Sciences and Biotechnology, University of Brescia, 25123 Brescia, Italy. Tumors are characterized by a high mitogenic activity of neoplastic cells and by their ability to invade surrounding normal tissue and to metastasize. This is paralleled by the disorganization of the cytoskeleton and of the extracellular matrix mediated by the release of proteolytic enzymes. The production of autocrine growth factors by cancer cells have been reported. Usually, rapidly growing transformed cells, which frequently secrete growth-promoting polypeptides, also produce high amounts of proteolytic enzymes. This rises the possibility that malignant cells produce autocrine growth factors able to regulate their proteolytic potential. On this basis, we have investigated the effect of the expression of high levels of basic Fibroblast Growth Factor (bFGF) on cell/matrix interaction and protease production in cultured cells. To this purpose, NIH-3T3 fibroblasts have been transfected with the expression vector pZipNeoSV(X) harbouring a 1.1 kb human bFGF cDNA coding for 18 kD-bFGF and its high molecular weight isoforms (22 kDand 24 kD-bFGF). A transfected clone (C2) has been obtained whose elevated levels of production of all bFGF isoforms have been confirmed by Northern blot, Western blot, immunocytochemistry, and in situ hybridization. These cells have been utilized to study the modifications of the interaction of extracellular matrix proteins (fibronectin) with their cell membrane receptors (beta-1 integrins) and with cytoskeleton

(actin). These modifications have been related to the production of serine proteases, like urokinase-type plasminogen activator.

P15.3

The Mechanism of the Abnormal Cytokeratin (CK) Expression in Hepatocyte. I. In HBV-Caused Chronic Hepatitis and Liver Cirrhosis of 315 Cases. Q. Su, Y.-F. Liu and Y.-G. Zhang Department of Pathology, Tangdu Hospital, The Fourth Military Medical University, Xi'an, P.R. China. An ABC immunohistochemical study of 315 cases was made to investigate the expression of cytokeratin 19 (CKt9) and cytokeratin 18(CK18) in the hepatitis and liver cirrhosis caused by HBV infection. It was showed that hepatocytes could express CK 19 (the abnormal CK expression) in 73 ~ of chronic active hepatitis (CAH) (80/110) and 81070 of cirrhotic livers (117/144), which could be of help in differentiating CAH from chronic persistent hepatitis, subtyping CAH (mild, moderate to severe type) and in determining the activity of cirrhotic livers. The relationship between the abnormal CK expression and the activity of the liver disorders was confirmed. The CK19 expression in hepatocytes were demonstrated to have the closest relations with piece-meal necrosis and the formation of "liver cell rossettes", and it was suggested to be one of the adaptive reactions to these pathologic changes.

P15.4 Mechanism of the Abnormal Cytokeratin (CK) Expression in Hepatocytes. II. An Immunohistochemical and Immunoelectron Microscopical Localization of CK19 and Laminin (LN) in CCiy-Caused Hepatitis and Liver Cirrhosis. Q. Su and Y.-F. Liu Department of Pathology, Tangdu Hospital, The Fourth Military Medical University, Xi'an, P.R. China. In this research, we made a study on the changes of CK expression in hepatocytes in the chronic liver injuries caused by CCL4 intoxification and the relationship between this change and the abnormal LN deposition in liver. It was shown that abnormal CK expression could also happen in CCL4-injured livers, in which the distribution patterns of the CKI9 positive hepatocytes were similar to those in alcoholic liver injuries. The abnormal CK expression began at the 8th day of the intoxification. Its level rose markedly soon after the peaks of the necrosis of hepatoeytes, and dropped with the recovery of the normal structures after the intoxification stopped, which suggested that the abnormal CK expression was closely related to the activity, and an adaptive response during the reconstruction after the destruction of the lobules. The histologic distribution patterns of the CK19 positive hepatocytes indicated that the deposition of LN (maybe with some other extracellular matrix components) on the surfaces of hepatocytes was a crucial

568 event in the changes in CK19 expression. In addition, we observed the ultrastructural and the antigenic determinant changes of the IF cytoskeleton of the hepatocytes and suggested a model of IF assembly.

P15.5

The Mechanism of the Abnormal Cytokeratin (CK)Expression in Hepatocyte. III. An Immunohistochemical Study in the Butter YellowInjured Liver of Rat. Q. Su and Y.-F. Liu Department of Pathology, Tangdu Hospital. The Fourth Military Medical University, Xi'an, P.R. China. We observed the CK expression of hepatocytes in butter yellow-injured livers in order to investigate the relationship between CKI9 expression in hepatocytes and oval cell proliferation. After 10 days of the intoxification the centrolobular focal necosis could be found occasionally; At the 30th day, many oval cells appeared in or around the portal tracts, and the CK19 positive hepatocytes could be seen in the same region; at the 60th and the 80th day, the oval cell proliferation could be seen throughout the whole lobules, and the CKI9 positive hepatocytes scattered within the whole lobules and had very close relation in their positions with strongly CK19 positive oval cells. It was also shown in the neighbouring sections that most of the CK19 positive hepatocytes contacted with the LN positive oval cells and were equipped with discontinuous or continuous LN positive basement membrane-like structures. The results demonstrated that the butter yellow-caused liver injuries could also induce the CK19 expression in hepatocytes which was mainly related to the oval cell proliferation. This confirmed the roles of oval cells in inducing CK19 expression in hepatocytes, and indicated that LN was one of the inducers in this process.

P o s t e r sessions P15.7

The Mechanism of the Abnormal Cytokeratin (CK) Expression in Hepatocyte. V. The Influences of the 9 Experimental Local Liver Injury Caused by Liquid Nitrogen Freezing on the CK Expression of Hepatocyte. Q. Su and Y.-F. Liu Department of Pathology, Tangdu Hospital, The Fourth Military Medical University, Xi'an, P.R. China. A new model of local freezing liver injury was prepared in rat, and with which we made a study on the influences of the sample necrosis of liver tissue and the necrotic products on the CK expression in the hepatocytes surrounding it. The freezing was carried out with liquid nitrogen in a triangle pattern on the phrenic surface of the mid-lobe in SD rats (6). At the 3rd day after the operation, the local necrosis could be seen and the framework of the necrotic hepatocytes existed even in the boundary between it and the liver tissue surrounding it, where there was a marked connective tissue increase. At the 7th day, there could be seen a membrane composed of proliferating connective tissue enclosing the necrotic tissue, surrounding which the hepatocytes had connected together to form a limiting membrane-like structure, but in some areas the lobules remained open and the hepatic plates tended to be separated by the connective tissue, the liver cells here changes markedly in its shape and elongated, which expressed CK19 in addition to more CKI8 than the hepatocytes far from the boundary. When the limiting membrane-like structure became continuous surrounding the connective tissue at the 10th and 30th day, CK19 expression stopped. This is a strong support to our hypothesis that the expression of "bile duct type" CKs in hepatocytes was one of the adaptive responses in the repair process of the lobular structure. The adaptive significance of the change was discussed,

P15.8 P15.6

The Mechanism of the Abnormal Cytokeratin (CK) Expression in Hepatocyte. IV An Immunohistochemical Study in Peritumorous Liver Tissue of Rat. Q. Su and Y.-F. Liu Department of Pathology, Tangdu Hospital, The Fourth Military Medical University, Xi'an, P. R. China. We observed the influences of the intrahepatically implanted tumors of FSK7901 (a hepatocellular carcimona cell line) and FSK7902 (a cholangiocellular carcinoma cell line) on the CK expression of the hepatocytes surrounding them. It was shown in 42 solid tumors that tumors of both FSK7901 and FSK7902 cells grew in an inflating pattern. At the boundaries the normal lobular structure was destructed, the involved hepatocytes were elongated or even enclosed by the tumor cells or connective tissues. These hepatocytes were shown to express CK19. The change of CK expression in hepatocytes was demonstrated to be associated with the sizes of the tumors. These results further confirmed the relationship between the CK19 expression in hepatocytes and the destruction of the lobular structures.

The Mechanism of the Abnormal Cytokeratin (CK) Expression in Hepatocyte. VI. A CK- and Laminin (LN)-Immunohistochemical Study of the Liver of Rat Embryo. Q. Su, Y.-F. Liu and M.-Z. Gung Department of Pathology, Tangdu Hospital, The Fourth Military Medical University, Xi'an, P.R. China. In this research, we investigated the relationship between the morphogenesis of intrahepatic bile ducts and the changes in CK expression, and the role taminin played in the process. At the 29th embryonic stage, LN-positive substances began to deposit around the portal vein branches, and the immature hepatocytes here remained CK19 negative, but the CK18 expression increased. Up to the 31st stage, there appeared some CK19 positive tubular structures around the portal vein branches, which had transitional relationship with the neighbouring immature hepatocytes and were equipped with continuous or discontinuous LN positive basement membrane-like structures. The CK19 expression in the tubular structures increased with the process of the isolation and the enclosing of them by the LN-positive basement membrane-like structures, until the postnatal stage. Around the large portal vein branches near the hepatic hilum, this process happened

P o s t e r sessions earlier than those around the smaller portal vein branches far from the hilum. We suggested that, the CK19 expression was the consequence of the differentiation of the immature hepatocytes around the portal vein branches towards biliary epithelial cells, and one of the very important events in the morphogenesis of the intrahepatic bile ducts. The LN deposition around the portal vein branches was thought to play a crucial role in this process.

P15.9

The Cytokeratin (CK) Expression in Primary Hepatocellular Cholangiocellular Carcinomas. Q. Su and Y.-F. Liu Department of Pathology, Tangdu Hospital, The Fourth Military Medical University, Xi'an, P.R, China. In order to evaluate the applied significance of the antibodies against the "bile duct type" CKs (CK7 and CKI9) in the diagnosis of hepatocellular and cholangiocellular carcinomas, we made an ABC immunohistochemical study on 47 cases of primary hepatoma using the antibodies against CK18 (HK2) and CK19 (RAK1 and K174). It was demonstrated that, almost all cancer cells in all 10 cases of cholangiocarcinoma could express CK19, in addition to CK18; There could be seen some CK19 positive cancer cells in 14 of 37 cases (38%) of samples of hepatocellular carcinoma. Therefore, CK19 expression was not a reliable index to differentiate hepatocellular carcinoma from cholangiocellular carcinoma. Whether there was abnormal CK expression did not relate to the level of the differentiation of hepatocellular carcinoma but was shown to be associated with the histologic type of hepatocellular carcinomas which indicated its differentiation direction. Most of the glandular or pseudoglandular nests of cancer cells were demonstrated to be CK19 positive, which indicated that there were indeed some cancer cells differentiating towards biliary epithelial ceils in this type of hepatocellular carcinoma. The LN-immunohistochemical staining results in the neighbouring sections showed that the CK19 positive cancer cell nests were also LN positive and equipped with LN-positive basement membrane-like structures, which indicated that the differentiation of the cancer cells at least in some cases of hepatocellular carcinoma was influenced or modulated by LN (and some other extracellular matrix components) in the interstitial tissue or produced by themselves.

569 It was showed that the rat hepatoma cell line FSK7901 and FSK7902 belonged to cholangiocellular and hepatocellular carcinoma, alternatively. Both in culture and in the intrahepatic solid tumors, most of FSK7901 cells were CK19 positive, but most of FSK7902 cells were CK19 negative, which accorded with duct epithelium and hepatocytes, alternatively. Both of the 2 human hepatoma cell lines (QGY7703 and SMMC7721) were showed to be hepatocellar carcinoma but partly with glandular differentiation in their subcutaneously implanted solid tumors. ABC staining demonstrated that most of the glandular-differentiating nests of cancer cells expressed CK19. All these results above supported our hypothesis that the CK expression of hepatomas related with its histologic pattern indeed.

P15.11

Role of Microfilament Proteins in Apocrine Secretion of Rat Coagulating Gland and Dorsal Prostate. M. Steinhoff, M. Baydilli, J. Seitz, A. Meinhardt and G. Aumtiller Inst. Anatomy and Celt Biology, 3550 Marburg, Germany. Transglutaminase (TGase) is a secretory protein from rat coagulating gland that is extruded in an apocrine fashion (1). As details of the apocrine secretion mechanism are not fully elucidated as of yet, the significance of the cytoskeleton during apocrine release has been studied immunohistochemically on semithin cryosections and glycol-methacrylate embedded tissues of untreated or hormone-treated rat coagulating gland. Male Wistar rats aged five months were castrated or treated with estrogens (0,003 mg/kg wt) for 1, 3, 7, 14 days. Some castrated animals were substituted with testosterone (6 ~g/kg wt). In sexually active untreated animals a specific TGase-immunoreactivity was found exclusively within the apical blebs. In secretory cells of castrated and E2treated rats a reduced number of apical blebs and decreased immunoreactivity for TGase could be verified. For actin filaments, a specific immunoreactivity in the terminal web as well as in finger-like protrusions of apical btebs could be clearly demonstrated. On the other hand, the apico-basal orientation of microtubules was never affected which speaks against an involvement of tubulin in this secretion mode. These results confirm our hypothesis of an involvement of actin in the hormone-regulated apocrine secretion mode of rat coagulating gland and dorsal prostate. (1) Seitz et al. (1990) Histochemistry 93: 525-530.

P15.10

P15.12

The Cytokeratin (CK) Expression in 4 Hepatoma Cell Lines of Rat and Human and their Implanted Solid Tumars.

lmmunophenotypes of Myoepitheliai Cells in Human Mammary, Sweat and Salivary Glands.

Q. Su, M.-Z. Gung and Y.-F. Liu Department of Pathology, Tangdu Hospital, The Fourth Military Medical University, Xi'an, P.R. China. In order to clear the relationship between the CK expression and the histologic patterns of hepatomas, we determined the histologic type of 2 rat hepatoma cell lines at first, and then made a CK-immunohistochemical investigation on 4 hepatoma cell lines and their solid tumors.

A. Tsubura, H. Senzaki and S. Morii Department of Pathology, Kansai Medical University, Moriguchi, Osaka 570, Japan. Adult human normal mammary, sweat and salivary gland tissues were examined immunohistochemically on methacarnfixed, paraffin-embedded tissue sections using anti-actin antibodies; polyclonal muscle and nonmuscle actins antibody and IA4 (a-smooth muscle actin), anti-keratin antibodies; 3 1 2 C 8 - 1 (Keratin(K) 14), LL002 (K.14), K S - 1 A 3 (K.13),

570 CK8.12 (K. 13 & 16), and 34BE12 (K.5,10 & 11), and antivimentin antibody; V9, followed by ABC procedures. Actinase pretreatment was necessary for antigen visualization with 312C8 - 1 or K S - 1A3. Myoepithelial cells in the mammary, sweat and salivary glands were preferentially labelled by actin and weakly by vimentin, and by all the keratins tested. Although myoepithelial cells of 3 glands possessed the same immunophenotypes of actin and vimentin, other epithelial cell components of these glands were variably labelled with antikeratin antibodies: K. 14 was stained in inner and outer ductal cells of the sweat gland, and ductal basal cells of the salivary gland; K.13 was stained in mammary luminal cell, eccrine secretory cells and salivary ductal basal cells; CK8.12 labelled mammary luminal cells, eccrine secretory and inner ductal cells, and salivary ductal basal cells; and 34BE12 labelled mammary luminal cells, inner and outer ductal cells of the sweat gland, eccrine secretory cells, and all ductal cells of the salivary gland.

P15.13

The Expression and Distribution of Vimentin during Degeneration and Regeneration of Rat Skeletal Muscle. R. Vater, M. J. Cullen and J. B. Harris Division of Neurobiology, University of Newcastle-upon-Tyne, U.K.

Desmin is an intermediate filament protein of molecular mass ~53kDa. It is a major component of the cytoskeleton of mature skeletal muscle fibres. Its primary role appears to be to maintain the lateral alignment of myofibrils by linking adjacent Z-discs and by attaching peripheral myofibrils to the plasma membrane. During early development, the appearance of desmin is preceded by vimentin, a closely related intermediate filament protein of slightly higher molecular mass (,'o55 kDa). We have examined the expression of vimentin in skeletal muscle of the rat during a cycle of degeneration and regeneration. Venom (15 ~g in 0.2 ml 0.9% w/v NaC1) of the Australian tiger snake Notechis scutatus scutatus was inoculated over the soleus muscle of one hind limb. The muscle fibres began to degenerate at 1 - 3 h, and were completely destroyed by 24 h. By 48 h immature myotubes containing occasional myofilaments could be identified. By 5 d fibres, though small, were filled with myofibrils. The muscle fibres were fully regenerated by 21 d. Vimentin was first identified using gold-conjugated antivimentin antibodies in the cytoplasm of many satellite cells of degenerating fibres at 12 h, and by 24 h the majority of such ceils were labelled. At 48 h, when myotubes were first formed, there was extensive labelling of cytoplasm around the regenerating myofilaments. Labelling became more discrete as the sarcomeres of adjacent myofibrils were aligned, but it was not possible to judge whether the localisation of anti-vimentin labelling preceded, followed or was simply associated with alignment. Anti-desmin labelling was not significant before 2d, and coexisted with anti-vimentin labelling between 4 and 14 d. By 21 d, anti-vimentin labelling could no longer be identified. Our results suggest that degeneration provides a stimulus for satellite cell proliferation, and this is associated with gene

Poster sessions activation and the expression of vimentin. The role of vimentin in both satellite cells and early myotubes is, at present, unclear.

P15.14 Immunomorphologie Study of Renal Glomeruli in the Vertebrates. V. B. Zaitsev Murmansk Marine Biological Institute of the Russian Academy of Sciences, 184631 Dalnye Zelentsy Murmansk Region, Russia.

A comparative immunomorphologic study was carried out on cryostate sections of renal tissue of plaice (Pleuronectes platessa L.) and mongrel rat, using specific antibodies against the proteins of intermediate filaments - - cytokeratins and vimentin. No cytokeratins were revealed in cells of renal glomeruli in both the animals under investigation. Indirect immunofluorescence of the polyclonal serum against vimentin showed brightly coloured of the renal glomeruli of plaice and weakly coloured ones of rat. At the same time cells of parietal epithelium of the Bowman's capsule showed trace or negative reaction. The electron microscopic control revealed a powerful development of intermediate filament system in the podocyte cytoplasm of plaice, and a dense microfilament network and plural bundles of microtubules in the podocyte cytoplasm of rats. Double staining of renal tissue of rats of different age groups (6 - 7 weeks and 1.8 years old) with use of antibodies against actin and anti-vimentin polyclonal serum reveals in young rats an intensive fluorescence in the determining of actin and the little one in the determining of the vimentin. Renal glomeruli of old rats demonstrate the strong vimentin activity and less actin one. This study permits to make a conclusion that during phylo- and ontogenesis of vertebrates morphological reconstruction of podocyte cytoskeleton takes place.

P15.15 Immunocytochemistry of Tubulin in Fish Scale Scleroblasts Reveals Principles of Collagen Pattern Formation. L. Zylberberg* and J. Bereiter-Hahn** *Universitd Paris, Laboratoire Anatomie comparde, 2 Place Jussieu, 75005 Paris, France; **Goethe Universitdt, Arbeitskreis Kinematische Zellforschung, Senckenberganlage 27, 6000 Frankfurt am Main, Germany.

Fish scales are characterized by a highly ordered threedimensional collagen lattice. In teleost scales, the collagen fibrils are organized in superimposed plies (a plywood-like structure) and synthesized by fibroblasts (called scleroblasts) which form an uninterrupted layer lining the basal surface. In order to analyse the cellular control of the collagen deposition, immunocytochemical and electron microscope techniques were carried out. Intracellular microtubules (Mts) were oriented in parallel with the innermost collagen fibrils. We hypothesized that this coalignment has a significant implication: The alignment of the collagen fibrils is controlled by Mts and thus Mts have to be submitted to periodic rearrangements during the formation of the plywood-like structure. The frequency of the Mts reorientation depends on the developmental stage of the scales. Undisturbed scales grow

P o s t e r sessions very slowly while regenerating scales show an increased synthetic activity. The most probable way of the Mts rearrangement consists in their disassembly and reassembly. Monoclonal antibodies specific for the aminoacid composition at the carboxyterminus of a-tubulin (YL 1/2 and 1D5) revealed only Mts with tyrosin at the carboxyterminus in

571 regenerating scales while in fully developed scales Mrs contained also detyrosinated a-tubulin indicating their higher stability. These observations support the hypothesis that Mts rearrangment is involved in the construction of the collagen plywood-like pattern in teleost scales.

P16. H I S T O C H E M I S T R Y IN C A R D I O V A S C U L A R R E S E A R C H P16.1

Creatine Kinase Cytochemistry of Ouabain Treated Hearts Premedicated with R59494. A. Bakker, F. Goossens*, T. Rada**, J. Slezak**, H. van Belle* and W. Jacob University of Antwerp (UIA), Dept. of Med, 2610 Wilrijk, Belgium. *Janssen Pharmaceutica, Dept. of Biochem, 2340 Beerse, Belgium. **Slovak Academy (SA V), Inst. for Heart Research, Bratislava, Croatia. The aim of our study was to determine whether in Langendorf hearts, premedication with R 59494 has an effect on the mitochondrial creatine kinase activity after ouabain treatment. The most widely accepted mechanism of ouabain is its inhibitory effect on the Na/K ATP-ase bound to the sarcolemma. This reduces the Na gradient across the cell membrane, hence also the N a / C a exchange coupled Ca efflux which finally results in an increase of intracellular calcium. Our experiments show that the first effect of ouabain treatment is an increase of the LVP, followed by arrythmias after 10' R 59494 at 4.10-7M does not prevent the LVP increase after ouabain but avoids the arrythmias. Mitochondrial creatine kinase, as explored by cytochemistry is a sensitive marker for the metabolical state of the myocardial cell. For this purpose, mitochondrial creatine kinase cytochemistry was performed. One group of Langendorff perfused rabbit hearts received 10 -7 M of ouabain for a few minutes to cause the increase of LVP. The other group received the same amount of ouabain during the same time after premedication with R 59494. The morphometric results of the surface density of reactive contact sites are 0.42 _+ 0.06 for the ouabain treated hearts and 0.44 _+ 0.06 for the premedicated group. This means that R 59494 has no inhibitory effect on the stimulation of the heart by ouabain.

P16.2

Alterations in Cardiomyocytes Induced by Sulfate Isoprenoline or Adriamyeine Cardiomyopathy. L B. Buchwalow, I. V. Lenandovski, I. N. Chernysh and V. B. Sadovnikov Research Institute for Biotechnotogy, Moscow, Russia. A comparative histological and histochemical investigation of changes in cardiomyocytes of the heart left ventricle (LV), sino-auricular node (SN) and antrioventricular node (AN) was carried out under conditions of sulfate isoprenoline (SI) and adriamycine (AD) cardiomyopathy. SI rendered muscle fibers thinner. Electron microscopy revealed a lack of H- and I-discs and in distinct Z-discs; the width of intercalated discs increased markedly in LV, moderately in AN and quite insignificantly in SN cardiomyocytes. The nuclei contained

condensed chromatin preferentially bound to the nuclear envelope. The nucleoli exhibited a nucleolonemal structure. Mitochondria (MCh) were swollen, contained dense inclusions in intracristal space and dense granules in the matrix. ATPase activity was shown to decrease significantly. AD induced distortions of inner membranes of some MCh and a decrease in the content of condensed chromatin seen only along the nuclear envelope. The results obtained show that AD cardiomyopathy leads to more pronounced alterations in cardiomyocytes, as compared to those induced by SI cardiomyopathy.

P16.3 Succinic Dehydrogenase Activity in Cardiac Myocytes and Correlated Ultrastructure in Iron-Deficient Rats. R. Coleman, M. Nahir, Z. Tanne, D. Shornrat and M. B. H. Youdim Bruce Rappaport Faculty of Medicine, Technion-lsrael Institute of Technology, Haifa 31096, Israel. Male Sprague-Dawley rats aged 3 weeks that were maintained on an iron-deficient diet for 4 - 5 weeks developed severe anemia, which was accompanied by marked cardiac hypertrophy (cardiomegaly). The major morphological and cytochemical changes were seen in the left ventricle. Succinic dehydrogenase activity was localized cytochemically and the colored formazan reaction product was quantified using scanning and integrating microdensitometry with a Vickers M86 microdensitometer. The myocytes of the left ventricle of the iron-deficient rats showed a marked reduction in SDH activity. This correlated well with ultrastructure, which showed disruption of sarcomeres, myofilament loss, myofilament discontinuities and marked interfibfillar edema. The interfibrillar mitochondria were enlarged and showed degenerative changes in the matrix including the formation of electron-dense bodies. When the severely iron-deficient rats were restored to a normal diet for two weeks, hemoglobin levels recovered, though the heart remained hypertrophied and the myocytes continued to show severe degenerative changes. Supported in part by grants from the Ross Research Laboratories (USA), the Paul and Bess Goldings and A. & E. Blum Funds for Medical Research.

P16.4 Intermediate Filaments and ATPase Activity in the Vascular Wall of Vertebrates. K. T. Dikranian, M. T. Trosheva and M. I. Petrov Medical University, Varna, Bulgaria. The vascular wall of the aorta and vena cava in different representatives of vertebrates was examined for ATPase

572 activity and cytoskeletal intermediate filaments (IF). Enzyme activity was studied by the methods of Padicula-Herman and Wachstein-Meisel. A streptavidin-biotin immunohistochemical method was applied to reveal desmin (D) and vimentin (V) IF. Endothelial cells of all vessels were V-positive, D-negative and exhibited high ATPase activity. Vascular myocytes (SMC) in lower vertebrates (pisces and amphibia) were also V-positive and D-negative, but showed low ATPase activity. Vascular SMC in aves and mammals were D-positive and V-negative and possessed high ATPase activity, similar to that of the endothelium. In cow vascular wall, D-positivity and high ATPase activity was mainly expressed in bundles of mosaicly arranged thick SMC fibres of the outer aortic media, as well as in the longitudinal fibres in vena cava caudalis. In higher vertebrates, SMC of vasa vasorum are both V- and D-positive and showed high ATPase activity. Our results demonstrate that D-immunoreactivity is mostly expressed in SMC of layers of high functional activity, which correlates with the intense ATPase reaction in these cells.

P16.5 The Effect of Hibernation on Substance P Immunoreactive Nerve Fibers of Frog Lymph Hearts. C. Alessandrini, A. Rebuffat, A. M. Pucci, M. Guarna and C. Fruschelli Histology and Gen. Embryology Inst., University of Siena, Italy. The present study was carried out in order to investigate the effects of hibernation on the distribution of the substance P immunoreactive (SP-IR) nerve fibres in the lymph heart wall. Preparations of frog lymph hearts (Rana aesculenta) were examined at different times throughout the entire year to determine whether there was any seasonal effect on the SP-IR nerve fibres in lymph heart wall. Immunocytochemistry was performed on cryostat sections employing a rat monoclonal antibody anti SP (SeraLab) and an indirect immunofluorescence method. The hibernating frog lymph hearts showed a different pattern of innervation from that observed in the nonhibernating frog lymph hearts. In the hibernating frog lymph hearts, few SP-IR nerve fibres of larger diameter in the adventitia and numerous SP-IR nerve fibers are present in the inner layer of the media and beneath the endothelium. These findings might be correlated with the different degree of contractile activity in lymph hearts of hibernating frogs.

P16.6 Peptidergic Innervation of Frog Lymph Hearts. A. Rebuffat, A. M. Pucci, C. Alessandrini, M. Guarna and C. Fruschelli Histology and General Embryology Institute, University of Siena, Siena, Italy. The present work was carried out in order to study the distribution of substance P immunoreactive (SP-IR), calcitonin gene related peptide immunoreactive (CGRP-IR) and neuropeptide Y immunoreactive (NPY-IR) nerve fibers in frog lymph hearts (Rana Aesculenta). Immunocytochemistry was performed on lymph heart cryostat sections employing a rat monoclonal antibody anti SP (SeraLab.), a rabbit polyclonal antibody anti CGRP (Chemicon Inc.) and a rabbit

P o s t e r sessions polyclonal antibody anti NPY (Chemicon Inc.) and an indirect immunofluorescence method. Numerous peptidergic nerve fibers are present in the media among the striated muscle cells and beneath the endothelium of frog lymph hearts. The most numerous nerve fibers are the SP-IR ones while the less numerous are the NPY-IR ones. Our findings suggest that lymph hearts possess a well-developed plexus of nerve fibers containing sensory neuropeptides. These nerve fibers might have a functional role in reflex regulation of the spontaneous rhythmica ! contractile activity of these organs.

P16.7 Serotoninergic Innervation of Dural Blood Vessels in Guinea Pig. A. M. Pucci, M. Guarna, C. Alessandrini, F. Crestini and C. Fruschelli Histology and General Embryology Inst., Univ. of Siena, Italy. The present study has been carried out in order to investigate the distribution and nature of 5-hydroxytryptamine immunoreactive (5-HT-IR) nerve fibers in guinea pig dural blood vessels. The immunocytochemical study was performed on whole mount preparations of dura mater. A rat monoclonal antibody anti 5-HT (SeraLab) and an indirect immunofluorescence method were employed. A moderate number of 5-HT-IR nerve fibers were observed in dural blood vessels only after an in vitro pre-incubation of dura mater in an oxygenated Krebs' solution containing 5-HT creatine sulphate (Sigma). These nerve fibers disappeared after chemical sympathectomy with 6-hydroxydopamine (6-OHDA). These findings suggest that 5-HT IR nerve fibers in guinea pig dural blood vessels might simply be noradrenergic nerve fibers which take up 5-HT.

P16.8 DO Lymphatie Vessels Possess a True Serotoninergic Innervation? M. Guarna, A. M. Pucci, C. Alessandrini, N. Volpi and C. Fruschelli Histology and General Embryology Inst., Univ. of Siena, Italy. The study has re-examined, by immunocytochemistry, a proposed serotoninergic innervation of mesenteric lymph collectors in guinea pig. Immunocytochemistry has been performed on mesentery whole mount preparations by using a rat monoclonal antiserotonin antibody (SeraLab) and an indirect immunofluorescence method. Various conditions of fixation and pre-incubation in vitro and in viva pre-treatment with inhibitors of 5-Hydroxytryptamine (5-HT) uptake were employed in order to test the effect on 5-HT immunoreactivity. Numerous 5-HT immunoreactive (IR) nerve fibers in the lymph collector wall are only observed after pre-incubation of mesentery and the accompanying ileum in an oxygenated Krebs' solution. Pre-treatment in viva with a selective inhibitor of 5-HT uptake (fluoxetine-Lilly) causes the total disappearance of 5-HT immunoreactivity in the lymphatic wall. These findings suggest that the majority of 5-HT IR nerve fibers in the lymph wall might simply be noradrenergic nerve fibers which take up endogeneous 5-HT released from the ileum wall.

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P o s t e r sessions

P16.9 Altered Expression of Endothelial and Muscle Cell Antigens in Atherosclerotic Plaques. G. Giordano-Lanza, S. Montagnani*, A. Bernardo, B. Gallicchio, F. Maiuri, R. Vitelli and R. Spera Second Medical School of Naples, *Medical School of Reggio Calabria, Italy. We used immunohistochemistry and histochemistry to study some cellular aspects in human carotic atherosclerotic plaques. It is well known that athero-sclerotic disease is characterized by different aspects like calcium deposits, infiltration of mononuclear cells and proliferation of smooth muscle cells in the vascular wall. Recently some authors proposed a role of immunocompetent cells in developing disease and demonstrated altered pattern of expression of typical endothelial and muscle cell markers. We studied 16 atherosclerotic plaques from human carotid arteries and our observation pointed out some evidence: (1) Significant amounts of T-cells were found in phlogistic infiltrate of plaques. T-cells are strictly correlated with well known macrophage cell population but also with dendritic-Tassociated Langerhans-like cells. (2)Frequently in our samples, smooth muscle cells are not only increased but also markedly positive for monoclonal antibodies against MHC class II(Ia)antigens, so suggesting they can play a role in antigen presentation to T-cells. (3) Other smooth muscle cell markers, like desmin, smooth myosin and actin appear regularly expressed also in HLA-Dr positive smooth muscle ceils. (4) Endothelial cells also exhibit alterations in surface markers expression.

P16.10 Fatty Acid-Binding Protein as an Immunohistochemicai Marker for the Detection of very Recent Myocardial Infarctions in Man. A. H. Kleind, J. F. C. Glatz ~, M. G. Havenith 2, F. A. van Nieuwenhoven ~, G. J. van der Vusse ~ and F. T. Bosman 3 Depts. of ~Physiology and 2Pathology, University of Limburg, Maastricht, and 3Department of Pathology, Erasmus University Rotterdam, Rotterdam, The Netherlands. Depletion of human heart type fatty acid-binding protein (H-FABP) from cardiomyocytes in infarcted areas with varying post infarction intervals was studied in 23 patients who died from a myocardial infarction (MI), as was clinically diagnosed. With antibodies to human H-FABP, formaldehyde fixed and paraffin embedded myocardial tissue sections were stained immunohistochemically, and findings were compared with those from nitroblue tetrazolium (NBT) macroenzyme staining of sections of the same MI. In 11 of the 23 patients, in whom MI was confirmed by NBT-staining, decreased or absent H-FABP immunostaining was observed. In addition, in the 12 remaining patients depletion of H-FABP could be demonstrated in areas that showed normal NBTstaining (indicating a post infarction interval of <4 hrs). In two control cases of non cardiac death no depletion of H-FABP was observed in different myocardial tissue sections, while macroenzyme staining with NBT was normal. In conclusion, these findings indicate that H-FABP immunostaining detects very recent infarction in human myocardium and can be applied in routine autopsy pathology. This study

was supported by StiPT, Executive Agency for Technology Policy, grant MTR88002.

P16.11 Assessment of Human Cardiomyocyte Plastic Function by Means of Silver-Stained Nucleolar Organizer Regions (AgNORs). N. N. Mamaev, A. Y. Gudkova, C. K. Amineva, and O. V. Kovalyeva Faculty Therapy Clinic, Pavlov Medical Institute; The Institute of Cardiology, St-Petersburg, Russia. The investigation was carried out on autopsied myocardiums obtained from eight previously healthy persons (HP) who had perished in accidents, from 26 deceased cases with arterial hypertension (AH), and from 31 deceased'bases with chronic ischemic heart disease (CIHD) as well as on cardiobiopsy specimens taken from 70 patients operated for CIHD (n = 23) and for cardiac valvular disorders (CVD, n = 47). Both in normal and in pathology cardiomyocyte nucleolar argentofilia (CNA) were relatively weak. The mean number of Ag-grains in nucleoli of HP ranged from 7.5 to 12.5 (8.0 + 0.4). In patients with heart septum defects, it did not differ from the controls (7.6 _+ 0.9), but it increased in AH, CVD (11.0+_0.7; P<0.01) and even in CIHD unless complicated with severe cardiac insufficiency (CI, 9.9 _+ 0.7). The lowest CNA was found in most therapeutic patients with severe CI. As for surgical patients, most of them showed significant decrease of CNA at the stage of cardioplegic arrest (CA), while 10 operated patients revealed unexpected elevation of CMC metabolic activity. Soon after cross-clamp release and myocardium rewarming CMC plastic function in all but five patients were markedly restored. Since the mentioned non-standard deviations of CNA occurred presumably in the patients with higher operative risk the results suggest that the approach might be very useful for current cardiovascular surgery.

P16.12 High Expression of Von Willebrand Factor and GMP-140 on Coronary Heart Disease (CHD) Platelet Membrane. N. L i and J. Li Department of Histology and Embryology, The First Military Medical College, Guangzhou 510515, P.R. China. With SZ-29, an anti-human von Willebrand factor (vWF) McAb, and ~25I-labelled SZ-51, an anti-human GMP-140 McAb, platelet membrane-bound vWF and GMP-140 in normal subjects and CHD patients were quantitated by an indirect immunofluorescent technique and radioimmunoassay. The quantity of platelet-bound vWF in CHD patients (n = 25) was 2.1453 x 104 per platelet, and the quantity in normal subjects (n = 23) was 0.2092 x 104 per platelet. The quantity of GMP-140 was 3962 + 1161 per platelet in CHD patients (n = 20) and 2208 + 672 per platelet in normal subjects (n = 20). Quantities of both proteins on the CHD platelet membrane were much more than those in normal subjects (P<0.01). Because these proteins were mostly expressed on activated the platelet membrane, the results

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Poster sessions

demonstrated that there were greater quantities of activated platelets in CHD patients' blood, which may result in thrombosis.

ultrastructural images in good correlation with biochemical and functional data, showed that Aprikalim and Nicorandil protected against myocardial ischemia-reperfusion damage.

P16.13 Histochemical Method for Cholinesterase in Heart's Neurotopographical Study.

P16.15 Cardiotin, a Recently Characterized Component in the Cardiovascular System.

H. D. Pauza, N. Pauziene and R. Stropus Kaunas Medical Academy, Lithuania. Topography of intracardial nervous apparatus (NA) has been insufficiently studied and therefore the topographical knowledge of this apparatus is not used widely during physiological experiments and cardiosurgical operations. Histochemical methods for cholinesterase opened the possibilities for topographical studies of the heart's intrinsic NA, which has allowed the observation of the topography of NA of all the heart's wall layers in vitro by stereoscopic microscopy. This method allowed us to research neurotopographically not only small mammals, e.g. mouse, rat or rabbit but the total hearts of big mammals - human, pig, deer - of different ages. The method for cholinesterase in the neurotopographical research of total hearts consists of two stages. In the first stage, the accessory tissues are removed from the heart's basis and the form and orientation of its flabby atrium and veins are restored. For transmural staining the heart is subjected to proteolitical enzymes and detergents at a temperature of 4~ for some time. In middle of the second stage the heart is proteolitically affected by long-term incubation in circulating Karnovsky-Roots' medium at 4~ The staining process of the nervous tissue is visually controlled and corrected by additional preparation during incubation.

G. Schaart ~, P. van der Yen2 and F. Ramaekers ~ tDepartment of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands; 2Department of Cell Biology & Histology, University of Nijmegen, The Netherlands. To understand the abnormal development of the mammalian heart, it is important to analyse the cell biological processes that underly the morphological changes during normal cardiogenesis and the formation of sarcomeric structures. In this respect specific cardiac muscle markers are indispensable. Using the monoclonal antibody technique, we have identified two structural components of the cardiovascular system, which we designated vasculin and cardiotin. Vasculin is a 59 kDa protein and can only be detected in smooth muscle cells. Cardiotin is a high molecular weight protein and is found in cardiac and skeletal muscle tissues. No reaction with these antibodies was found in other human tissues. Cardiotin is localized around the myofibrils. The cardiotin antibody is cross-reactive in heart tissue of different species i.e. rabbit, dog, goat, monkey and human. In vitro studies have shown that cardiotin is also expressed in differentiating human skeletal muscle cell cultures. Cardiotin expression seems to be diminished in myofibrils during cardiomyopathy. This indicates that this component may be a relevant constituent in normal heart function and that the antibody to cardiotin may become a useful tool in the diagnosis of congenital myopathies.

P16.14 K+-Channel Openers Protect the Myocardium Against Ischemia-Reperfusion Injury. L. M. Popescu, S. Musat, O. C. Trifan, M. Popescu, M. Leabu, A. Popescu and M. E. Hinescu Division of Cell Biology and Histology, Davilla University of Medicine, Bucharest 35, P.O. Box 35-10, Romania. We studied the presumptive cardioprotective effects of two K + channel openers, Aprikalim (RP-52891) and Nicorandil, on isolated rat heart (Langendorff preparation: 10 min equilibration; 20min perfusion _+ drug; 30 rain global normothermic ischemia; 30 min reperfusion), by correlating cytochemical, ultrastructural, biochemical and functional data. The myocardial damage was evaluated by: l) enzyme ultracytochemistry (lactate dehydrogenase, succinate dehydrogenase, cytochrome oxidase, catalase, transaminases); 2) Ca ~ cytochemistry (oxalate trapping followed by X-ray microanalysis); 3) biochemical data (lactate dehydrogenase, creatine kinase, transaminases, peroxidatic activity released in perfusate and electrophoretic pattern of proteins in perfusate); 4) ultrastructural morphometry; 5) functional parameters (left ventricular developed pressure, left ventricular pressure variance, coronary flow). Perfusion with Aprikalim or Nicorandil (1/~M) for 10rain before ischemia clearly protected the myocardium against ischemia-reperfusion damage. The effect of these K + channel openers is quite specific, since Glybenclamide (4/~M), an antagonist of K § channels, prevented the protection. Cytochemical and

P16.16 Adenylyl Cyclase and G-Proteins Localization in Heart Tissue. W. Schulze, M. Vannauer, I. Kiittner, L. Will-Shahab, K. Spicher* and W. Rosenthal* Max-Delbrfick Center of Molecular Medicine, 0-1115 Berlin and *Inst. Pharmacology, Free Univ. I4/-1000Berlin 33, Germany. Hormone sensitive adenylyl cyclase (AC) system is comprised of three types of components: the receptors, the regulatory GTP binding proteins (G-proteins) and the AC. The study describes the localization of G-proteins by immunogold method and the co-localization of AC in heart tissue of laboratory animals by metal precipitation techniques. For G-proteins localization rat heart tissue was fixed 20 min in 3% formaldehyde and embedded in Lowicryl K4M. The UV polymerization temperature was -25~ Ultrathin sections from left ventricular papillary muscle were incubated with affinity purified anti G ....... and anti G~I....... peptide antisera (Hinsch et al. FEBS Lett. 238, 191, 1988) and visualized by gold (14 nm) labelled protein A. Gold particles were found to be associated with the sareolemma of the cardiomyocytes and with the cell membranes of the endothelial cells. No labelling was seen inside the cells (e.g. SR membranes). AC activity was demonstrated with adenylyl imidodiphosphate as the substrate and lead, cerium or strontium salts as the capture reagent. In rabbit, guinea pig

P o s t e r sessions and rat cardiomyocytes from left ventricles AC was found to be localized at the plasma membrane including parts of the intercalated discs and the T-tubule membranes. After severe damage following long lasting ischemia AC activity was additionally seen at the junctional SR.

P16.17 Cytochemical Localization of H202 in Isehemic and Reperfused Myocardium. J, Slezdk, N. Tribulovfi and P. K. Singal Institute for Heart Research SAS, Bratislava, CSFR; St. Boniface Res. Ctr., Div. of Cardiovasc. Sci., Winnipeg, Canada.

Oxygen derived free radicals if accumulated have been implicated as a major cause of damage during reoxygenation/ reperfusion of ischemic myocardium. Beside the activity of different oxidases, different reactions of antioxidative defense system involve the intermediate production of H202. Although immediate relative toxicity of H202 is less than toxicity of highly reactive 9OH and 902 radicals, under certain conditions it can accumulate and greatly damage almost all constituents of cardiac tissue. Furthermore, the major danger associated with the accumulation of H202 is the production of highly reactive unstable . O H radicals in Haber-Weiss or Fenton reactions. Appropriate cytochemical demonstration of subcellular sources of free radicals can help to elucidate controversies concerning importance and time sequence of myocardial reperfusion injury. The direct method of H202 localization using cerium chloride with specific substrates and inhibitors was used in present study. Fifty microns thick, shortly prefixed tissue sections were incubated in medium containing substrates and inhibitors to express specific oxidase activities. OsO4 or glutaraldehyde postfixed sections were routinely dehydrated and embedded in Epon. Unstained ultrathin sections of osmicated and nonosmicated tissues were studied in electron microscope. Specific fine granular precipitate was localized in significant amount only in reperfused tissue with highest amount confined to glycocalyx of myocytes and abluminal surface of the endothelial cells. Very fine needle like or granular reaction product was also randomly distributed throughout the myocytes and endothelial ceils. Luminal side of endothelial cells did not reveal strong reaction.

P16.18 Distribution of Peptide-Containing Nerves in Cardiac Valves of Rats with Different Ages. 1-1. Su, Q. M a t , Y. Zhang and W. Huang Department of Histology and Embryology, Fourth Military Medical University, Xi'an 710032, P.R. China.

The mammalian cardiac valves are innervated by adrenergic and cholinergic nerves. However, detailed information about distribution of peptide-containing nerves has not been described. In this work free-floating immunostaining with the ABC method combined with the glucose oxidase-DAB-nickel enhancement was used to demonstrate NPY-, VIP-, CGRPand SP-immunoreactive nerve fibres in the cardiac valves of rats aged 1, 8 and 18 months. The results showed that the atrioventricular valves were richly innervated by positive fibres, The sequence of the density of four peptide-containing

575 nerves was NPY > VIP > CGRP > SP. More peptidecontaining fibres were found in the right valve than in the left one. In the semilunar valves, however, only NPYimmunoreactive fibres were seen. Two origins of the fibres were noticed to enter into the atrioventricular valves: (1) from the cardiac wall to which the valves were attached, and (2) from the chordae tendineae in which the nerve fibres were extended from the papillary muscles and travelled along its longitudinal axis. In the atrioventricular valves of rats aged 1 month the fibres tended to be thicker near the basal portion of the leaflets than those found in older animals. The nerve fibres ran toward the margins of the valves as far as 2/3 wide of the leaflets. The fibres from two origins were not in connection with each other. With the rat growing older the fibres were extended up to about 4/5 wide of the leaflets. The fibres originated from the cardiac wall were intersected with those from the chordae tendineae in rats aged 8 and 18 months. Our findings add new data to the innervation of rat cardiac valves, but the role of these nerves remains to be examined.

P16.19 Gap Junction Distribution in and around the Mammalian Sinoatrial Node Studied by Immunohistoehemistry. I. ten Velde, A. de Mazi~re, B. de Jonge, D. Gros* and H. J. Jongsma Department of Physiology, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands, and *Lab. Biologie de la Diff#renciation Cellulaire, LA CNRS 179, Universitdd'AixMarseille II, 13288 Marseille Cedex 9, France. The distribution of gap junctions in and around the sinoatrial node is being studied by immunofluorescent labelling of connexin (CX) 43, the major cardiac gap juntional protein, to establish how the sinoatrial node is electrically connected to the atrium. Antipeptide-antibodies raised in rabbit against a C-terminai domain (residues 314-322) of rat CX 43 (El Aoumari et al., 1990, J. Membrane Biol. 115, 2 2 9 - 4 0 ) are used on sections of guinea pig right atrial preparations. CX43 is abundantly present in the right atrium but can almost not be detected in the more central sinoatrial node region. The reason for this finding might be either that gap junctions in the central sinoatrial node are so small that we cannot detect them with the method we use or that gap junctions in this region contain connexins different from CX43. Preliminary results of immunoelectron microscopy with gold labelling on Lowicryl sections of rabbit sinoatrial tissue show a few small labelled gap junctions between typical nodal cells, as well as larger labelled gap junctions between cells with transitional or atrial characteristics, indicating at least the presence of CX43 in the node. However, in all cell types some small ( 5 0 - 130 nm) gap junctions devoid of label are found. With the labelling efficiency reached up till now, it cannot yet be concluded that CX43 is absent from these junctions. Three-dimensional schematic images of the CX43-distribution by immunofluorescent labelling are obtained from serially sectioned right atrial preparations. Identification of the sinus node region is based on the histological appearance on van Gieson stained sections. The unlabelled region appears to be larger than the histologically defined central nodal area. A band of peripheral or atrial cells running perpendicular to the

576 crista terminalis is seen along the endocardial side of the nonstaining region. Between this heavily labelled band and the region apparently devoid of it a small zone of intermediate labelling is present, which may represent the region where a gradient of coupling is located. Currently the labelling patterns of central, peripheral and atrial cells are studied at high magnification using confocal laser scanning microscopy after lectin and CX43 double staining.

P16.20 Sensitivity of the Heart Conducting Tissue to Ischemia. Histo- and Cytochemical Investigation.

Poster sessions studied in relation to titin. Tropomyosin followed upon tifin with respect to its exclusive expression in the heart anlagen and its organization into a striated pattern. Myosin and desmin were organized into cross-striated patterns after titin and tropomyosin. Actin, keratin and vimentin were distributed in cytoplasmic filaments in the embryologic stages we investigated. Since the first contractions are already detected in 3-somite embryos, we conclude that the organization of titin, tropomyosin, myosin and desmin into a striated pattern does not seem to be essential for the initiation of mucle cell function in the heart anlagen. The sequence of expression and organization of the proteins we investigated is species dependent and varies in time in different regions of the developing heart.

N. Tribulovd, J. Slez~ik, T. Ravingerov~i and I~.

Okruhlicov~i Institute for Heart Research SAS, Bratislava, CSFR.

The cardiac conductive system has been considered to be more resistant to ischemia than working myocardium. However, some recent ultrastructural reports cast doubts on the idea of its generally higher tolerance to ischemia. The aim of the present work was comparative ultrastructural, histochemical and cytochemical study of conducting and working myocytes of the hearts submitted to 30 and 60 min of ischemia followed by 30 min of reperfusion. Enzymes glycogen phosphorylase, LDH, SDH, fl-HBDH, ATPase, AMPase, AlP, ACHE were analyzed histochemically and AC, GC, K-pNPPase, G6Pase, 5NC, SDH, ACHE, AMPase cytochemically. Under ischemia, enzyme activity of glycogen phosphorylase and LDH determined histochemically were markedly reduced in working myocardium but they were only slightly changed in conducting myocytes. On the cytochemical level the reactions for AC, GC and pNPPase were completely absent in severely damaged myocytes, however they were positive in structurally less damaged conducting cells. The activities of G6Pase and SDH persisted even after 60 min ischemia in working as well as in Purkinje fibres. It can be concluded that ischemia-related histo and cytochemical changes were more pronounced in working than in conducting tissue. Therefore the conducting myocytes are more resistant to ischemia than working myocardium.

P16.21 Expression and Organization of Muscle Specific Proteins during the Early Developmental Stages of the Rabbit Heart. F. T. L. van der Loop', G. Schaart 1, F. C. S. Ramaekers'

and C. Viebahn 2 JUniversity of Limburg, Department of Molecular Cell Biology & Genetics, CARIM, Maastricht, The Netherlands; 21nstitute of Anatomy, University of Bonn, Bonn, FRG.

The expression and intracellular distribution patterns of muscle-specific proteins were studied during rabbit embryo development ( 7 - 1 3 dpc) using muscle-specific monoclonal antibodies. From our panel, titin appeared to be the first muscle-specific protein to be exclusively expressed in the embryonic rabbit heart. Upon cellular differentiation, titin reorganizes from dot-like aggregates into a cross-striated pattern via a transiently filamentous distribution. The expression and organization of the other muscle proteins was

P16.22 Concomitant Changes of Glycogen Phosphorylase Activity and Ultrastructure of Ischemic Rat Heart. W. M. Frederiks, H. Vreefing-Sindeldrovd and J. P. M. Schellens Laboratory of Cell Biology and Histology, University of Amsterdam, Academic Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Glycogen phosphorylase activity was tested as an enzyme histochemical parameter for irreversible cell damage during in vitro ischemia of rat heart to be correlated with ultrastructural changes. After ischemic periods ranging from 0 - 2 4 0 min, activity of glycogen phosphorylase was determined by cytophotometric measurement of glycogen synthesized by conversion of glucose-l-phosphate. The amount of endogenous glycogen was taken as control. Electron microscopic specimens were sampled after 0, 30 and 60 min of ischemia. After 20 rain of ischemia glycogen phosphorylase activity and endogenous glycogen were reduced by 25% and 350/0 respectively. After 60 rain of ischemia glycogen phosphorytase activity was decreased to 25o/0 of the control value, and after 120 rain activity could not be detected anymore. Endogenous glycogen was not further reduced during prolonged ischemia. Ultrastructural analysis after 30 min of ischemia showed that in some of the myocytes part of the mitochondria contained flocculent densities, which are indicative for irreversible cell damage. After 60 min of ischemia damage of the plasma membrane and flocculent densities in mitochondria were observed in all myocytes. It is concluded that glycogen phosphorylase activity can be used as sensitive parameter for irreversible damage of cardiac myocytes.

P16.23 Uitracytochemical Verification of H § -K + -ATPase Activity in the Rat Right Atrial Muscle Cells. V. S. Zinchuk and A. V. Bulavka Department of Pathology, Kiev Medical Institute, Ukraine.

In order to study the mechanism of action of the atrial natriuretic factor [ANF] within the atrial muscle cells, we looked at the enzymatic properties of H § -K § -ATPase, which is responsible for the secretion. Tricine-buffered, cerium-

Poster sessions based method was undertaken to evaluate enzyme cytochemical localization. Reaction product [RP] of enzyme as fine, uniform and crystalline electron dense precipitate was traced on the atrial mucle cell sarcolemma, its vesicles, T-tubule membrane and sarcoplasmatic reticulum. At high power, RP was located along the internal side of sarcolemma. Enzyme activity was markedly diminished by using omeprazole. Based on our observations, we propose the possibility that

577 atrial muscle cells have a very special enzyme cytochemicat organization. ANF within them appears to provide a new dimension in balancing of water and salt content. Ultrastructural localization of ouabain-insensitive. H+-stimulated K+-pNPPase lets assume a transporting mechanism via atrial muscle cell sarcolemma. Enzyme cytochemistry will be very helpful for understanding of ANF metabolic activity. This perfectly facilitates the precise localization of cerium phosphate.

P17. I M M U N O H I S T O C H E M I S T R Y OF ENZYMES P17.1

Megakaryocyte Precursors in Chronic Myeloproliferative Disorders and in Acute Megakaryoblastic Leukaemia. A Clinicopathological Study of Seventeen Cases. J. L. Ivdnyi, A. Kiss and B. Telek 2nd Dept. Med. Univ. Med. School, Debrecen, Hungary. Identification of megakaryocyte precursors (megakaryoblasts) with enzyme cyto- and histochemical/immunohistochemical methods in bone marrow smears and trephine biopsy specimens was performed from patients with blastic phase of chronic granulocytic leukaemia (CGL, four cases), from chronic megakaryocytic-granulocytic myelosis (CMGM), a subtype of chronic myeloproliferative disorders (three cases) and from ten cases of acute megakaryoblasfic leukaemia. In the b|ast cells acid phosphatase and nonspecific (naphthyl acetate) esterase were always demonstrable, whilst antibodies against beta-thromboglobuline, von Willebrand antigen (polyclonal antibodies), GpIIIa (CD41) in circulating megakaryoblasts (with flow cytophotometry) and of bone marrow were detectable only in various percentages (immunofiuorescence and A P A A P methods). The greatest bone marrow reticulin content was visible in acute megakaryoblastic leukaemia cases. Although different clinicopathological entities, the same representative phenotype (megakaryoblasts) of these haematological malignancies produced a short survival of patients in every group (5.1 vs 5.0 and 3.5 months).

P17.2

lmmunohistochemical Localization of Alkaline Phosphatase in Rat Hepatocytes. [4. Chida Department of Pathology, Kitasato University School of Hygienic Sciences, Sagamihara, Kanagawa 228, Japan. Changes in localization of alkaline phosphatase in rat hepatocytes by two experimental treatments- partial hepatectomy and bile duct ligation were examined by means of immunohistochemical techniques. The rat livers were excised 24 hr after partial hepatectomy or bile duct ligation and small tissue fragments were fixed in the fixative solution consisting of 4~ paraformaldehyde and 0.25~ glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 1 hr at 4~ The tissues were further fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 5 hr at 4~ At the level of light microscope, frozen sections were cut in a cryostat and stained

by use of peroxidase-labelled antibody method and immunogold silver staining. At the level of electron microscope, the free-floating frozen sections were stained with peroxidase-labelted antibody method and embedded in Epoxy resin after osmification. Moreover, the fixed tissue fragments were embedded in Lowicryl K4M and then ultrathin sections were stained with immunogold staining. After partial hepatectomy, a high level of positive reactions were recognized along cell borders between adjacent hepatocytes. On the other hand, after bile duct ligation, a moderate level of positive reactions were observed along the entire hepatocyte surfaces. On the examination with electron microscope, after partial hepatectomy, reaction products were seen in abundance on the surfaces of microvilli of bile canaliculli enlarged along cell borders between adjacent hepatocytes. Meanwhile, after bile duct Iigation, immunoreactions appeared not only on the bile canalicular membrane but also on the sinusoidal and lateral membranes.

P17.3

Immuno-Eiectron Microscopic Localization of Fatty Acid fl-Oxidation Enzymes. N. Usuda and T. Nagata Department of Anatomy and Cell Biology, Shinshu University School of Medicine, Matsumoto 390, Japan. The localization of fatty acid /3-oxidation enzymes was detected immunohistochemically in peroxisomes and mitochondria of hepatocytes at the electron microscopic level. Rat liver tissues of both normal and DEHP (di-(2ethylhexyl)phthalate)-treated animals were used for the study. Tissues were processed by 3 procedures, i.e. 1) fixed in buffered glutaraldehyde & paraformaldehyde and embedded in Lowicryl K4M, 2) rapidly frozen and freeze substituted and embedded in Lowicryl K4M, 3) rapidly frozen and cryosectioned. They were stained immunochemically using the antibodies for peroxisomal enzymes: acyl-CoA oxidase, bifunctional protein, 3-ketoacyl-CoA thiolase and mitochondrial enzymes: very long-chain acyl-CoA dehydrogenase, trifunctional protein, acyl-CoA dehydrogenase, acetoacetylCoA thiolase, acyl-CoA hydrogenases, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase with the protein A-gold technique. As the results, the gold particles were observed over peroxisomes or mitochondria. They were localized in the high density area in the peroxisomal matrix and along the inner membrane in the mitochondrial matrix. These results show the

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578 different localization of fatty acid/~-oxidation enzymes in two kinds of cell organelles.

P17.4

Immunohistochemical Study with Monoclonal Antibody to Human GST-e. M. Ohyama, M. Shiozawa and S. Also Department of Anatomy, Keio University, Tokyo, Japan. Glutathione S-transferases (GSTs) are a group of enzymes that play important roles in the biotransformation of xenobiotics, carcinogens and other toxic compounds. In mammals, the enzyme is widely distributed in many tissues as liver, placenta, lung and kidneys. In human tissues, GST isozymes are classified into three groups with different isoelectric points (pI values); the acidic, near neutral and the basic forms. Human liver has several forms of the isozymes. Among them, GST-e is one of the basic forms of the enzymes with pI 8.8 and occupies a large amount of hepatic GSTs. In this study, we prepared monoclonal antibodies specific to human GST-~ and, with this, investigated the distribution of this isozyme in human liver. To obtain the monoclonal antibody, we purified GST-e from human liver and immunized Balb/c mice with this antigen. Then hybridomas were prepared by fusion of mouse spleen cells and mouse myeloma cells (SP2/0-Ag 14), and were selected in HAT medium. Screening of specific antibody production was performed by enzyme-linked immunosorbent assay. After twice limiting dilution and agarose cloning, one hybridoma secreting specific monoclonal antibody (IgGl(r)) was cloned. In immunohistochemical study, the cytoplasm of human hepatic parenchymal cells was stained homogeneously by the prepared monoclonal antibodies. The nucleus and cell membrane was not immunostained in the hepatic cell. In human full term placenta, where the acidic isozyme of GST (GST-n) is located, the monoclonal antibodies did not show any positive immunostaining.

whereas the cells of the zona fasciculata and the zona reticularis were unstained. A faint positive staining for CA I could also be detected in the cells of the zona glomerulosa. The control stainings with preimmune and anti-CA VI sera were negative. The presence of CA II in the cells of the zona glomerulosa may be linked to the regulation of secretion or biosynthesis of mineralocorticoids.

P17.6

Location of a Membrane-Bound Carbonic Anhydrase Isoenzyme (CA IV) in the Reproductive Tract of the Male Rat. S. Parkkila ~, A.-K. Parkkila ~, K. Kaunisto ~, A. Waheed 2, W. S. Sly2 and H. Rajaniemi ~ 1University of Oulu, Department of Anatomy, SF-90220 Oulu, Finland. 2Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University Medical Center, School of Medicine, St. Louis, MO 63104, U.S.A. The acidification of rat epididymal fluid during its passage through the epididymis, a process thought to account for sperm quiescence in the cauda epididymis, has been tentatively attributed to bicarbonate reabsorption. As it has been suggested that CA IV participates in this bicarbonate reabsorption in kidney tubules, its location was studied in the reproductive tract of the male rat using specific polyclonal antiserum to rat CA IV and immunohistochemical techniques. CA IV-specific staining was detected in the ductal epithelium of the corpus and caput epididymis. The staining was most distinct in the brush border of the epithelial cells. The testis and the epithelium of the cauda epididymis, ductus deferens, seminal vesicle and ventral prostate failed to stain. The presence of CA IV in epididymis could be confirmed by immunoblotting which revealed 39K and 29K polypeptides. The results show that the ductal epithelium of the rat epididymis contains CA IV, the presence of which is probably linked to acidification of the epididymal fluid and thereby to the regulation o f sperm motility.

P17.5

Location of a High Activity Carbonic Anhydrase Isoenzyme (CA II) in the Zona Glomerulosa of Human Adrenal Gland. A.-K. Parkkila, S. Parkkila and H. Rajaniemi Department of Anatomy, University of Oulu, SF-90220 Oulu, Finland. Carbonic anhydase (CA) catalyzes the interconversion of carbon dioxide and bicarbonate. Accordingly, it is involved in physiological processes such as ion transport, regulation of acid-base balance, transport of carbon dioxide from cells and the conversion of carbon dioxide to bicarbonate to be utilized for biosynthetic reactions. In this study, the cytoplasmic isoenzymes (CA I and II) were purified from human erythrocytes using inhibitor affinity chromatography and ion exchange chromatography and polyclonal antibodies were raised to them. The enzymes were localized in the human adrenal gland using specific antibodies and immunohistochemical techniques. Specific staining for CA II was found in the cortical adrenal cells both with the fluorescence and the peroxidaseantiperoxidase complex method. The positive staining was localized to the cytoplasm of the cells of the zona glomerulosa,

P17.7

Light- and Immunoelectronmicroscopic Localization of Cytochrome P-450 Isoenzymes of the CYP4A Gene Family in Various Organs of Nafenopin Treated Male Rats. E. Persohn, H. Thomas and F. Waechter Ciba-Geigy Limited, Cell Biology, CH-4002 Basel, Switzerland. The cytochrome P-450 superfamily of genes code for a group of enzymes that catalyze the oxidation of numerous lipophilic endogenous and foreign compounds. The families 1, 2, 3 and 4 appear to be involved to a significant extent in the metabolism of xenobiotics. Among them, the CYP4A isoenzymes have been shown to be inducible by peroxisome proliferators (e.g. nafenopin) in rodents. In the present study we have investigated the expression of CYP4A proteins using monoclonal antibodies diagnostic for CYP4A1 (clol), CYP4A2/A3 (clo6) and CYP4A1/A2/A3 (clo4) in liver, kidney, ileum and lungs of rats treated with nafenopin by immunohistochemistry and immunoelectron microscopy. P-450s, reactive with clo4, clo6, and clol (except lung) are expressed in all organs. In the kidney CYP4A protein

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579

expression is restricted to the proximal convoluted segment. Although there is a visible immunoreaction in control animals, the treated animals show a much stronger immunoreaction in various organs indicating an induction of CYP4A proteins by nafenopin. At the subcellular level, clo4 and clo6 reactive P-450s are expressed in the SER of various cells. The results show the heterogenous distribution of the P450 isoenzymes CYP4A in various organs and cells and for the first time their location at the subcellular level.

P17.8 Immunolocalization of Cathepsin-D in Human Inflamed Gingiva. C. L. Ribaux*, A. Joffre*, D. J. Hartmann** and

C. Chavrier* *Facultd d'Odontologie, Lyon; **[nstitut Pasteur, Lyon, France.

Cathepsin-D, one of the most important lysosomal aspartic peptidase, is distributed in mammalian tissues, and can degrade type I and type III collagens in acidic conditions. Cathepsin-D activity was found in gingival fluid and in inflamed human gingival tissue of patients with advanced periodontal destruction. However, the localization of Cathepsin-D in gingival tissues has never been described. Therefore the present study was undertaken to examine the Cathepsin-D distribution in human gingival tissues, by using the immunohistochemical procedure. Samples were obtained during periodontal surgery, performed on patients showing high degree of gingival inflammation. Tissues were immediately washed and fixed, quickly frozen and routinely prepared for immuno-peroxidase procedure. The preparations were incubated with purified

P18. HISTOCHEMISTRY

OF THE NERVOUS

PIS.1 Motoneuron Plasticity Induced by Dorsal Rhizotomy. P. Ambrogini*, R. Cuppini, G. Fulgenzi and

P. Del Grande* Istituto di Scienze Morfologiche* Istituto di Anatomia e Fisiologia, UniversiM di Urbino, Italy.

Deafferentation causes a sequence of modifications in motoneuron synaptic inputs (S. Wang, M. Goldberger, M. Murray, J. Comp. Neurol. 304: 525-568, 1991). Moreover anatomical plasticity of motoneurons depends on its own activity (A. L. Connold, G. Vrbova, Neurosci. 34:525 - 532, 1990). In order to study the possible influences of sensorial inputs on motoneuron plasticity, the intramuscular motor axons and the neuromuscular junctions of the extensor digitorum longus (E.D.L.) muscle have been observed after homolateral dorsal rhizotomy in one month old rats. The animals were sacrificed at different times until twelve weeks from surgery. Unrhizotomized control rats were also sacrificed. Left EDL muscles of normal and deafferented rats were stained with Pestronk and Drachman's technique (A. Pestronk, D. Bo Drachman, Muscle Nerve 1: 7 0 - 7 4 , 1978) that demonstrates the neural processes through a silver impregnation and the endplate zones through a histoehemical reaction for the acetylcholine-esterase content. The presence

polyclonal rabbit anti-Cathepsin-D antibody (Andujar et al., Matrix 9, 1989), revealed with goat anti-rabbit IgG peroxidase conjugate, and subjected to the DAB. Post-fixation was made with osmium tetroxyde and the prepared tissues were embedded in Epon 812. Controls were incubated with PBS or non-immune rabbit serum instead of the primary antibody. With light microscopy, the Cathepsin-D was localized in the cytoplasm of fibroblasts and inflammatory cells. Electron microscopy revealed positive labelling in tysosomelike vesicles, distributed in the cytoplasm of fibroblasts and macrophages.

P17.9

Immunohistochemical Study on the Distribution of H,K-ATPase in Rabbit, Rat and Human Distal Colons. M. Takeguchi, S. Asano and N. Takeguchi Fac. Pharmaceu., ScL, Toyama Med. & Pharmaceu. Univ. Toyama, 930-01, Japan.

Using monoclonal antibody HK4001 that can completely and effectively inhibit the gastric H +, K +-ATPase activity, we have shown the presence of H § K+-ATPase in the crypt cells of rabbit distal colon (Gastroenterology,1990, Takeguchi, M. et al.): that is, HK4001 stained the crypt cells of rabbit distal colon. Monoctonal antibody HK4013 inhibits 70% of gastric K*-ATPase activity. But this antibody did not stain the crypt cells. We got also similar evidence in rat distal colon. But all these four antibodies stained crypt cells of human distal colon. We used a laser scanning confocal imaging system to obtain more detailed distribution and found that the H +, K+-ATPase in the crypt cells was not uniformly distributed.

SYSTEM of multiply innervated endplates and of terminal and nodal sprouts has been considered as an index of rearrangement phenomena. Among these parameters, nodal sprout occurrence resulted increased, and it reached the highest values on eight weeks after operation, when sometimes two sprouts arising from the same node of Ranvier were visible. Twelve weeks after deafferentafion the motor innervation pattern appeared like the controls.

P18.2 Subeellular Localization of Cytochrome B561 in Dog Splenic Nerve: Comparison with Synaptophysin. IV. G. Annaert, J. Quatacker, I. Llona, J. Pinxteren and

W. P. de Potter Lab. Neuropharmacology, Department of Medicine, University of Antwerp (UIAL Universiteitsplein 1, B-2610 Antwerpen (Belgium).

Both immuno-EM and fractionation techniques were used to study the subcellular localization in dog splenic nerve (control and ligated) of cytochrome b561 (CYT), a LDV-antigen, and to compare it with synaptophysin (p38), a SSV-antigen. Ultrastructural analysis of control splenic nerve segments revealed several organelle-types, like LDV, axonal reticulum (AR) elements and multivesicular bodies (MVB), all of which

580

Poster sessions

could be labelled with anti-CYT. Upon a 24 hrs ligation, LDV and AR were found densely packed in the proximal first segment of the ligation, while MVB accumulated almost exclusively distally from the ligation. No significant immunolabelling in these segments was found for p38. Similar results were obtained by immunoblotting. Ligation studies, performed at different time intervals (6, 12 and 24 hrs) showed a co-accumulation of both NA and CYT proximally of the ligation, and only of CYT distally. No accumulation on either side of the nerve crush was observed for p38. Instead, p38-containing membranes moved away from the side of the nerve crush as ligation-time increased. In adrenergic nerve axons, MVB account for the retrograde transport of retrieved LDV-membranes in the nerve terminal to probably the lysosomal compartment in the cell bodies. Both LDV and AR contribute in the anterograde transport of CYT but not of p38. Instead we propose that p38-containing membranes show a reversal of anterograde transport following a nerve crush. These results point to a different localization of p38 in adrenergic nerve axons compared to markers for LDV.

P18.3 Localization of Vasoactive Intestinal Peptide lmmunoreactive (VIP-IR) Structures within the Central Nervous System (CNS) of the Japanese Quail (C o r t u r n i x

japonica ).

N. Aste, G. C. Panzica, G. Cellino and C. Viglietti-Panzica Department of Human Anatomy and Physiology, Torino (Italy). VIP-IR structures have been detected within the CNS of vertebrates including birds. Nevertheless very few studies are concerned with the whole brain and the majority of them were performed after intraventricular colchicine injections. Recently this drug has been demonstrated to activate mRNA transcription for several neuropeptides in neurons that normally do not express them. For these reasons we have studied the VIP-IR neurons and fibres distribution in the whole brain of untreated adult quail. Paraffin sections of SUSA fixed brains were processed for immunocytochemistry (biotin-avidin technique) employing two antisera raised respectively against pig and chicken-VIP (Sanchez-Franco, Madrid, and Sharp, Edinburgh). The two antibodies gave a comparable pattern of immunoreactivity. Cell bodies were detected only within the organ of the lateral septum and in the caudal portion of the tuber. Using the chicken-VIP antibody also the paraventricular organ was stained. Dotted and varicose fibres were particularly dense in the paraolfactory lobe, in the lateral septum, in the tuberal region, in the external portion of the median eminence, in the nucleus of the solitary tract, and within the IX and X cranic nerve nuclei. Scattered IR-fibres were observed throughout the diencephalon and mesencephalon. Very few varicose fibres were observed within the raphe nuclei and the reticular substance of the ports. The pattern of cell and fibre distribution in the quail was comparable with that described previously in other avian species.

P18.4 Testosterone Effects on Neuropeptide Y Immunoreaetivity (NPY-IR) in the Medial Preoptic Nucleus (POM) of the Quail. N. Aste, G. C. Panzica, A. Fasolo ~ H. Vaudry*, J. Balthazart** and C. Viglietti-Panzica Dept. Human Anatomy & Physiol. and ~ Animal Biology, Torino, Italy; *Lab. Molec. Endocrinol., Rouen, Mont-SaintAignan, France; **Lab. Gen. Comp. Bioch., Lidge, Belgium. The quail POM shows high levels of NPY-IR, and its volume and cytoarchitecture are dependent on testosterone (T) levels. To investigate if the peptidergic supply of the nucleus is also influenced by T we performed a semi-quantitative analysis of NPY-IR. Adult quail of both sexes were either gonadectomized (CX) or gonadectomized and treated with T or left intact. Paraffin 10/am-thick sections were processed for NPY immunocytochemistry. The analysis was performed on the section showing the highest level of NPY-IR by an image analyzer developed in our laboratory. The POM, drawn on corresponding adjacent Nissl section, was divided in different portions of known area. The percentage of the sampling area covered by immunoreactive structures (fractional area: FA) was determined employing the threshold method after shading correction of the digitized image. The ratio of the number of threshold pixels to the number of pixels in the sampling area gave the value of the FA. The average of the individual field values was considered as the F A of each nucleus. The different fields within the nucleus were also analyzed separately according to their position (dorsal, central, and ventral, or lateral and medial groups). The data revealed a decrease in NPY-IR in CX males but statistical significance was reached only in the analysis of the lateral region. These data show that T can modulate the NPY-IR within the POM and that the analysis of values related to the whole nucleus can hide localized changes in immunoreactivity.

P18.5 Porcine Enteric Neurons Projecting to the Cranial Mesenteric Ganglion (CrMG), as Revealed by Retrograde Tracing Experiments. M. Barbiers, J.-P. Timmermans, W. Stach j, D. Adriaensen, M. H. A. de Groodt-Lasseel and D. W. Scheuermann Laboratory of Cell Biology and Histology, Department of Morphology, University of Antwerp, Groenenborgerlaan 171, B2020 Antwerp, Belgium and ~Institute of Anatomy, University of Rostock, Germany The retrograde tracers Fluorogold and Fast Blue were used to investigate projections from porcine enteric neurons to the CrMG. Two to four weeks after injection of the tracer into the CrMG, labelled cells were found in the myenteric and outer submucous plexus of duodenal, jejunal, ileal, caecal and colonic segments. The cells were mainly located in ganglia lying on the side of the mesenteric attachment and had a multidendritic appearance. The highest number of labelled cells was seen in the colon. Part of these colonic neurons were immunoreactive for CGRP or serotonin. Compared to the data obtained for small laboratory animals, similarities but also significant differences seem to be present in these intestino-intestinal reflex pathways.

Poster sessions

P18.6 Caleitonin Gene-Related Peptide and Substance P Immunoreaetivity in the Normal and Arthritic Knee Joint of the Mouse. P. Buma ~, C. Verschuren ', H. Versleyen ~, P. van der Kraan

and A. B. Oestreicher 3 ~Institute of Orthopaedics, Section Histomorphology, ~Laboratory for Experimental Rheumatology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. 3Rudolf Magnus Institute for Pharmacology, University of Utrecht, 3584 CH Utrecht, The Netherlands.

The localization of substance P (SP) and calcitonin generelated peptide (CGRP) fibres was studied in the knee joint of the mouse. The aim of this study was to describe the normal distribution of nociceptive fibres in detail and to obtain more insight into the changes in innervation which may be associated with degenerative processes in the joint. Degenerative arthrosis was induced by a single subpatellar intra-injection of bacterial collagenase. The distribution of fibres containing CGRP and SP was studied after staining with the peroxidase-antiperoxidase preembedding immunocytochemical method. Special attention was given to control experiments on the mouse brain as a reference for the effect of EDTA on the immunostaining. A rich innervation of thin varicose CGRP and SP immunoreactive fibres was found extra- and intra-articularly. After collagenase-induced osteoarthrosis the overall distribution of CGRP and SP fibres appeared to be the same as in the control joints. However, around the cruciate ligaments, in the synovium and other soft tissues around the patella and menisci, the CGRP and SP innervation was severely damaged or completely destroyed. With an antibody to the growth associated protein (GAP-43/B-50), signs of degenerated axonal profiles were observed. At other peripheral locations, such as the muscles, the GAP43/B-50 distribution was normal. Thus, the present study provides detailed information on the localization of neural nociceptive structures and the role of nerve fibres in pain perception. Knowledge on the changes which occur during arthrosis may give more insight into the presenting clinical symptoms.

PI8.7 IgG-Like Immunoreactivity in Rat Neurons In Vivo and In Vitro. J. Dai* and L. Ru Department of Neurosurgery. The Second Affiliated Hospital of Heng Yang Medical College. Hengyang. P.R. China. Department of Neurobiology. Tong Ji Medical University, Wuhan. P.R. China.

We studied the neurons in the central and peripheral nervous system for the presence of endogenous IgG. The representative brain, spinal cord, spinal ganglia, superior cervical ganglia sections of the normal and Freund's complete adjuvant (FCA) treated rats and Cultured rat spinal ganglia neurons were examined, using PAP immunohistochemical technique. The specific staining for IgG was observed in the cerebral Cortex, caudate-putamen, globus Pallidus, hippocampus, amygdala, thalamus, hypothalamus, cerebellum, brainstem, spinal cord, spinal ganglia and superior cervical ganglia. The destruction of the IgG-like immunoreactive (IgGLI) neural cell bodies and Fiber terminals in these areas have

581 been described. In addition, the intensity of the IgG-LI Product in most area in the FCA-treated rats increased obviously compared with that in the normal rats. Cultured spinal ganglia neurons prepared from newborn rats also contained IgG-LI products, but satellite cells did not. These results indicated that the neurons may have the function of synthesizing. Secreting (releasing) Immunoglobulins. Furthermore, this study is possible to give a new way of thought for elucidating the immune response mechanisms in the nervous system and explaining the immunopathologic mechanisms of neurological diseases.

P18.8 Origin of Brain Maerophages and Mieroglia in Mouse Central Nervous System, Using Combined NonRadioactive In Situ Hybridization and Immunoperoxidase Techniques. C. J. A. de Groot, W. Huppes*, T. Sminia, G. Kraal and

C. D. Dijkstra Dept. of Cell Biology, Div. Histology, Vrije Universiteit, van de Boechorststraat 7, 1081 BT, Amsterdam, *ITRI-TNO, Rijswijk, The Netherlands.

The origin of brain macrophages and microglia is still being debated. We have investigated the bone marrow origin of brain macrophages and microglia in the mouse central nervous system (CNS). Therefore we used bacteriophage lambda transgenic mice as donors for bone marrow transplantations in recipient mice of different ages. Bone-marrow chimeras were made, using transgenic mice with forty copies of bacteriophage lambda. The detection of a transgenic signal is achieved, after hybridization with a photobiotinylated DNA lambda probe on 8 lain cryostat sections. Using streptavidin alkaline phosphatase as a conjugate, we were able to obtain a purple signal, detectable for light-microscopy. This technique was combined with previous immunoperoxidase staining with moabs to identify brain macrophages (MOMA-1; Kraal et al., 1986) and microglia (Mac-l-a; Perry et al., 1985) to determine the phenotype of bone marrow-derived cells in the CNS. A large number of MOMA-l-positive brain macrophages, located in the leptomeninges and ventricles displayed the transgenic signal. However, only a few Mac-l-a-positive microglia were double-labelled. It can be concluded that: 1) brain macrophages should be considered as a distinct cell type of bone marrow origin 2)the majority of 'resting' microglial cells are of local, presumably neuroectodermal origin.

P18.9 Texture Features for the Determination of Alterations in Cytological Properties of Neurones. R. B. de Heus and P. C. Diegenbach Department of Experimental Zoology, University of Amsterdam, Kruislaan 320, 1098 SM, Amsterdam, The Netherlands.

Changes in Nissl substance organization in neurons that occur after axotomy, pharmacological treatment or following alterations in functional activity, have been correlated with changes in protein metabolism. Therefore in the present study structural changes of the Nissl substance in motoneurons, after spinal cord transection, have been quantified by using texture features. Texture features, i.e. parameters that can be

582 calculated from the texture of a digitized image, have already proven their usefulness in the quantification of structural changes of nuclear DNA contents. The selected motoneurons innervate the white musculature, of the European eel, are relatively large and occupy a constant position within each spinal segment. Consequently, because of their form and location they are readily recognisable from fish to fish. Microscopical images were digitized with a CCD camera attached to a Quick Capture Frame Grabber fitted in a Nubus slot on a Macintosh II computer. Frame grabbing and subsequent image analysis was controlled by the "gImage" program (developed by one of us; details available on request). With image analysis techniques and using a multivariate statistical approach we were able to discriminate, with high accuracy (up to 90%), between motoneurons belonging to control animals, fish that underwent cordotomy or silver eels. These subtle alterations of the Nissl substance organization, which are evident with image analysis, might be correlated with changes in protein metabolism and are already detectable one day after cordotomy. Texture features could be used with the advantage for estimating small changes in cellular substance that are invisible or not obvious to the human eye. Our results indicate that the use of texture features could be applied in neurobiology.

P18.10 Immnnohistochemical Localization of Neuropeptides in the Human Nucleus Cuneatus. M. Del Fiacco and M. Quartu Dept of Cytomorphology, University of Cagliari, Italy. Spinal ganglia neurons utilize numerous neuropeptides as transmitters or modulators of sensory stimuli. Contrary to the abundance of neuropeptide-containing nerve structures in the dorsal horn laminae, a relatively scarce peptidergic innervation is detectable in the dorsal column nuclei (Ljungdahl et aL, Neuroscience 3, 1978; Tamatani et al., Brain Res. 495, 1989; Conti et al., Neuroscience 34, 1990; Fabri et al., Neuroscience 34, 1990; Taber-Pierce et al., Neuroscience 15, 1985). We have previously reported on the peculiar localization of substance P (SP)-like immunoreactive material in the human fasciculus and nucleus cuneatus (NC) (Del Fiacco et al., Brain Res. 264, 1983; Neuroscience 12, 1984). Here we provide evidence for the presence of calcitonin gene-related peptide (CGRP)-, somatostatin (SOM)-, galanin (GAL)-, peptide histidine-isoleucine amide (PHI)-, and enkephalin (EK)-like immunoreactivity (LI) in the human nucleus cuneatus and compare their localization with that of SP-LI. Autoptic specimens of medulla oblongata, obtained within 35 hours post-mortem, are examined by the indirect single and double staining immunofluorescence technique. All peptides are localized to beaded nerve fibres, and dotlike or plexiform nerve terminals. A few SOM-IR perikarya are also observed. The immunoreactive elements appear restricted to discrete areas of the NC, placed close to the dorsal edge of the nucleus, or more or less completely isolated from it and embedded in the fasciculus. Double immunofluorescence shows that CGRP-, SOM-, GAL-, PHI, and EKLI are located in the same regions that contain SP-LI. Moreover, each peptide is distributed in a laminar pattern strikingly similar to that detectable in laminae I and II of the

Poster sessions dorsal horn (Schoenen et al., Neurology 35, 1985; FrancoCereceda et al., Peptides 8, 1987) and of the spinal trigeminal nucleus (Pioro et al., in: The Human Nervous System, ed. G. Paxinos, Academic Press, 1990; Quartu et al., in: Calcitonin Gene-Related Peptide, ed. Y. Tach~ et al., The NYAS, 1992). This work was funded by the Italian M.U.R.S.T. and CNR.

P18.11 Peptide-Immunoreactive Neuronal Structures in the Human Celiac/Superior Mesenteric Ganglionic Complex (CSM). M. Del Fiacco, A. Floris, M. L. Lai and M. Quartu Dept of Cytomorphology, University of Cagliari, Italy. This study extends previous work on the localization of neuropeptides in human sympathetic ganglia (Helen, P. et al., Neuroscience 12, 1984; Del Fiacco M. et al., Brain Res. 321, 1984; Levanti, M. C. et al., Bas. Appl. Histochem. 32, 1988) by providing evidence for the presence of somatostatin (SOM)-, vasoactive intestinal polypeptide (VIP)-, peptide histidine-isoleucine (PHI)-, and galanin (GAL)-like immunoreactivity in the human CSM, and by comparing their localization with that of substance P (SP). Autoptic specimens of CSM were obtained from subjects at perinatal and adult life stages, within 35 hours post-mortem, and examined by the indirect immunofluorescence technique. All peptides were localized to nerve fibres and terminals; SOM- and VIP-like immunoreactive (LI) cell bodies were also observed. A high number of VIP-, PHI- and SOM-LI varicose and non varicose nerve fibres were seen running in thick bundles, and arranged in plexuses among and around the principal ganglionic perikarya. As a whole, GAL-LI structures appeared to be less abundant than the other peptide-LI elements. Double immunostaining showed that a high proportion of PHI- and GAL-LI structures also contained SP, whereas the coexistence of SOM and VIP with SP was less frequently observed. As to the origin of the peptide-containing fibers, data obtained in the laboratory animals induce to suppose that a conspicuous proportion of the peptide-positive elements also containing SP is of sensory origin. Furthermore, preganglionic sympathetic neurons and intramural enteric ganglia may well represent an important source of the peptidergic innervation observed. The results obtained point out that the human CSM receives a complex input of peptide-containing elements and suggest a role for them in the functional regulation of neuronal activity within the prevertebral sympathetic postganglionic neurons. This work was funded by the Italian M.U.R.S.T. and CNR.

P18.12 A Combined NADH-Diaphorase and Acetylcholinesterase Enzyme-Histochemicai Assay: An Easy-to-use Method for the Demonstration of Nearly all Enteric Nerve Cells in the Fixed Small Intestine of Fetal and Postnatal Pigs. E. F. de Ridder, M. J. de Smet and A. L. Weyns Lab Vet Anat & Embr, Dept Morphology, Fac. of Meal., State Univ. Cent. Antwerp, RUCA, Slachthuislaan 68, B-2060 Antwerpen, Belgium.

Wanting to count the total number of enteric nerve cells in

Poster sessions whole mount preparations of formaldehyde-fixated fetal porcine small intestine, we encountered several problems, such as: the need for an optimal tissue-penetration (immunohistochemistry, e.g. for NSE), the need for fresh tissue in, for example, the NADH-diaphorase-method and above all that the techniques we knew obviously did not show all neurons. We performed a NADH-diaphorase enzymhistochemical assay (cfr Thomas, t981) on fixated material with success, but still numerous holes in the ganglia indicated non-stained neUl~OllS.

We then tried out acetylcholinesterase enzyme-histochemical assays according to Hanker (1973) and according to Baljet et al (1975). The Baljet-technique showed too much background-staining of glial and smooth muscle cell nuclei. The Hanker-technique showed a better signal but was more labourous and still did not show the requested staining ~pecificity and intensity. tn our laboratory, we developed a technique inbetween of the two mentioned above. Although the staining specificity and intensity were alright that time, still not all neurons were marked. The best results until now were obtained by combining the two enzyme-histochemical methods (NADH-diaphorase and acetylcholinesterase). In these preparations no holes were left in the enteric ganglia, suggesting that apparently all nerve cells were marked. By the time this abstract was written, the actual counting still had to begin.

P18.13 Analysis of Cell Differentiation and Turnover in the Olfactory System by a Novel Double Staining Technique for Gene Expression and DNA Synthesis. A. Fasolo, S. Biffo, I. Perroteau, L. Verdun and

M. Sasso~ Pognetto Dip. Biotogia Animale, Via A. Albertina 17, 10123 Torino, Italy.

A novel histochemical procedure for the simultaneous detection of m-RNA expression (by in situ hybridization with digoxigenin labelled riboprobe) and DNA synthesis (by immunohistochemicai detection of bromodeoxyuridine -BrdU- incorporation) was used for evaluating the correlation between the onset of calmodulin (CAM) m-RNA expression and the birth date or the turnover rate of olfactory neurons. This technique results in good cellular resolution, highly sensitive BrdU immunocytochemical detection and permits a long term pulse and chase with BrdU. During postnatal development of the olfactory system, we show that CaM mRNA expression occurs in tufted and granule neurons five days after their -putative- final mitotic division. We also evaluated in the adult olfactory neuroepithelium the differential kinetics of proliferation, differentiation and survival of primary olfactory neurons either in the presence or absence of their target. By double staining methods for calmodulin m-RNA and BrdU incorporation we show that in the target deprived side olfactory neurons are completely eliminated by 35 days after the BrdU pulse. In contrast, in the not target deprived neuroepithelium many olfactory neurons double labelled for catmodulin m-RNA and BrdU incorporation were seen 50 days after BrdU pulse. (Supported by CEE B/SC1 CT900551, CNR and MURST).

583

P18.14 Differentiation of Dopaminergic Neurons in the Mediobasal Hypothalamus an Immunocytochemical and Radioautographic Study. S. O. Fetisov, M. V. Ugrumov, At P. Popov and

J. Thibault* Institute of Developmental Biology, Russ. Ac. Set., Moscow, Russia; *College de France, Paris, France.

Earlier studies showed that dopamine (DA) synthesized in the neurons of the mediobasal hypothalamus (MBH) is responsible for the regulation of prolactin secretion by the adenohypophysis. The present study evaluated the differentiation of dopaminergic (DA) neurons of the MBH in rats both in vivo and in the graft introduced to the infundibular recess of the 3rd cerebral ventricle. Immunocytochemistry of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC), the key enzymes of the DA synthesis, as well as the radioautography of the [3H]dopamine uptake were used to solve this problem. TH-immunopositive (IP) neurons were first observed in the MBH in fetuses, four days before birth. These uni- and bipolar neurons, small in size, possessed scanty cytoplasm with poorly developed organelles: Golgi complex, granular endoplasmic reticulum, etc. Further differentiation of the neurons both in vivo and in the graft was manifested in their enlargement, sending axons either to the target neurons or to the hypophysial portal circulation, expressing AADC, etc. After birth, DA axons gave rise to the specialized contacts: a) symmetric and asymmetric synapses with dendrites and cell bodies in the arcuate nucleus; b) axo-axonic contacts in the median eminence; c) synaptoid contacts with basal processes of tanycytes; d) axo-vascular contacts with the primary portal plexus. According to our radioautographic data, the membrane mechanism for DA transport was functionally active at the level of the axonal terminals as early as during the intrauterine development, while at the level of the cell bodies it became evident only after birth. The special morphometric analysis showed no principal differences in size, form, space orientation and ultrastructure of the DA neurons differentiated either in vivo or in the graft. Thus, the DA neurons of the MBH underwent similar differentiation both in vivo and in the graft showing the predominant genetic determination of their phenotype expression.

P18.15 Synapses of GABA-Containing Elements with Other Identified Structures in the Inner Plexiform Layer of the Anuran Retina. R. Gdbriel Deportment of Anatomy and Histology, The Flinders University of South Australia, GPOBox 2100~ Adelaide, 5001, SA, Australia.

Synapses of GABA-immunoreactive (IR) amacrine and bipolar ceils (AC & BC) with other GABA-eontaining structures as well as with serotonin (5HT)- and tyrosine hydroxylase (TH)-IR amacrine cell processes and retrogradety filled ganglion cell (GC) dendrites were examined~ GABA-IR AC dendrites occupied both pre- and postsynaptic positions at all BC terminals, including the GABA-IR BC terminals. Contacts with BCs mostly occured in sublaminae (SL) 1 & 2 of

584 the inner plexiform layer (IPL). GABA-GABA ACs represented 11~ of all GABAergic synapses. GABA-IR AC dendrites synapsing on or receiving synapses from TH-IR dendrites were also frequently observed both in SL1 and 5 of the IPL. Some of the 5HT-IR amacrine cell dendrites were IR also for GABA. Both types of 5HT-positive profiles received inputs from and provided outputs to GABA-containing elements. However, double-labelled profiles have never been in presynaptic position to BC terminals. GABA-IR AC dendrites synapsed on retrogradely filled GC dendrites preferably in SL3, 4 & 5. Some of the GC dendrites that received GABA-IR AC synapses were also IR for GABA. The results show, that (1) BCs are under significant GABAergic control; (2) the GABA-IR ACs are heterogenous and that the GABA-GABA AC interactions which may lead to disinhibition of GCs, are frequent; (3) 5HT- and TH-containing ACs are able to influence the GABAergic neurons and vice versa; (3) a subset of 5HT-IR ACs contains GABA, and that this population is also linked to other GABAergic neurons; (4) ON-centre GCs receive more substantial direct GABAergic inhibition; and (5) GABA-containing GCs are controlled by direct GABAergic synapses from ACs that may be able to remove the inhibitory component from the projections to the visual centres.

P18.16 Seasonal Changes in Acetylcholine Level and Activities of Cholinergic Enzymes in the Alimentary Tract and Heart of the Edible Frog, Rana esculenta L. R. Gdbriel ~* and D. Budai 2 ~Department of Zoology, Attila Jdzsef University and 2Central Research Laboratory, University School of Medicine, Szeged, Hungary. *Present address: Department of Anatomy and Histology, The Flinders University of South Australia, Adelaide, A ustralia. The level of acetylcholine and the activity of cholineacetyltransferase and acetylcholinesterase enzymes were measured in the stomach and intestinal musculature and in the heart muscles of frog from the hybernation (January-stage 1) through the low activity (April-stage 2) to the highest motility level (July-stage 3). Identical parameters were measured from the brain homogenates of the same animals for control. The acetylcholine level doubled in the stomach from stage 1 to 2, then little change occurred. In the intestine, the acetycholine level has changed little during the stages, while slightly decreasing levels were found in the heart from stage 1 to 3. Significant increase in acetylcholine levels were found in the brain during the period observed. The level of cholineacetyltransferase increased in all tissue samples during the time. The acetylcholinesterase activity was not significantly different during the stages in the stomach and intestine. In the heart, a 2.5 fold increase was measured in April compared to January, followed by a sharp decrease to the half of the April by July. The activity of this enzyme increased continuously from January to July. The results show that these cholinergic parameters change differentially in different organs of in this poikiloterm animal during the year, resulting in a fine control of the viscera, probably in cooperation with other autonomic transmitter systems.

P o s t e r sessions

P18.17 Fine Structure of Galanin-Like Immunoreactive Neuronal Elements in the Rat Preoptic Area and Median Eminence in Comparison with that of LHRH-Like Immunoreactive Elements. Y. lbata, Y. Ichitani, M. Tanaka* and H. Okamura Dept of Anatomy and *Anesthesiology, Kyoto Pref. Univ. of Med., Kyoto 602, Japan. Galanin-like immunoreactive neurons are distributed throughout the preoptic area (POA). LHRH-like immunoreactive neurons are also distributed in the P O A particularly in its ventrolateral portion, and L H R H and galanin are reported to coexist in some of the neurons of this area. In this present paper we studied the fine structure of galanin-like immunoreactive neurons in the P O A by immunoelectron microscopy in comparison with that of LHRH-like immunoreactive neurons. Galanin-like immunoreactive neurons showed numerous shapes such as oval, triangle and fusiform although almost of LHRH-Iike immunoreactive neurons showed fusiform shape. In electron microscopic level, galanin-like immunoreactive neurons showed welt development of cell organellae such as rER and mitochondria. In addition, dense granules (about 100 nm in diameter) were disseminated throughout the cytoplasm. Galanin-like immunoreactive axons were detected to make synaptic contacts with non-immunoreactive dendrites particularly in the lateral part of the POA. Nonimmunoreactive axons were also found to make synaptic contacts with galanin-like immunoreactive dendrites of dendritic spines. We also scrutinize the fine structure of galanin-like immunoreactive endings in the external layer of the median eminence particularly in its lateral parts in which LHRH-like immunoreactive endings are mainly distributed.

P18.18 Degeneration of the Nigral Dopamine Neurons after 6-Hydroxydopamine Injection into the Monkey Neostriatum. Y. Ichitani, M. Tanaka*, H. Okamura and Y. Ibata Department of Anatomy and *Anesthesiology, Kyoto Prefectural University of Medicine, Kyoto 602, Japan. Nigrostriatal dopamine (DA) neuron system plays an important role in modulating voluntary movement, and the impairment of this neuron system causes severe movement disorders like Parkinson's disease. We previously reported that 6-hydroxydopamine (6-OHDA), a neurotoxin which selectively destroys catecholamine (CA) neurons, injected into the rat striatum caused degeneration of the nigral DA neurons. In the present study, we investigated whether a similar phenomenon can be observed in monkeys. Japanese monkeys (Macaca fuscata) were given unilateral 6-OHDA injection into the neostriatum (head of the caudate nucleus and the putamen, total dose: 1.8 mg) using stereotaxic apparatus. After 1 - 2 months, animals were perfused and then brain sections were treated for ABC immunohistochemistry of tyrosine hydroxylase (TH), which was employed as a marker for CA neurons. The density of TH-immunoreactive terminals on the 6-OHDA injected side of the neostriatum greatly decreased. At the same time, there was a great decrease in the number of TH-immunoreactive neurons and dendrites

Poster sessions on the injected side of the substantia nigra (SN) compared with that on the control side. Nissl staining of the same region showed a severe cell loss in the pars compacta of SN. These results suggest that 6-OHDA taken up from dopaminergic terminals in the neostriatum may produce degeneration of the nigral DA neurons in the monkey.

P18.19 Relationship Between Galanin and Estrogen Receptor Containing Neuronal Structures as Revealed by Immunocytochemistry. L I(alld, C. W. Coen*, C. Fekete, B. Flerk6 and Z. Liposits Department of Anatomy, Electron Microscopic Laboratory, University Medical School, Pdcs, Hungary and *Division of Biomedical Science, King's College, London, England. A great number of estrogen receptor expressing neurons, and galanin immunoreactive structures have been demonstrated in the rat preoptic area (POA). The high density of galanin binding sites in this region suggests that galanin may affect preoptic neurons as a neurotransmitter or modulator. To determine whether estrogen receptor immunoreactive (ER-IR) neurons are postsynaptic targets of galanin-IR terminals, immunocytochemical double labelling was carried out in ovariectomized rats at the light and electron microscopic Jevels. In the periventricular and medial preoptic nuclei the distribution of galanin containing neuronal structures, detected by the black, silver precipitate, overlapped that of ER-IR cell nuclei, visualized by the brown diaminobenzidine chromogen. Semithin sections revealed that the contour of E R 4 R neurons were surrounded by GAL-IR axon terminals. At the ultrastructural level, these juxtapositions have been proved to be symmetric axo-sornatic synaptic connections. These findings indicate that in addition to the circulating estrogens, galanin containing afferents may also influence - via synaptic mechanisms - - the function of ER-expressing cells of the preoptic area.

P18.20 Histochemistry of Lectins in Lafora Type Myoclonus Epilepsy. B. Krijne-Kubat ~, L. Gerhard ~, K. Kubar V. Reinhard ~ Inst. of Neuropathology, Essen, Germany I, Inst. of Pathology, Nijmegen, The Netherlands? Progressive myoclonus epilepsy with Lafora inclusion bodies (LME) is a rare hereditary metabolic disease characterized by polyglucosan inclusions in the neurons of the central nervous tissue, heart, liver, striated muscles and sweat glands. We investigated the myocardium of four patients with LME by means of histochemistry and a panel of eight sugar-binding glycoproteins (lectins). The histochemical investigation revealed medium size branched polyglucosan, containing neutral mucopolysaccharides (MPS) with varying resistance to amylase digestion, and acid MPS with varying content of sulphate groups. In one case frozen material was available in which no lipids but small amounts of proteins were demonstrated. The dehydrogenases SDH, LDH, fl-HBDH and NADH: TR as markers of myocardial metabolism showed a decreased activity, most pronounced in SDH, indicative of a

585 severe metabolic impairment. Immune histochemistry of the lectins PNA, SBA, BSA I, DBA, UEA I, Con A, BPA and WGA, and the specific sugar inhibition tests demonstrated D-galactosamine, /3-D-galactose (1 - 3) galaetosamine, a-L-fucose, D-glucosamine, a-D-glucose, a-D-mannose, D-galactose and sialic acid in the polyglucosan chains. We discuss the implications of the findings with regard to the composition of the stored material and the underlying metabolic error.

P18.21 UItrastructural Analysis of Serotonin-Immunoreactive (5-HT-IR) Axons in the Rat Mesencephalic Trigeminai Nucleus (Me5). R. S. B. Liem, J. C. V. M. Copray and J. D. van Willigen Dept. of Neurobiology, University of Groningen, The Netherlands. The mesencephalic trigeminal nucleus (Me5) contains primary afferent neurons innervating the masticatory muscle spindles and periodontal mechanoreceptors. They are involved in the jaw-jerk reflex and in the control of mastication. Recently, we have demonstrated the presence of synaptic input on Me5 neurons. To establish the 5-HT component of this input, a light and electron microscopic immunocytochemical method was employed. The aim was to gain insight in the fine structure and distribution of 5-HT-IR fibres and terminals in the rat Me5. Two types of 5-HT-IR terminals were distinguished: (1) 5-HT-IR terminals, containing small round (0 = 2 0 - 4 0 urn) vesicles and (2) 5-HT-IR terminals with pleomorphic and granular (0 = 5 0 - 160 am) vesicles. Examination of 355 serotoninergic terminals revealed that 93% formed synaptic contacts with dendritic shafts: 68% on small and 25070 on large dendrites. The remaining 7% formed axosomatic synapses. The results suggest that: (1) primary afferent neurons of Me5 receive synaptic input from terminals that show immunoreactivity for 5-HT. (2) The main targets of 5-HT-IR terminals are small dendrites; most likely these are distal dendrites originating from multipolar Me5 neurons. (3) 5-HT input in Me5 may affect the transfer of sensory signals from jaw-muscle spindle proprioceptors and periodontal mechanoreceptors and so ultimately influences orofacial motor activities.

P18.22 Immunocytochemicai and Golgi Study of the Substantia Nigra in the Normal and Pathological Infant Brain. E. G. Markova*, V. Howard**, D. van Velzen** *Brain Research Institute Russian Academy o f Medical Sciences, per. Obukha 5, Moscow, 103064, Russia; **University of Liverpool, P.O. Box, 147, Liverpool, L69 3BX, England. As it is known the substantia nigra (SN) is the largest dopaminergic (DA) center of the brain and plays an important role in the regulation of motor functions and homeostatic mechanisms. The aim of this study was to analyse the neurons and astrocytes of the SN in the infants with the diagnosis of intrauterine growth retarded (IUGR) syndrome using Golgi and TH-, GFAP-imrnunocytochemical methods. For quantitative estimation of DA-neurons and GFAP-posifive astrocytes a new method of grey level image analysis was used.

586 This method is effective for the extraction of low contrast objects located at uneven background. The Golgi-impregnated neurons of the SN were analysed in three dimensions with the aid of the microscope computer system "Orthoplan-3D". A set of metric and topological parameters characterising the shape and size of the somata and also the dendritic pattern of neurons was estimated. The results have shown the hypertrophy and intensive GFAP-reacfion of astrocytes. On the basis of quantitative data obtained it was revealed the retarded development of the dopaminergic neurons of the SN in the IUGR cases compared to the normally developed infant brain.

P18.23 Subcellular Localization of Low-Affinity Nerve Growth Factor Receptor-Immunoreactive Protein in Injured Cerebeilar Purkinje Cells of the Adult Rat. R. Martihez-Murillo, L. Caro and J. Rodrigo Dpt. Chemical Neuroanatomy, Cajal Institute, CSIC, A vda. Doctor Arce 37, 28002-Madrid, Spain.

Purkinje cells in the neocerebellum of normal adult rats rarely express low-affinity nerve growth factor receptor immunoreactivity, as revealed by immunocytochemistry using the monoclonal antibody 192- IgG. However, a number of these cells were found to significantly increase their expression of low-affinity nerve growth factor receptor immunoreactive protein in response to injury. Identified stained structures by light microscopy were analyzed under the electron microscope. In reactive Purkinje cells, immunoreactivity occurred in subcellular organelles involved in synthetic processes (the Golgi apparatus and the rough endoplasmic reticulum) and in recurrent collaterals of Purkinje cell axons. Thus, it is suggested that up regulation of low-affinity nerve growth factor receptor synthesis may be essential in regulating Purkinje cell plasticity during adulthood. Research supported by CAM (C179/91) and FIS (92/0269).

P18.24 Vasoactive Intestinal Polypeptide (VIP) Immunoreactive- and Binding-Sites in the Hamster and Guinea-Pig Seminal Vesicle. G. Rodrigues", M. S. Pinh&*, L. Mata~ and S. Gulbenkian a "Department of Cell Biology, Gulbenkian Institute of Science, Oeiras, Portugal, *Faculty of Veterinary Medicine, Lisbon, Portugal. The distribution of VIP immunoreactivity and VIP bindingsites was studied using light microscope immunohistochemical and autoradiographic techniques, respectively. For immunostaining, the tissues were removed from adult animals, fixed in Zamboni's, processed for cryostat sectioning (15 tam thick) and stained using an indirect immunofluorescence procedure. For autoradiography, 15 taM thick frozen sections were incubated with 0.125 nm '25I-VIP either in the absence or in the presence of 1.250 taM unlabelled VIP to assess either specific or non-specific binding, respectively. The sections were then covered by dipping in LM-1 emulsion and exposed for 4 - 5 days. In guinea-pig seminal vesicle, VIP immunoreactivity was found in both muscle and mucosal layers whereas in the

Poster sessions hamster gland it was predominantly located in the mucosal layer. VIP specific binding-sites were detected in the secretory epithelium of both species. It was also observed that the density of labelling was relatively higher in the guinea-pig than in the hamster epithelium. The present results provide morphological evidence that VIP-containing nerves may play an important role in the regulation of secretion in seminal vesicle epithelial cells of both the hamster and guinea-pig.

P18.25 Immunohistochemical and Ultrastructural Studies of the Development of Astrocytes in the Midbrain of the Lizard Gallotia galloti. M. M. Mayor*, C. Yanes**, R. R. Sturrock t, J. de Barry ~t

and G. Gombos t* *Dept. de Morfologra (Histologfa). F.C.M.S. Univ. Las Palmas de G.C. lslas Canarias, Spain. **Dept. de BiologFa Celular. Facultad de BiologYa. Univ. La Laguna. Tenerife. Islas Canarias, Spain. *Dept. of Anatomy and Physiology. Univ. of Dundee. Scotland. U.K. ~Centre de Neurochimie. C.N.R.S. Strasbourg. France.

Astrocyte development was investigated electron microscopically and immunohistochemically (Glutamine synthetase) in the midbrain of the lizard from E32 to adult. With E.M. very immature glioblasts were present until hatching. From E34 until adult could be identified by dark and early glioblasts. The radial glia was observed at E35. Astroblasts which could be identified by the characteristic rough endoplasmic reticulum appeared at E35. With increasing age the quantity of gliofilaments in astrocyte cytoplasm increased. Astrocytes in the adult white matter contained very large amounts of gliofilaments and some glycogen granules, whereas those in grey matter contained many fewer gliofilaments. During ontogeny, Glutamine synthetase (GS) immunoreactivity appeared in radial glial processes at E35, then increased until hatching and decreased until adult, but it did not disappear. Labelled scattered star and pear-shaped cell bodies became progressively more numerous and immunoreactive and their processes formed perivascular end feet. At E35 one of the earliest signs of differentiation is the appearance of the endoplasmic reticulum with floculent material, feature observed with E.M. and GS, that verify the nature of this marquer in cells that begin to differentiate. With two techniques verified that the radial gila is abundant during development and decreased progressively in adult. In the same way in reptilia the "sulcus" produced astrocytary cells from E40 until adult. These facts could be to indicate the procedure of two types of astroglial cells from the radial gila or from "proliferative zones" called "sulcus".

P18.26 GABAergic Projection from the Nucleus of the Optic Tract to the Superior Coiliculus. A Combined Traeing and Immunocytochemical Study. B. Nunes Cardozo and H. van der Want The Netherlands Ophthalmic Research Institute. Amsterdam, The Netherlands.

The nucleus of the optic tract (NOT) receives direct input from the retina and is primarily known for its role in gaze

Poster sessions stabilization. Slow movements of the visual surround lead to series of slow compensatory eye movements which are interrupted by fast conjugated eye movements, essentially saccades to reset the eye position. This is called the optokinetic reflex (OKR). The superior colliculus (SC) is known for the generation of saccades. Pharmacologically it has been shown that GABA plays an important role in the slow phase of the OKR in the NOT. Electron microscopical immunocytochemical studies have demonstrated that GABAergic neurons in the NOT resemble the description characteristic for projection neurons. The present study was undertaken to examine a possible GABAergic projection from the NOT to the SC which might be involved in the fast phase of the OKR. Retrograde tracing of cholera toxin conjugated to 7 nm gold particles and GABA post embedding colloidal gold immunocytochemistry showed that the projection the NOT to the SC comprises a number of neurons which are GABAergic. GABAergic projection neurons and a population non-GABAergic neurons are inhibited by GABA. Furthermore it is shown that some GABAergic and some non-GABAergic neurons make synaptic contacts with retinal terminals. The presence of a dual projection from the NOT the SC suggests an inhibitory and an excitatory influence on the fast resetting component of the OKR.

P18.27

Localization of Intracellular Neuronal Proteins (B-50 and MAP2) by Pre-Embedding Double Immunolabelling Detected with Gold Probes. A. B. Oestreiche/, M. van Lookeren Campagne2, A.

Marquart ~, W. H. Gispen x and A. J. Verkleij 3 ~Rudolf Magnus Institute, 3Department of Molecular Cell Biology, University of Utrecht, Utrecht, and 2Netherlands Institute for Brain Research, Amsterdam, The Netherlands.

The ultrastructural localization of B-50/GAP-43 and MAP2 was studied in cultured hippocampal neurons. Neurons were fixed in PGC buffer (4~ paraformaldehyde, 2 or 0.05o70 glutaraldehyde (G1) dissolved in cacodylate at pH 7.6). The first incubation (overnight) was with mouse monoclonal MAP2 immunoglobulins (IgGs). Detection was with a goat anti-mouse IgGs coupled to 1-nm gold which was silver enhanced. Second, an overnight incubation with rabbit B-50 IgGs followed, detected by goat anti-rabbit IgGs coupled to 5-nm gold. After postfixation in 2.5o70 Gt and 0.5% OsO4, cells were examined and photographed for LM, contrasted in 0.5 % uranyl acetate and embedded in Epon. Neurons cultured for 8 days in vitro extend a network of dendrites and axons. The dendritic marker MAP2 was readily observed in LM due to silver enhancement. In EM, MAP2 was located on microtubules inside dendrites. The B-50 labelling with the 5-nm gold was only observable by EM. B-50 was predominantly found at the plasma membrane of axons and, sometimes, at the cytosolic face of 100-nm electron lucent vesicles (elv). In single immunolabelling for B-50 in combination with silver enhanced 1-nm probe, B-50 was detected more frequently on elv. The use of 0.05~ Triton X-IO0 to promote penetration did not increase B-50 immunolabelling. In conclusion, we show that fixation of cultured neurons in PGC buffer allows the LM and EM detection of intracellular

587 antigens by pre-embedding immunolabelling using 5-nm and silver enhanced l-ran gold probes. Under these conditions, use of the larger diameter (5-nm) gold probe appears to restrict the B-50 detection to the plasma membrane.

P18.28 The Ultrastructural Evidence of GABAergic Neurons Participating in Transmission and Integration of the Proprioception of Fifth Nerve of Rat. P. Luo and J. Li Neuromedieine Center of Pearl River Hospital, Guangzhou 510282, P.R. China.

The present study was designed to explore the GABAergic neurons in the dorsomedial part of trigeminal principal sensory nucleus (Vpdm) and the caudolateral part of supratrigeminal nucleus (Vsup CL) are in charge of transmission and integration of proprioception of the fifth nerve by using the methods of Ricin transganglionic degeneration, HRP retrograde tracing combined with antiGABA immunocytochemistry. The trigeminal mesencephalic neurons(Vme) innervating jaw-closing muscle spindles were transganglionic degenerated by Ricin injection of masseteric nerve. HRP-labelled, GABA-positive and HRP-GABA double labelled neurons were observed simultaneously in the Vpdm and Vsup CL after HRP injection of ventroposteriomedial nucleus of thalamus and anti-GABA immunocytochemical staining. Under electron microscope, the degenerated teminals from the Vme synapsed with HRP-GABA double labelled soma and dendrites of the Vpdm and Vsup CL. The degenerated terminals and GABA-positive terminals containing clear round vesicles did also formed synapses simultaneously with HRP-GABA double labelled, GABApositive or non-labelled dendrites respectively. Some complicated synaptic complex, axo-axo-dendritic synaptic triad among these degenerated terminal, GABA-positive terminal and dendrite and HRP-GABA double labelled terminal and dendrite were also encountered. The present results provide strong evidence that the GABAergic thalamic projecting neurons in the Vpdm and Vsup CL directly charge of transmission of proprioception of the fifth nerve in the rat. GABAergic interneurons in the neuropil of the Vpdm and Vsup CL play an extensive integration role to the proprioceptive afferents. The integration mechanisms included inhibition, disinhibition, recurrent inhibition and presynaptic facilitation.

P18.29 An Anatomical Evidence of GABAergic Neurons Involved in Transmission and Integration of Nociceptive Information of Jaw-closing Muscles. P. Luo and J. Li Neuromedicine Center of Pearl River Hospital, Guangzhou 510282, P.R. China.

An anatomical evidence of GABAergic neurons in the caudalis of trigeminal spinal tract nucleus (Vc) involving in transmission and integration of the nociceptive information of jaw-closing muscles was provided in the rat by using combined Ricin transganglionic degeneration, HRP retrograde transport and immunocytochemieal study. After Ricin injection of the

588 masseteric nerve, the minor ganglionic neurons innervating nociceptive afferent of jaw-closing muscles were degenerated. HRP-labeled, GABA-positive and HRP-GABA double labelled cell bodies were observed in lamina I and II of the Vc. In the neuropil of laminal and II of the Vc, degenerated terminals from the minor ganglion synapsed with large and middle sized dendrites of HRP-GABA double labelled neurons. Degenerated terminals formed axo-dendritic synapses with GABA-positive or non-labelled dendrites. GABA-positive terminals also made axo-dendritic or axosomatic synapses with H R P labelled, GABA-positive a n d / o r HRP-GABA double labelled soma and dendrites. The present ultrastructure evidence demonstrated that GABAergic thalamic projecting neurons of the Vc directly control transmission of nociceptive messages afferent of the jawclosing muscles. Meanwhile, GABAergic interneurons contributed to integrate this transmission via presynaptic and postsynaptic inhibitory mechanisms.

P18.30 The Distribution and Morphology of GABAergic Trigemino-Thalamie Projecting Neurons in the Trigeminal Sensory Nuclear Complex (TSNC) of Rat. P. Luo and J. Li Neuromedicine Center of Pearl River Hospital, Guangzhou 510282, P. R. China. The distribution and morphological characteristics of GABAergic trigemino-thalamic projecting neurons in the TSNC were examined in the rat by using a double labelling technique o f H R P retrogradely transport combined with antiGABA immunocytochemistry. After HRP injection contralaterally into the ventral posteriomedial nucleus of thalamus and anti-GABA immunocytochemical staining, HRP-labelled, GABA-positive and H R P - G A B A double labelled neurons were observed in the TSNC extensively. However, different distributional and morphological features and proportions of HRP-GABA neurons were noted in each nucleus of TSNC. In the caudalis of trigeminal spinal tract nucleus (Vsp), 38.6% of HRP-GABA neurons were mainly distributed in the lamina I and II which had fusiform and triangular cell bodies and long dendrites. In the interpolaris, 40~ H R P - G A B A neurons were mostly distributed in the ventrolateral part and had triangular perikarya. A small amount of HRP-GABA cells were merely located in the ventral part of oralis of the Vsp. 20-30~ HRP-GABA neurons were predominantly located in the dorsomedial part of the principal sensory nucleus which had fusiform, triangular and multipolar perikarya. It was demonstrated that there are two kinds of GABAergic neurons in the TSNC, GABAergic local circuit interneurons and projecting neurons. They might be in charge of integration and transmission of the pain, temperature, touch, depressor and proprioceptive information afferents of fifth nerve. The findings provided for the first time, a new anatomical evidence for GABAergic neurons as long distance projecting neurons.

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P18.31 A Protein Immunologically Related to H + - K +ATPase in the Human Brain. K. Renkawek, G. J. C. G. M. Bosman, F. P. van Workum, T. J. F. van Uem and J. J. H. H. M. de Pont Institute of Neurology and Department of Biochemistry, University of Nijmegen, The Netherlands. The H + and K § transporting ATPase from parietal ceils in the gastric mucosa (H +/K § EC 3.6.1.36) exchanges protons for potassium ions upon hydrolysis of ATP. A polyclonal antibody was raised against pig gastric mucosa H+/K+-ATPase. This antibody recognizes the asubunit (Mrs95 kD) and the 3-subunit ( 6 0 - 8 0 kD) of H+/K+-ATPase, but does not react with kidney Na+/K +ATPase or with sarcoplasmic Ca 2+-ATPase. Human temporal and hippocampal cortex were obtained 4 - 8 h post mortem, fixed in formalin-sublimate for 24 h, and embedded in paraffin. Incubation of 4 tam sections with the anti-H+/K +ATPase followed by the ABC detection method, revealed immunoreaction in large pyramidal cells and small neurons of all cortical layers. There was no reaction in glia cells or in mesenchymal tissue. In the hippocampus, reaction was present in the CAI sector and in the fascia dentata. Strong reaction was found in the entorhinal cortex. Immunoblots of snapfrozen tissue homogenates showed one immunoreactive protein (Mrs80 kD) in the cytosol fraction. The identity of these proteins is the subject of further investigations.

P18.32 Subcellular Localization of Inositol 1,4,5Trisphosphate Receptor in the Rat Cerebellum, Vestibular Complex and Cartwheel Cells of the Dorsal Cochlear Nucleus. J. Rodrigo ~, J. M. Polak 2, K. Mikoshiba 3 and R. Martinez-Murillo ~ ~Dpt. Chemical Neuroanatomy, Instituto Cajal, CSIC, Spain. eDpt of Histochemistry, Hammersmith Hospital. London. UK. 3Dpt. of Molecular Neurobiology. The University of Tokyo, Japan. The subcellular localization of inositol 1,4,5-trisphosphate receptor protein, P400, was investigated using the monoclonal antibody 4 C l l , following the avidin-biotin peroxidase procedure. Under the electron microscope, the immunoreactive product in the perikaryon of the Purkinje cell, ectopic Purkinje and cartwheel cells was associated with the smooth endoplasmic reticulum, the nuclear envelope, the outer surface of some mitochondria and subsurface systems. The recurrent preterminal axons and axon terminal boutons of Purkinje cells in the granular cell layer as well as the terminal fibres in the cerebellar and vestibular nuclei also showed immunoreactivity related to stacks of smooth endoplasmic reticulum. Results of the present study suggests that this receptor protein which is involved in the release of Ca2 +, may also be involved with neurotransmitter release. Research supported by CAM (C179/91) and FIS (92/0269).

Poster sessions P18.33 Monoclonal Antibody ICO-10 against Human T Cell Differentiation Antigen. M. L. Savich, L. E. Zavalishina, A. U. Barishnicov, I. B. Bukhualov and G. V. Vikha Scientific-Production Association "'Biotechnologia'" Moscow, Russia.

The tumors of sympathetic nervous system will average about 10070 of malignancy in childhood. One of the markers of these tumors is Thy-1 antigen. It is expressed about 79~ the cases of neuroblastomas, all neurosarcomas, neurolemmomas, teratoblastomas, about 80070 the cases of the metastatic neuroblastomas. We have developed a strategy for production of highly purified monoclonal antibody ICO-10 IgM class against Thy-1 antigen. Approximately 8 mg of 19s IgM free of contamination was isolated per ml ascite. Fragments with real. wt 140 kD were generated by incubating the intact antibody with pepsin and purified by gelfiltration on the column with Ultrogel AcA 34. Approximately 2 mg of fragments is isolated per 10 mg IgM. The fragments are stable. We have obtained its conjugates with fluorescein and peroxidase. We use this reagents for making up the immunological atlas of the different neuroblastomas.

P18.34 Validation of the Neural-Cell Adhesion Molecule (NC A M ) as a Marker for Peripheral Nerve Fibres in Normal Human Liver and its Application to the Study of Intra-Lobular Innervation. J.-Y. Scoazec, L. Racine, A. Couvelard, A. Moreau, D. Bernuau and G. Feldmann Laboratoire de Biologic Cellulaire, INSERM U327, Facultg de Mgdecine Xavier Bichat, 16, rue Henri Huchard, 75018 Paris France.

Clinical and experimental data suggest that the lobular innervation of human liver might participate in the control of sinusoidal blood flow and in the regulation of certain hepatocyte metabolic functions. So far, the study of intralobular innervation of the liver has been hampered by the lack of adequate markers. In this study, we show that N-CAM, a membrane protein involved in cell-cell adhesion within the central and peripheral nervous system, is a sensitive and specific neural cell marker and we use it to study the distribution of intra-lobular innervation in the normal human liver. The study was performed on liver samples obtained from 15 patients subjected to hepatic resection for small cancers. Samples were taken at a distance from the neoplastic lesions, in macroscopically normal areas. Three different antibodies against N-CAM were tested by light and electron microscopic immunohistochemistry: Leul9 (BectonDickinson), VCI.1 (Sigma) and H 2 8 - 1 2 3 (Immunotech). Their reactivity was compared to that of monoclonal antibodies directed against other neural cell markers: (a) S100 protein, (b) neurofilaments, (c) neuron-specific enolase (NSE). In portal spaces, the strongest reactivity was obtained with the clone Leul9. This antibody labelled large nerve bundles and the periarteriai and peribiliary plexus. The other anti-N-CAM antibodies tested gave only a faint labelling of those structures. Only the largest nerve bundles reacted with antibodies against neurofilaments. Reactivity with antibodies

589 against S100 protein and NSE was very faint. In the lobule, Leul9 decorated isolated nerve fibres, distributed along the perisinusoidal space or between adjacent hepatocytes. Most of those fibres were not decorated by the other antibodies tested in the study. Immunohistochemical ultrastructural examination showed that most intra-lobular nerve fibers were amyelinic. The density of intra-lobular nerve fibres labelled by Leul9 was variable from one patient to another, but was constantly heterogeneous within the lobule: 8.7 _+ 2.3 nerve fibres were counted by 0.3 mm 2 field in the periportal zone and 1.8 ___ 1.2 in the centrilobular one. In conclusion, N-CAM is a sensitive marker, useful for the study of the intra-lobular innervation of human liver. This marker shows that the density of nerve fibres is heterogeneous within the lobule: intra-lobular innervation predominates in the periportal region. This anatomical variation is a further argument supporting the existence of different microenvironments within the hepatic tobule: it might be involved in the maintenance of the metabolic heterogeneity of hepatocytes.

P18.35 Direct Retinal Projection to the VIP Neurons in the Rat Suprachiasmatic Nucleus. M. Tanaka, Y. Ichitani, H. Okamura and Y. Ibata Dept. of Anesthesiology and Anatomy, Kyoto Prefectural University of Medicine. Kyoto 602, Japan.

The suprachiasmatic nucleus (SCN) is considered to be a circadian oscillator of mammalian. It is well known that vasoactive intestinal peptide(VIP) immunoreactivity and mRNA in the SCN demonstrates diurnal variation under daily light-dark cycle. In this study, we have elucidated the connection between optic nerve terminals and VIP containing neurons in the SCN. Cholera toxin B subunit(CT) as anterograde tracer was injected into right eyeballs of the SD rats 2 days prior to sacrifice. After colchicine treatment into the lateral ventricle, rats were perfused with a fixative, and coronal hypothalamic sections were cut with a vibratome. Double labelling light and electron immunocytochemistry was performed to identify optic nerve terminals and VIP neuronal elements using anti-sera against CT and VIP. Optic nerve terminals were visualized by silver-gold (SG) intensification. In light microscopy, the distribution of black dots of optic nerve terminals intermingled with brown colored VIP neurons in the ventrolateral SCN. The ultrastructural examination revealed that CT positive nerve endings were easily recognized in the ventral SCN because SG intensified particles were found on them specifically. They made synaptic contacts with VIP immunoreactive dendrites and perikarya although far less in the latter. In conclusion, we have confirmed direct input from optic nerve afferents to VIP neurons in the SCN as axodendritic and axo-somatic synapses.

P18.36 Monocional Antibodies Specific to the Phyiogenetically Stable Neuronal Structures of the Rat Brain. N. A. Veretennikov Brain Research Institute of the Russian Academy of Medical Sciences, Moscow, Russia.

We have raised MAbs against the proteins extract of the fixed gray matter of the brain stem of the cat with the aim of finding

590 ligands that recognize particular cytological neural structures. The splenocytes were fused with myeloma cells by using conventional techniques. Supernatants of the hybridoma microcultures were screened by indirect immunoperoxidase technique of cryostat 15 mkm thick horizontal sections of fresh rat brain. The MAbs to neural cat's antigens 1A4 and 3Ell (two of the several MAbs obtained) stain the specific antigens of the rat brain. 1A4 recognizes only the large motoneurons of nucleus hypoglossus and Purkinje cells (premotor). 1A4 stains only the membrane structures of the cytoplasm of the cell bodies without staining the surface membranes, nuclei and processes of the mentioned neurons. 3Ell stains presynaptic boutons (densely and uniformly stained mass of small dots) on the surface of dendrites but not cell bodies in phylogenetically old cortical regions: in cerebellar (granular layer) and all layers of Ammon's horn. The restricted distribution of the antigen 1A4 within motor and premotor neurons suggests the antigen molecule is most likely involved in functions specific to the large effector neurons and the distribution of 3El 1 may point to existing of molecules specific to other structures and functions remaining undetermined.

P18.37 An Immunocytochemical Study of Substance P System in the Brain of the Japanese Quail (Coturnix

japonica). C. Viglietti-Panzica, N. Aste, G. Cellino, C. Andreone*, A. Fasolo* and G. C. Panzica Dept. Human Anatomy & Physiology and *Dept. Animal Biology, Torino, Italy. The topographical localization of Substance P immunoreactive (SP-IR) system in the brain of the Japanese quail (Coturnix japonica) has been studied by biotin-avidin immunocytochemistry on SUSA fixed paraffin sections from adult birds of both sexes. SP-IR neurons are clustered in the telencephalic accumbens nucleus (n.) and close to the occipitomesencephalic tract. Dispersed neurons are localized close to the third ventricle (medial preoptic n., paraventricular n., periventricular grey) and throughout the tuberal and infundibular regions. Very few and scattered SP-IR cells are observed along the mesencephalic central grey and within the reticular formation. In contrast to the limited distribution of cell bodies, SP-IR fibres are present in several brain regions, where they can locally reach very high levels of immunoreactivity. In the telencephalon, the archi- and paleostriatum, and the lateral septum are highly innervated, whereas the hyperand the ecto-striatum showed only few, scattered fibres. The diencephalic fibre system is mainly localized periventricularly and in the external layer of the median eminence. Many SP-IR fibres are observed within the mesencephalon (EdingerWestphal n., ventral tegmental area, optic lobe) and the pons (locus coeruleus, subcoeruleus n., raphe n., nuclei of the V and VII encephalic nerves). In the medulla, SP-IR fibres are observed within the nuclei of the IX and X encephalic nerves, the nucleus of the solitary tract, and in the inferior olivaris n. The data reported here confirmed and extended results previously obtained in the brain of other avian species suggesting a major role of SP in the regulation of the basal ganglia and of several catecholaminergic cell groups of the brain stem.

Poster sessions

P18.38 Origin of the Dopaminergic Projection to the Rat Prefrontal Cortex using Gold Conjugated Choleratoxin-B Subunit and Tyrosine Hydroxylase Immunohistochemistry. K. Yagita, H. Okamura and Y. Ibata Department of Anatomy, Kyoto Prefectural University of Medicine, Kamikyo-ku, Kawaramachi-Hirokoji, Kyoto 602, Japan. Choleratoxin-B subunit conjugated to colloidal gold particles (ChTx-Au) is a sensitive retrograde neuronal tracer that has a characteristic of small diffusion at injection site and minute incorporation by passing through axons. In this study, we examined the topography of the midbrain dopaminagic (DA) neurons projecting to the prefrontal cortex (PFC) using the combination of the ChTx-Au and tyrosine hydroxylase immunohistochemistry. Four days after the ChTx-Au (0.02 ul) injection into the adult Wistar rat PFC, animals were fixed and processed for silver enhancing method and ABCimmunohistochemistry visualized with DAB. Double-labelled cells (black silver dots over brown DAB-stained cytoplasm) were detected in the ventral tegmental area (VTA) and in midbrain raphe nuclei, but not in the substantia nigra. Since double-labelled cells were detected more frequently detected in the midbrain raphe nuclei (interfascicular nucleus, rostral linear nucleus of raphe, caudal linear nucleus of raphe and dorsal raphe nucleus), than in ventral tegmental area (VTA) in all of the cases examined, this study strongly suggests that DA inputs to PFC originate mainly in midbrain raphe nuclei.

P18.39 Myelinization and Development of Oligodendrocytes in the Telencephalon of Gallotia galloti: Immunochemical and Uitrastructural Studies. C. Yanes ~, M. Monz6n Mayor2, R. R. Sturrock 3, J. de Barry4 and G. Gombos" ~Dpto. M. y Biologfa Celular, Fac. Biologfa, Univ. La Laguna, Tenerife, Spain. 2Dpto. Morfologib, F. C. M. S., Univ. de Las Palmas de G. C, Las Palmas, Spain. J Dept. of Anatomy and Physiology, Univ. Dundee, Scotland, United Kingdom. 4Centre de Neurochimie C.N.R.S., Strasbourg, France. We have performed immunohistochemical and ultrastructural techniques in the telencephalon of the lizard Gallotia galloti, to study the localization and chronologycal appearance of Myelin Basic Protein (MBP) and oligodendrocytes. MBP-like immunoreactivity was present in different degree of intensity in many nerve fibers (isolated in tracts and in commisurae). During ontogeny the earliest MDP-like immunoreactivity appeared in the few fibers (lfb) at E40, proceeding caudo-rostrally and from ventral dorsal (alar) regions. Accumulation of MBP continued after hatching, oligodendrocytes cell bodies were not immunopositive. On the other hand with Electron Microscopy. Oligondendroblast were present from E37, the Active Oligodendrocytes could be found from E38 and they are numerous at hatching. The three types of Oligodendrocytes (Light, Medium and Dark) first classified by Mori and Leblond (1970) in rat, could also been identified in the lizard midbrain (Monz6n et al., 1990) and now in telencephalon. Light Oligondendrocytes were present at all ages from hatching, Medium Oligodendrocytes and Dark Oligodencrocytes first appeared from postnatal specimens.

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591

1Mori S. & Leblond C. P. (1970). J. Comp. Neurol. 170: 33 - 4 1 . 2Monzdn Mayor M., Yanes C., James J. L. & Sturrock R. R. (1990). J. of Anat. 170: 4 3 - 4 9 .

P18.40

Inhibition of Ca + +-ATPase Activity in the Guinea Pig Cerebral Cortex as a Result of the X-ray Influence. A. V. Bulavka and Vo S. Zinchuk Research Institute for Neurosurgery and Department of Pathology, Medical Institute, Kiev, Ukraine. It is well understood that cerebral cortex is sensitive for the X-

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P19.1

Quantitative Measurements of Antiporter Mediated Cell Rounding by Image Analysis Using Both the LM and SEM Modes. B. H. Bay, K. H. Sit, Y. G. Chan and H. L. Chan Department of Analomy, Faculty of Medicine, National University of Singapore, Singapore 0511. Monolayer anchored cells can be induced to retract by direct ionic motivation of the N a + / H + antiporter via downgradient exchanges with Na + bicarbonate-saline buffer (Sit and Wong, Tissue & Cell 2 1 : 3 8 8 - 4 0 0 1989; Sit et al. Tissue & Cell 22: 785 - 802, 1990). In this study, we used an on-line Quantimet 520-plus image analysis system to quantitate the cell profile area and perimeter under both light microscopy and scanning electron microscopy. For LM measurements, parallel cultures of Chang liver cells in 25 cm 2 culture flasks are fixed in 3 % trichloroacetic acid (TCA) and stained in aqueous Giemsa dye before quantitation. For SEM measurements, the cells are grown on 5 • 8 mm 2 glass slides and fixed in 5o7o glutaraldehyde in 0.1 M cacodylate buffer and osmicated in 2% osmium tetroxide before drying in a Balzer's Critical Point Dryer. After coating with 20 nm of gold, the specimens are examined at 20 kV in a Stereoscan 200 SEM and measurements are done at a magnification 700 X (Sit et al. Scanning Microscopy (in press) 1992). In both modes of measurement a biexponential decrease in cell area is demonstrated.

P19.2 Effect of Muscle and Heart Type Lactic Dehydrogenase Inhibitors in Liver and Kidney. An Image Analysis Study. V. Bertone, I. Freitas and P. Griffini Dept. of Animal Biology and CNR Center for Histochemistry, University of Pavia, Italy. Histochemistry is a valuable tool for monitoring the enzyme activity of individual cells in organs with metabolic zonation. Lactate dehydrogenase (LDH) has typical activity zonation in mouse liver and kidney. LDH5 predominates in liver and LDH3, LDH2 and LDH1 in kidney. Using PVA for

ray activity. In this respect we have studied Ca + +-ATPase localization activity as a key factor of the cellular metabolism. Our ultracytochemical procedure suggested incubation of the tissue under study at physiological pH with cerium as a capturing agent. Animals were radiated by 2.58 mKI/kg and 180.6 mKI/kg. The decrease of the enzyme activity was prominent mainly within pre- and postsynaptic plasma membranes in association with synaptic vesicles and postsynaptic densities. In the second case, plasmatic membranes substrate hydrolysis was inhibited a little stronger. The detection of the enzyme activity shows a great sensitivity of Ca++-ATPase to experimental radiation influence. The stage development of the pathological process makes it feasible to admit cerebral cortex metabolism violation after the X-ray alteration.

CYTOMETRY preserving soluble enzyme forms, diffuse (Di), granular (Gr) and mixed (Di + Gr) mono- and diformazans were observed. The staining intensity and subcellular aspect of the final reaction product in different areas were assessed with image analysis. The Gr particles were coarser in liver than in kidney. Dark Di + Gr predominated in liver, whereas kidney showed lighter D i + G r . Lipid extraction in acetone decreased the reaction intensity and the granules dimensions, indicating enzyme inhibition and partial loss into the solvent. Periportal and pericentral areas in liver showed a D i + G r product whereas midzonal areas a Gr pattern. Glomeruli were slightly positive (Gr product). Renal tubules showed zonal distribution of formazan in terms of staining intensity and D i / G r ratio. Urea (inhibitor of M-LDH) caused in both organs a slight staining decrease with higher incidence upon the Gr product, thus allowing the visualization of more cells with Di pattern in kidney. Pyruvate (inhibitor of H-LDH), caused an almost total inhibition of Di both in liver and in kidney. The latter was almost negative whereas liver retained moderate Gr activity. These results agree with Freitas et al. hypothesis that Di represents soluble LDH isoenzymes (solubility being promoted by H-subunits) and Gr visualized isoenzymes bound to membranes through M-subunits, with different kinetic and functional properties. (MURST 60% funds).

P19.3 Use of Leukogate (LG) in the Analysis of Lymphocytes from Burned Patients. D. G. Burleson, K. M. Wolcott, A. D. Mason Jr. and B. A. Pruitt Jr. US Army Institute of Surgical Research, Fort Sam Houston, Texas 78234 - 5012, USA. Immunophenotypic analysis is improved by LG, a technique using anti-CD14 and anti-CD45 antibodies to define light scatter gates (ScG) to improve NK and B cell determinations. LG works well in most samples, but not in samples from trauma patients, in whom lymphopenia, granulocytosis and monocytosis are common and nonspecific antibody binding by nonlymphoid cells (NLym) makes LG discrimination of cell types difficult. To assess LG in samples from such patients, peripheral blood monocytes from burned patients were

592 isolated and stained with monoclonal antibodies. Coulter Cyto-Trol cells were used as controls. Ungated data were collected in list mode using a FACSTAR Plus flow cytometer. ScG were set using LG, restrictive ScG settings (RScG) to limit false positives to less than 1~ or LG plus an independent 90 ~ ScG setting ( L F + 90). All three methods gave equivalent results in controls. In the patient samples, Lg gave excessive false positives, falsely increased cell percentages (CP) with antibodies that cross reacted with NLym, and falsely low CP for those that did not. Many LG analyses could not be evaluated because of excessive NLym contamination. RScG produced few false positives, but excluded some B and NK cells. LG + 90 gave more accurate CP for B and NK cells and fewer false positives than LG. The use of a 90 ~ ScG setting to exclude NLym increases the accuracy of LG analysis in samples of cells from patients who have sustained severe trauma.

P19.4

Flow Cytometric Two-Parameter Analysis of Cellular DNA and Nonprotein Thiols. T. Heiden*, M. Edgren** and B. Tribukait* *Department of Medical Radiobiology, Karolinska Institute, Box 60212, S-10401 Stockholm, Sweden. **Department of Tumour Biology II, Karolinska Institute, S-10401 Stockholm 60, Sweden. The cellular tripeptide glutathione can protect from damages from ionizing radiation or from a variety of clinically useful cytotoxic drugs. There is evidence that the radiation and alkylating agent resistance of tumor cells in cancer patients can correlate with an increased glutathione content. The objective of this investigation was to establish a flow cytometric method for two-parameter analysis of cellular DNA and glutathione content. Nonprotein thiols, which are essentially represented by glutathione, were stained using mercury orange according to O'Connor et al. (1988) and DNA was stained with DAPI. By means of this method, the nonprotein thiols of aneuploid tumor cell populations can be analyzed separately from diploid cells and erythrocytes for a better characterisation of the relevant cells. A good specificity of the glutathione measurement and a reasonable precision of the DNA analysis were obtained.

P19.5 Avidin-EITC as an Alternative to Avidin-FITC in Fluorescence Labelling Experiments and Confoeal Scanning Laser Microscopy. R. Hulspas, P.-J. Krijtenburg, J. F. Keij and J. G. J. Bauman ITRI-TNO, Dept. of Molecular Pathology, P.O. Box 5815, 2280 HV Rijswijk, The Netherlands. In double labelling experiments, where fluorescein-5isothiocyanate (FITC) is combined with another fluorochrome (e.g. Texas Red), it is essential that both fluorochromes are optimally excited and detected. Expecially, in confocal microscopy the simultaneous detection of two fluorochromes is often suboptimal. Moreover, FITC bleaches very rapidly, due to high intensity laser excitation. Consequently, in confocal microscopy one can easily fail to detect small FITC labelled targets. We studied the possibility to use eosin-5-

P o s t e r sessions isothiocyanate (EITC) as an alternative to FITC in confocal microscopy. Upon excitation with 514 nm on a Bio Rad MRC 600, avidin-EITC is more efficiently excitated and detected than avidin-FITC. The low quantum efficiency of avidinEITC, however, resulted in detection of equal FITC and EITC fluorescence. More importantly, the fading characteristics of avidin-EITC compare very favourably to those of avidinFITC. This makes avidin-EITC a very useful conjugate in experiments where fluorescent labelled targets are detected by means of confocal scanning laser microscopy.

P19.6

A New Method to Analyse the Content of Active-Calmodulin. M. Fan, S.-H. Liu and S.-D. Gan Institute of Basic Medical Sciences, 27 Tai Ping Road, Beifing 100850, P. R. China. There are some methods to analyse calmodulin by means of enzymology, immunology and biochemistry. Recently, we developed a new method to analyse the content of activecalmodulin with fluorescence staining and FACS measurement. Trifluoperazin, a chelate for caimodulin and a fluorescence substance, can bind calcium activated calmodulin irreversibly under ultra-violet radiation and give out yellow fluorescence as irritated with 4 5 0 - 4 8 8 nm light. Cultured HL60 or hippocampus cells were put into fixation solution (formaldehyde: acetone, 1 : 1) for 15' and collected by centrifugation 3600 rpm for 5'. After treated with trypsin and filtered through a 200 mesh sift, the cells were stained with 0.1 mM trifluoperazin for 15'. Washed with PBS for three times, the samples were fully ready for FACS. Result indicated that cellular fluorescence intensity and dispersion degree were good enough for analysis, and nearly same results were observed in samples made O, 1, 2, 3 and 4 weeks ago.

P19.7

Expression of Membrane Phospholipases C and D in Granulocytes and Monocytes After Immune Stimulation. S. Murea, V. Cialficu and L. M. Popescu Division of Cell Biology and Histology, Davilla University of Medicine, Bucharest 35, P.O. Box. 35-10, Romania.

Phospholipases C (PLC) and D (PLD) expression in different leukocytic populations after immune stimulation is controversial. Stimulated (with 10 -7 M f-Met-Leu-Phe and immune complexes) and non-stimulated peripheral granulocytes and monocytes from healthy human donors were incubated with rabbit IgG-anti PLC and rabbit IgG-anti PLD antibodies in different concentrations ( 0 . 2 - 5 taM) and stained with PElabelled goat anti-rabbit serum. The distribution of PLC and PLD in these populations was determined on a FACStar Plus system (Becton-Dickinson). For both enzymes, the binding curves of the antibodies were nearly identical, with a maximum at 3.5/aM. Immune-stimulated and non-stimulated granulocytes, as well as monocytes, showed no difference in anti-PLC and anti-PLD binding.

Poster sessions

P19.8 Topological Regulation of CD4 and Phosphoinositidase C by TPA and Interferon-Alpha. S. N. Constantinescu, C. Cernescu and L. M. Popescu Division of Cell Biology and Histology, Davilla University of Medicine, Bucharest 35, P.O. Box 35-10, Romania. TPA induced a dose-dependent (10-200 nM) and timedependent (15 rain- 12 h) internalization of cell-surface CD4 on lymphoblastoid CEM-CM3 cells as shown by f l o w cytometry (FC), using Phycoerythrin-labelled Leu-3A. FC using a rabbit antibody directed to cell-membrane phosphoinositidase C (PLC-PI) and fluorescent conjugate showed that TPA similarly down regulated PLC-PI expression on CEMCM3 cells, lymphoblastoid WIL-2NS cells and HeLa-S3 cells, but not on human fibroblasts. The effect of TPA was inhibited by protein kinase C (PKC) inhibitors H-7 (20 laM) and staurosporine (1 nM) and was not mimicked by phorbols which cannot activate PKC. The topological regulation by TPA of both CD4 and PLC-PI on CEM-CM3 cells could not be inhibited by cytochalasin B (40/ag/ml), known to block only receptor-independent endocytosis. Interferon alpha (IFN) induced a low level of CD4 internalization on CEMCM3 ( 1 0 - 1 2 % ) and induced down-modulation of PLC-PI only on HeLa-S3 and fibroblasts. Immunoelectronmicroscopy using 10 nm colloidal gold showed that 1 h treatment of human fibroblasts with 3,000 IU/ml IFN induced the internalization of PLC-PI, while IFN did not induce the internalization of phospholipase A2 or ferritin-labelled Concanavalin A. Although PKC seems to be implicated in down-regulation of both CD4 and PLC-PI, the specific mechanisms of TPA or IFN effects seem different in details.

P19.9 Membrane Potential Changes During the Cytotoxic Process. K. Radosevic, T. C. Bakker Schut, M. van Graft, B. G. de Grooth and J. Greve University of Twente, Enschede, The Netherlands. The changes in the membrane potential of interacting NK and K562 cells were studied using standard flow cytometry, slitscan flow cytometry and quantitative fluorescence microscopy. As a membrane potential indicator we have used DiBAC (3). Increase in the DiBAC (3) fluorescence indicates depolarization of the cell membrane while a decrease in the fluorescence indicates hyperpolarization. By labelling the NK cells with an additional probe (TR-18 or DiL) prior to incubation with the target cells we could identify three populations in the flow cytometric measurements: single NK cells (TR-18/DiI +, small scatter signals), single K562 cells (TR-18/DiI-, large scatter signals) and conjugates formed by NK and K562 cells (TR-18/DiI+, large scatter signals). By using standard flow cytometry we detected an increase in DiBAC (3) fuorescence of a fraction of conjugates during the cytotoxic process. Using a recently developed slit-scan technique, which enables us to monitor the signals of each cell in a conjugate separately, we determined that this increase was mainly due to depolarization of the target cell. This was confirmed by means of quantitative fluorescence microscopy. Depolarization could be an early sign of target cell damage because the cell membrane remained impermeable to propid-

593 ium iodide. Our results also indicate that depolarization of the NK cell occurs as a result of its cytotoxic activity.

P19.10 Optico-Structural Machine Analysis of D N A and Some Enzymes in Lymphocytes During Diurnal Variations and Experimental Autoimmune Processes. M. V. Robinson, A. V. Shurlygina and V. A. Trufakin Institute of Clinical and Experimental Lymphology, Novosibirsk, Russia. The investigation of DNA and some hydrolytic and oxidative enzymes was performed on the automatized cytophotometric complex "Protva" connected with EVM. Diurnal fluctuations of DNA and different enzymes have been studied in thymus, spleen, blood, their fluctuations have been revealed. Synthesis of DNA was maximal in thymocytes at night, the peak of mitotic activity was in the morning. The maximal activity of blood lymphocyte dehydrogenases and adenylatecyctase was at the morning-day period. The activity of the latter enzyme in thymocytes was maximal at night. There is an active proliferation in the thymus at the morning, at the evening-night period metabolic activity of the migrating cells was low. During autoimmune experimental processes, induced by injection of syngeneic warmed erythrocytes, the content of DNA increased in T- and B-lymphocytes, some changes in the activity of enzymes were observed. There were changes in correlative relations and variability of the lymphocyte enzymes in control and experimental groups. The extent of autoimmune processes correlates with the enzyme spectrum of lymphocyte. So the metabolism and DNA content of immunocompetent cells changed during day time and experimental autoimmune processes and determine the course of physiological and pathological conditions.

P19.11 High Resolution DNA/Protein Flow Cytometric (FC) Analysis in Human Lung and GI Tract Tumors. M. L. Trinca*, M. Persiani*, W. G6hde ~ F. Salvati t, A. Manganelli* and L. Teodori* *Div. of Toxocology, ENEA-Casaccia, Rome; +Div. of Pneumology, Forlanini Hospital, Rome; *Inst. Lagrange, Rome, Italy," ~ of Radiobiology, Mfinster Univ., Germany. To improve the diagnostic and prognostic value of FC, dualparameter measurements have been carried out on some 250 samples from lung and GI tract tumors, stained with DAPI and SR 101 for DNA and protein content analysis and simultaneously determined in a 256 • 256 channel matrix. Biparametric demonstrated that cells with abnormally high or tow protein content (red fluorescence) which is indicative of unbalanced growth were often observed in malignant tumors. The use of dual-parameter analysis allowed the recognition of aneuplod tumor-cell lines not detectable by one-parameter FC analysis thus indicating that the frequency of FC determined diploid tumor is lower than that previously described by DNA monoparametric analysis. The recognition of aneuploid subpopulations in histologically negative one-parameter FC "diploid" samples assumes a remarkable diagnostic value in

594 clinically dubious cases. The results have also shown the presence of multiple protein subpopulations in clones with the same ploidy value, indicating a higher level of cellular heterogeneity than that demonstrated by DNA monoparametric measurements. In the case of lung tumors a relationship between protein distribution and morphological feature was observed. In fact in the case of epidermoid tumors, histograms with low protein content were related to tumor cells producing corneal pearls and associated to low Ki 67 levels (5% to 15%); conversely, cases with high and very high protein level were related to tumor cells not producing pearls and associated to Ki 67 values ranging from 15% to 55%. The adenocarcinoma group showed a more heterogeneous protein pattern distribution. However, some protein distribution were typical for the papillary and mucinous forms (lower protein content) with respect to simple adenocarcinoma tumors (higher protein content). All cases of SCLC analysed were associated to a high Ki 67%. Relationship with DNA protein content and morphology in GI tract tumor were not performed yet.

P19.12 Interlaboratory Image Analysis Study on DNA Measurements On Behalf of CAAC, Breast Cancer Project Group. F. B. Thunnissen, I. A. Ellis, U. Jfitting and G. Burger Dept of Pathology, University of Limburg, PO Box 5800, Maastrict, The Netherlands. In the European Concerted Action on Analytical Cytology group a multilaboratory study on DNA measurements was performed. Smears prepared from suspension of liver tissue were stained with two different Feulgen procedures. Three successive rounds were scheduled. From each slide 200 diploid, 100 tetraploid and 50 octaploid cells were randomly measured by means of the image cytometry system present in the laboratory. The features integrated optical density (IOD) and AREA were reported. In the three rounds the number of participating laboratories was 11, 14 and 9, respectively. The coefficient of variation (CV) after measuring more than 1600 nuclei in one specimen

P o s t e r sessions the first round was 4.4%. This gives an indication of the intra specimen variation. The interlaboratory variation expressed as the CV of IOD for the three rounds varied from 2 - 17%. In all laboratories the CV of the 2c peak was higher than the CV of the 4c or 8c peak. Sequential plotting of normalized IOD values yielded useful information about intrameasurement variation. After calculation of the 4c/2c and 8c/2c ratios, most of the instruments showed a good linearity. Comparison of measurements in specimens stained in the participating and central laboratory revealed similar CV values. Intercomparison of DNA measurements performed on different instruments is well feasible.

P19.13 Quantitative Immunofluorescence Image Analyses of Prostate Specific Antigen (PSA) and DNA in Prostate Adenocarcinoma. N. Wang, Y. Pan and B. Tribukait Department of Medical Radiobiology, Karolinska Institute, Box 60212, S-10401 Stockholm, Sweden. Serum PSA has become an important indicator of tumor development and response to treatment in human prostate carcinoma. PSA originates from prostate ductal and aciner epithelium but is normally not excreted to serum. In tumors, PSA is increasingly released from the tumor depending on the degree of differentiation. The aim of this investigation was to quantify PSA at the tissue level. Tissue sections from the benign and the malignant prostate were stained for PSA with Avidin-Biotin and Texas red and for corresponding DNA of the cell nuclei with DAPI. A high sensitive cooled CCD camera with low background and a dynamic range of 0 - 2 ~6was used to quantify the amounts of PSA and DNA fluorescence. PSA as related to DNA decreased from 1.89 +_ 1.20 in benign tissue (n = 25) to 1.15 __ 0.22 (n = 4) in well differentiated tumors and to 1.2 +_ 0.9 (n = 10) and 0.79 _+ 0.28 (n = 15) in moderately and poorly differentiated tumors, respectively. Combined immunofluorescence and DNA image analyses provide quantitative information of cellular constituents in tissue sections.

P20. A P P L I C A T I O N S OF MICROWAVE T E C H N O L O G Y P20.1 Microwave Oven Irradiation vs Trypsin Digestion for Antigen Unmasking in Fixed, Paraffin Embedded Material. G. Cattoretti, F. Dominoni, F. Fusilli and O. Zanaboni Dept. of Pathology, Istituto Nazionale Tumori, Milano, 20133 Italy. Optimal immunodetection of antigens in aldehyde fixed, paraffin embedded tissues requires epitope unmasking by mild proteolysis. The mechanism of the antigenic retrieval is not known. Recently, Shi et al. (1991) reported that microwave (MW) irradiation in a heavy metal solution allows antigen retrieval by breaking the formali-induced cross linkages and subsequent modification of the tertiary protein structure. We compared trypsin and MW treatment on formalin- and

Bouin's-fixed, paraffin embedded tissues with more than 150 antibodies. Four patterns of reactivity were obtained: 1) de novo staining of previously unreactive antibodies by either MW or trypsin, not both 2) staining increase selectively with MW or trypsin 3)enhancement with both treatment 4 ) n o effect. MW and trypsin treatment could not be combined because of the extensive damage to the sections. Salt composition and molarity of the MW solution were of outmost importance to reveal antigens such as Ki-67 and CD2. In 20 cases the sequence of the epitopes on the primary protein structure was analyzed for the presence of trypsin cleavage sites and of aminoacids reactive with formaldehyde. No correlation was found between the ability of MW or trypsin to retrieve antigenicity and the epitope structure.

Poster sessions P20.2 The Effect of the Preservation of Mineral Elements on the Fine Structure of the Noradrenaline Granules in the Chromaffin Cells of the Adrenal Medulla of the R a t , 7". Daimon Department of Anatomy, School of 34edicine, Teikyo University, Itabashi-ku, Tokyo, Japan. Freeze-substitution and electron microscopic cytochemistry using microwave irradiation were successfully used to preserve the mineral elements in the noradrenaline (NA) granules. A remarkable ultrastructural preservation of the NA granules was also observed. The adrenal medulla was frozen on the liquid nitrogen-cooled copper mirror immediately after microwave irradiation (50 msec) which prevented the formation of large ice crystals. The specimens were then freeze-substituted with an acetone-OsO4 mixture or with acetone, embedded in resin and cut on to ethylene glycol with no water. The granules of the NA cells freeze-substituted with an acetone-OsO4 mixture were totally filled with an electron opaque material, while these were a bull's eye appearance with a dense core surrounded by an electron lucent gap and a membrane in the cells fixed by the conventional preparative method using glutaraldehyde and OsO4 in buffer. Freezesubstitution of the NA ceils only with pure acetone revealed also the presence of an electron opaque granules of which the major elemental constituents were Na, Mg, P, S, C1, K and Ca by X-ray microanalysis. The presence of cationic metallic ions in the NA granules were confirmed even in the cells fixed by the cytochemical method of glutaraldehyde-K oxatate followed by OsO4-K antimonate during microwave irradiation which accelerated the chemical reaction.

P20.3

The Use of Microwave Oven Improves Temporal Bone Research. S. Hellstrdm, C. Johansson, A.-L. Grehn and M. Nilsson Dept of Otorhinolaryngology, University Hospital of UmeS, S90185 Urneh, Sweden. The microwave oven can be used for virtually all procedures in histological processing; e.g. fixation, dehydration, embedding, staining and immunotechniques (1). In the present study on rat temporal bones the microwave oven was used for fixation and decalcification in order to prepare tissue for localization of hyaluronan by the hyaluronan-binding protein (HABP) probe (2). Rats were killed by an overdose of pentobarbital, the temporal bones dissected free and immediately transferred in saline to the microwave oven. In a fixative containing 2% glutaraldehyde and 0.5O7o formaldehyde the temporal bones were processed in the microwave oven at a setting of 700 W and 45~ After rinsing in buffer the temporal bones were transferred to an 10~ EDTA solution and microwaved at 450~ W up to 35~ and then the effect was switched to 150 W at which the bone was kept until softened (20 to 24 hrs). After dehydration and paraffinembedding the paraffin-sections were reacted with the HABP-probe and further processed for the avidin-peroxidase technique. The histoprocessing with the microwave oven was extremely time-saving, in particular the decalcification, and resulted in

595 an excellent preservation of the ear tissues. A strong reaction for hyaluronan was demonstrated at several locations of the ear; the pars flaccida, the Eustachian tube, the round window membrane, the middle ear muscles and in the spiral ligament of the cochlea. The study showed that the microwave oven histoprocedures have many advantages in temporal bone research. 1. Boom M. E. & Kok L. P. Microwave cookbook of pathology. The art of microscopic visualization. Leiden. Coulomb Press, Leyden; 1987. 2. Hellstr6m S, Tengblad A, Johansson C, Hedlund U, Axelsson E. An improved technique for hyaluronan histochemistry using microwave irradiation. Histochem Journal 1990; 22: 6 7 7 - 682.

P20.4 Extending the Limits of Tissue Preparation by Microwaves - - The Basement Membrane. D. Hopwood and N. Kanjaria Department of Pathology, Ninewells Hospital and Medical School, Dundee, UK. When human duodenal biopsies are heated to 60 ~ in phosphate buffered saline in a microwave oven, the enterocytes lift off the villi in sheets to reveal the underlying basement membrane. This is penetrated by a number of pores or fenestrae. These obviously form a potential route between the lamina propria and the interepithelial celt space, through which T lymphocytes, macrophages and other inflammatory cells may pass, Duodenal biopsies from 23 patients were prepared by this technique and the basement membrane analysed morphometrically. The pore density was 22371/mm 2 of basement membrane SD 7500 ie about 6%. The pore areas varied from 0.7 - 7.5 ~m 2. A second biopsy from an adjacent site was assessed histopathologically for duodenitis. T cells were stained immunohistochemically and their volume density estimated at 16~ of the epithelium.

P20.5 Improvement of Immunohistochemistry for Quantitative Microscopy Using Microwave Irradiation. 1(. Kayser, H.-J. Gabius, J. Lfibcke and M. Tacke Abs Pathologie, Thoraxklinik L VA Baden, Amalien Str. 5., D6900 Heidelberg, Germany. The background and the specificity of immunohistochemistry and glycohistochemistry can be significantly improved by application of microwave irradiation: After incubation with the first antibody or biotinytated probe, the slides are transferred into glass containers filled with 200 ml PBS buffer for washing. These slides are irradiated with microwaves in a conventional microwave oven at 150 W for 1 rain. The same procedure is performed after incubation with the second antibody. Quantitative assessment of the background staining in comparison to the signal of the binding sites revealed an improvement of the signal/noise ratio by a factor 5 - 1 0 according to the antibody/probe used. The technique is especially useful for quantitative assessment of binding sites

596 such as hormone receptors, cytokine receptors, and tumor markers. The improvement was observed in both formalinfixed, paraffin-embedded specimens and fresh frozen tissue.

P20.6 Microwave Irradiation Staining Method for Biological Electron Microscopy. V. Mizuhira, H. Hasegawa and M. Notoya Shionogi Co. & Ltd., Kanzakigawa Research Laboratory, Toyonaka, Osaka 561, Japan.

Ultrathin sections which were fixed with microwave irradiation (MWI) method were mounted on a grid. A filter paper is laid on the bottle of the dish containing some water (to keep moisture during the staining with uranyl acetate). Small staining dishes of plastic bottle-caps are arranged in the Petri dish (on a wet filter paper). Next, 2070 uranyl acetate water solution is poured into the staining dish. A section of the grid is into the uranyl solution, finally the Petri dish is covered. The temperature of uranyl solution on the dish is set at 50~ and irradiation time is also set for 5 - 8 min of the computerized control-box using an intermittent irradiation (5 sec irradiation/10 sec rest). The grids are rinsed with distilled water, and move to the next lead staining. Soda-lime granules are placed at the bottom of the Petri dish. The staining dishes are placed on the soda-lime. Lead hydroxide solution is poured into each dish, and the grids were laid in them. The MWI is switched on as described above. Temperature was the same, but staining time was a little short, 3 - 5 min under intermitted condition. The grids were vacuum evaporated with carbon, after rinse, observed with electron microscope. Excellent results were obtained with good staining with electron opacity. 1 gm thick sections were compared the results under X-ray microanalysis, MWI-stained, hot stained with hot staining solution, and conventional staining methods. The results were very clear that X-ray intensity ratios of U.La and Pb.La were 30 : 10 : 3. This serial procedures enabled very easy observation of the extremely fine structures of all cellular elements, membraneous, secretion of synaptic vesicles, triad structure with Cabinding/releasing proteins, "feet" structure, neurofilaments, or tubules, ribosomes and associating messenger RNA filaments, and ATP-synthethase particles arranged on the mitochondrial crystae.

P20.7 Tissue Fixation Method by the Aid of Microwave Irradiation (MWI) - - Centred to the Calcium Ion Detection by the Combination with X-ray Mieroanalysis. V. Mizuhira, H. Hasegawa and M. Notoya Shionogi Co. & Ltd., Kanzakigawa Research Laboratory, Toyonaka, Osaka, Japan. For the detection of calcium ion localization in cells, fresh tissue blocks were fixed with a fixative containing 3 0 - 60 mM of potassium oxalate (K-OX) instead of CaC12, at pH 7.4 for 2 0 - 30 sec with MWI, then the blocks were left in the fixative for 3 0 - 6 0 rain at room temperature, and subsequently were postfixed with 1% osmium tetroxide containing 2% potassium

Poster sessions antimonate (K-Sb) at pH 7.4 adjusted with KOH or acetic acid, for two hrs at room temperature. They were then subjected to the sampling procedures, embedded in Epon. During the postfixation procedure, Ca-oxalate which was caused by the prefixation procedure was substituted with K-Sb to the Ca-antimonate, Ca[Sb(OH)6]2. The theoretical ratio of molecular weight (WT, 070) Ca(l): Sb(2) = 14 : 86 (070), and the chemical binding molar ratio (AT, %) was C a ( l ) " Sb(2) = 3 4 : 6 6 , and we obtained the same ratio under the energy dispersive type X-ray microanalysis using EDAX-PV-9800 computer combined analyzing system from a nonstained ultrathin section. There could be seen beautiful high resolution electron microscope images in all specimens, brain, skeletal or cardiac muscular cells, or endothelial cells of the blood capillary. In the cholinergic or amine type synaptic vesicles in the presynaptic button were very clear, and there was easily seen an image of the synaptic vesicles opening to the postsynaptic knob. Furthermore, very fine precipitates of Ca-antimonate (Ca-Sb) were observed in- and outside of the synaptic vesicles. The EDX analysing data indicate that exists calcium ion there. At the same time, very fine Ca-Sb precipitates were clearly distributed inside the pinocytotic vesicles of the endothelial cells of blood capillaries. A large amount of Ca-Sb precipitates was distributed in the sarcoplasmic reticulae of the intercostal or cardiac muscle cells.

P20.8 Microwave Stabilization without Chemical Fixation. G. Pons, E. Oberholtzer and S. Tognozzi Dipartimento di Biologia Animale, Facolta "di Scienze MFN, Universita "di Torino, v. Accademia Albertina 17, I 10123 Torino, Italy.

Microwave irradiation (MW) has been widely used as an alternative and rapid method of tissue fixation: it produces a very good tissue preservation for Light and Electron Microscopy. MW irradiation has been shown to facilitate fixatives diffusion processes involved in cell fixation, by a rapid and uniform heating of tissue specimens. These investigations were performed to evaluate whether it was possible to use MW irradiation for tissue stabilization without chemical fixatives and whether, at these conditions, it was possible to obtain enzyme activity preservation too. We used commercially available MW ovens (Philips M704 and M902, 2450 MHz). The biological specimens (kidney, liver, intestine of rabbit and Xenopus laevis), trimmed to 1 - 2 mm 3, were kept in glass vials and immediately irradiated, at various power conditions (600, 750, 850 W), in 3 ml of cacodylate buffer, 0.1 M pH 7.4, for 1 5 - 3 0 sec. In order to check operative conditions, temperature was measured by a digital thermometer dipped in the buffer solution. The specimens, after MW irradiation, were processed and embedded in polar resins (Glycol Methacrylate: Technovit 7100 Kulzer) for histochemistry (LM) or in Epon for ultrastructural studies (EM). Samples, so stabilized, showed a good preservation of cell morphology and, also, of enzyme activity. This work was supported by Italian MURST (60~

P o s t e r sessions

P20.9 Immunohistochemical Localization of Peptidergic Hormones in Insects: Rapid Fixation, Washing and Immunolabelling in a Water-Cooled Microwave Oven. H. M. Smid Wageningen Agricultural University, Department of Entomology, PO 8031, 6700 EH Wageningen, The Netherlands. Microwaves (MW) are now increasingly used for the acceleration of histochemical procedures. In our laboratory for insect endocrinological research, the microwave oven not only speeded-up histoprocessing protocols, it also significantly extended our technological possibilities and has lead to some new insights. This was made possible by the development of a special cooling-device, which was assembled in the microwave oven cabinet. This cooling-device, basically a small water jacket perfused with tap-water and surrounding a vial with the tissues to be irradiated, was capable of controlling the temperature of these tissues during constant irradiation at full MW power. The water-cooled vial with the tissues can be filled diverse media, e.g. fixatives and antibody dilutions for fixation and whole-mount immunotabelling. With the use of a perforated vial, the water-cooling device is converted into an efficient washing device. The here-described MW set-up has been used for some years now in our laboratory, and has proved to be reliable. It enabled us to process entire insects for LM serial sections, which provided us with some essential new information on the location of peptidergic hormones in peripheral endocrine centres.

P20.10 Microwave-Stimulated Antigen Retrieval. A New Method Facilitating Immunohistochemistry of Formalin-Fixed, Paraffin Embedded Tissue. A. J. H. Suurmeijer Martini Hospital, Groningen, The Netherlands. Immunomicroscopy is a powerful tool in pathology. Unfortunately, the reactivity of certain monoclonal antibodies, e.g. antibodies reactive with intermediate filament proteins, is reduced or lost by (prolonged) formaldehyde fixation. In this context the recent discovery of Shi et al. that the negative effect of formaldehyde fixation on immunostaining can be eliminated by microwave heating of tissue sections immersed in metal salt solutions is of great importance as far as diagnostic accuracy is concerned. Testing other metal salts for antigen retrieval capacity, we found that strong enhancement of immunostaining could also be obtained with aluminium chloride. Immunostaining results appeared to depend on the concentration and pH of this metal salt solution. Concerning immunoreactivity for keratins and vimentin, the results

597 obtained with 4 per cent aluminium chloride (pH 3.1) proved superior to those achieved with commercially available Antigen Retrieval solution containing lead thiocyanate (BioGenex Laboratories). Possible mechanisms involved in microwave-stimulated antigen retrieval are (a) Breakage of formaldehyde cross-links between antigenic determinants and blocking proteins, (b) Extraction of soluble blocking proteins, and (c) Unmasking of these epitopes due to loss of three dimensional protein structure (denaturation).

P20.11 Microwave-Enhanced Silver Staining of Degenerating Neuronal Processes. B. Van Deuren, J. Van Reempts and M. Borgers Department of Morphology, Life Sciences, Janssen Research Foundation, Beerse, Belgium. A simple and rapid staining technique for degenerating neuronal processes and axon terminals in 100 tam vibratome sections of rat brain is presented. The method employs microwave irradiation to reduce silver impregnation times. Rat brains were perfusion-fixed with a mixture of 2.5% glutaraldehyde and 2% formaldehyde. Hundred tam vibratome sections, mounted on gelatine-coated slides were treated with a 5% silver nitrate solution while irradiating for 40 sec at 700 Watt, immediately followed by 5 rain at 150 Watt. The temperature of the silver solution did not exceed 70~ After microwave incubation the sections were rinsed in 1% acetic acid for 1 rain. Sections were then rinsed twice in distilled water and further subjected to silver enhancement for 2 to 6 min. Counterstaining was carried out with azure-cosine or cresylviolet after differentiation in ethanol 100%. For electron microscopy, free floating 200 tam sections were silver stained as mentioned above, postfixed in OsO4, dehydrated in graded ethanol series and embedded in Epon LX. Areas of neuronal degeneration were characterised by the presence of numerous black dots, clearly differentiated from unstained grey matter and lightbrown white matter. Localisation and density of silver deposits depended upon the type of insult. Subtle areas of retrograde neuronal degeneration were also detected. Calcification areas appeared as large black globules. Electron microscopy confirmed that silver deposits were largely confined to degenerating presynaptic terminals. However, a considerable number of dendritic processes also showed deposits inside the cytosol or the mitochondria. This staining procedure is very well suited for thick unembedded sections of whole rat brain. The high contrast makes it easy to quantify the degree of damage in several experimental models and can therefore be a helpful tool in the neuropathology laboratory.

598

Poster sessions

P21. LOCALIZATION OF IONS P21.1

Ultrastructural Detection of Calcium and Magnesium in the Nucleus and Chromatoid Body of Mouse Spermatids by Means of Electron Spectroscopic Imaging and Electron Energy Loss Spectroscopy. M. Biggiogera, V. B. Rouelle-Rossier and S. Fakan Centre of Electron Microscopy, University of Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland. We have studied at the ultrastructural level the presence of calcium and magnesium in the chromatoid body (CB) of mouse spermatids. By means of electron spectroscopic imaging (ESI) and electron energy loss spectroscopical (EELS) analyses on pyroantimonate fixed material, we have shown that the granules representing the end product of the reaction contain calcium and magnesium. These granules are associated with the CB and are also present within the nuclei and in association with the nucleolus. Some of the granules contain both calcium and magnesium, while others contain only one of the two elements. In the absence of pyroantimonate fixation very few spots containg calcium can be localized by ESI and EELS. The pyroantimonate technique, although effective in blocking diffusible divalent cations, is not specific for calcium and reveals also magnesium. P21.2

Localization of Calcium I n S i t u under the Influence of Estradioi in Rat Vaginal Epithelium. S. B. Relia and P. D. Gupta Centre for Cellular & Molecular Biology, Hyderabad 500 007, India. Calcium plays a key role in regulating many vital cellular processes in the cell. Perturbations in calcium levels or changes in intracellular distribution of calcium is a reflection of altered physiological state. We have shown that estradiol induces uptake of calcium in vaginal epithelial cells within 15 rain of its administration, calcium levels decline by 30 rain. and remain at around control levels for next 12 h. Intracellular calcium distribution was assessed by electron microscopy by in situ precipitation of calcium after perfusing sodium oxalate prior to tissue fixation. Calcium oxalate was localized as electron dense precipitate on mitochondria, intermediate filament bundles, keratohyaline granules in the cytoplasm; nucleolus and inner nuclear membrane in the nucleus. After 15 rain. of estradiol injection, the electron density was high at all the above mentioned sites and electron density reduced considerably after 30 min. EM studies using oxalate method in neonatal rats 0 - 3 0 day revealed that estradiol induces calcium uptake in rats only above 30 days of age.

P21.3 Intravital Ca + § Cytochemistry - - 4-D Observation by Confocal Microscopy. T. Takamatsu, H. Kawachi, T. Minamikawa and S. Fujita Department o f Pathology, Kyoto Prefectural University of Medicine, Kawaramachi, 602 Kyoto, Japan. Fluorescence confocal microscopy with scanning incident laser

beam excitation has definitive advantages over conventional microscopy. (1) High resolution along z-axis, that is, along depth of the material under a microscope. (2) The resolution along focal plane that we conventionally call resolution of a microscope, is also greatly improved; theoretically the resolution is twice improved. (2) For the confocal laser scanning microscopy (CLSM), fluorescent materials are best suited. We can utilize advantages of fluorescent microscopy fully in CLSM. CLSM, however, so far has some disadvantages: Mechanical scanning takes time, usually 1 to 10 seconds is required to make one image. This is a serious disadvantage when the CLSM is applied to a rapidly changing cellular processes. We adopt different modes of scanning, i.e. scanning in a limited window, or scanning by a single scan line. The former mode enables us to obtain an image every 20 to 50 msec, and the latter enables us to record changes that occur within a few msec. By injecting Fluo3 into a living cardiac muscle cell and exciting with 488 nm laser beam, we can measure Ca ++ concentration in quantitative terms. By using this technique, we could observe dynamic behaviour of Ca ++ wave propagation with in a cell. When 2 cells are isolated as a pair, the Ca + + wave can be observed passing across the opposed cell membrances with propagation velocity unchanged. This can be abolished by enflurane. The Ca ++ wave obviously propagates through gap junctions by the mechanism of CICR (Ca ++ included Ca ++ release).

P21.4 Intake of Low Lead Doses in the Tissues of Juvenile and Adult Carp (Cyprinus carpio L.). N. Kralj-Klovubar Faculty of Natural Sciences, Department of Zoology, Zagreb, Croatia. Larvae after hatching, 30-day old early juveniles, 1-year old juveniles and adult carps (Cyprinus carpio L.) were treated with Pb (NO3)/ added into aquarium water in low concentrations of 25, 50, 100 and 200 ~g3 pb2/1. Timm's histochemical method was used for observation of lead accumulation in brian, liver and kidney. All larvae perished after being treated over a period of 7 days, but no lead was found in them. In the brain of late juvenile and adult carps lead was detected in capillary walls after a 7-day treatment, whereas in early juveniles no lead was detected even after a 21-day treatment. The test results suggest a difference in the degree of differentiation of brain capillaries related to the growth factor and thereby a difference in the function of the blood-brain barrier. Low lead concentrations in the early growth stage cause brain damage without bringing about visible pathological changes. In the liver of late juvenile and adult carps lead inclusions are found in hepatocytes from the 14th day of treatment onwards. Inclusions are most abundant in juveniles, whereas their number is decreasing with adults, perhaps as a result of intensified appearance of bile secretion. Lead inclusions in kidney are found only in exceptional cases in few tubules. The analytical method applying the

P o s t e r sessions atomic absorption spectrophotometry has shown that lead values in brain, kidney and liver are too Iow to be detected by the apparatus. Higher lead values have been found in muscle tissue only.

P21.5 The Goigi Apparatus - - A Compartment of the Intracellular Ca z+ Pool. D. Onicescu, C. Vidulescu and M. Popescu Division of Cell Biology and Histology, Davilla University of Medicine, Bucharest 35, P.O. Box 35-10, Romania. The precipitation reaction with potassium oxalate was used to investigate the ultrastructural localization of Ca2 § in atrial myocardocytes and in Schwann cells of non-myelinated nerve fibres. Chelation with EGTA was used to probe the presence of Ca2 § in the electronoopaque deposits. In both cell types the oxalate reaction was intense in the classically described Ca2 + deposits: the perinuclear cisterna, the endo(sarco)plasmic reticulum, the mitochondrial matrix, but also in a novel localization: the eisternae of the Golgi stack in all cis to trans positions, suggesting that Golgi Apparatus is a major intracellular Ca2 + pool. These data are challenging for further work to assess the functional significance of the presence of Ca2* in the Golgi cisternae.

P21.6 Histopathology, Histochemical Localisation and Quantification by Image Analysis of Mercuric Chloride in Insect Cells. H. Raes* and W. de Coster** *Laboratory of Zoophysiology & **Centrefor Image Analysis, K. L. Ledeganckstraat 35, University of Gent, B-9000 Gent (Belgium).

Introduction: We present the ultrastructural effects on insect cells of short-time exposure to sub-lethal HgC12 concentrations. Hg accumulation was studied both qualitatively and quantitatively by autometallography and automatic image analysis, Materials and Methods: All Experiments are performed on Aedes albopictus (Diptera) cells. The cells cultured on coverslips and in culture tubes, were exposed for 24 h to 0, 0.1, 0.2, 0.4, 0.8 ppm HgC12. Autometallography was done directly on coverslips and on semi-thin resin sections of cell pellets. The silver grains in the latter were quantified by automatic image analysis. Cells on coverslips were also proceeded for TEM after silver enhancement. Results and Discussion: Silver-grain localisation shows that for 0.1 & 0.2 ppm, Hg is accumulated exclusively in the lysosomes. For 0.4 and 0.8 ppm small grains are also scattered over the cytoplasm and the nucleus. Quantification of silver grains on resin sections is possible in a reproducible way, provided the material is processed under strictly standardised circumstances. The results are expressed as percentages of area covered by silver-grains to total cell-surface. Hg accumulation is less with 0.8 than with 0.2 and 0.4 ppm. Ultrastructural changes include: the cytoskeleton, extrusion of membrane whorls, dilatation of the R.E.R. and degeneration of mitochondria and nucleus.

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P21.7 Histochemical and Immunohistochemical Localization of Copper, Iron and Metallothioneins in the Liver and Kidney of LEC Rats. Y. Sumi, S. Kawahara U, Y. Kikuchi 2, J.-I. Sawada 2, T. Suzuki and K. T. Suzuki 3'4 St. Marianna Univ. Sch. of Med., Kawasaki,; ITsukuba Univ., Tsukuba; 2Natl. Inst. of Hyg. Sci., Tokyo; 3Chiba Univ. Chiba; *Natl. Inst. for Environ. Studies, Tsukuba, Japan. The LEC (Long-Evans with a cinnamon-like coat color) rat is a new mutant strain with hereditary hepatitis associated with severe jaundice. Recently, it was reported that this hepatitis might be caused by abnormal deposition of copper in the liver. Tissues were therefore obtained from two female LEC rats (body weight 129 and 119 g, 18 weeks after birth) with very severe jaundice. The tissues were fixed in formalin, embedded in paraffin, and then sectioned. Copper was stained with pdimethylamino-benzylidenerhodanine and iron was stained with hexacyanoferrate. Metallothioneins were immunostained using a monoclonal antibody against metallothioneins according to the avidin-biotin-peroxidase complex method. Intense staining for copper and iron was limited to Kupffer's cells in the liver, but no staining was seen in hepatocytes, even though they appeared to be in various stages of degeneration. In the kidney, both iron and copper reaction products were localized in the epithelial cells of urinary tubules. Intense staining for metallothioneins was observed in Kupffer's cells and in epithelial cells of urinary tubules. These results suggest that hepatitis in the LEC rat may be associated with abnormal deposition of both iron and copper.

P21.8 Semiquantitative Analysis of Cadmium Accumulation in Rat Kidney Proximal Tubules. A. Tanimoto, T. Hamada, H. Teramoto, S. Iwai and O. Koide University of Occupational and Environmental Health, Japan.

We have previously demonstrated that subcutaneous injection of cadmium (0.6 m g / k g / d a y , 6 times/week) induced apoptosis and regeneration of proximal tubular ceils at outer stripe over 4 weeks exposure in rats, The apoptotic cells and regenerating ceils contained detectable amount of cadmium by means of oxine-fluorescence technique. In the present study, we evaluated the intensity and distribution of cadmium in renal tissue using an image analyser (IBAS, Carl Zeiss). Fresh frozen sections of rats with 6 weeks exposure stained by oxine were photographed by using of a fluorescent microscopy (M80, Carl Zeiss). The photographs were submitted for image analysis and relative grey value was measured in the apoptotic cells and regenerating proximal tubules. The apoptotic cells showed most intense fluorescence and grey value. The fluorescent strength of the proximal tubules in the straight portion varied tubule by tubule. Among them, the tubules with sloughted apoptotic cells in the lumen had lower grey value than those without apoptotic cells (p<.0001). The convoluted proximal tubules also contained cadmium, and showed rather homogenous distribution. The glomeruli and interstitium had no oxine reaction. The various fluorescent intensity seen in the proximal tubules in straight portion may result from marked cell regeneration subsequent to cell deletion by apoptosis.

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P22. I M M U N O H I S T O C H E M I S T R Y OF T H E D I F F U S E N E U R O E N D O C R I N E SYSTEM

P22.1 lmmunocytochemical Study of the GonadotropbinReleasing Hormones, LHRH I and LHRH II in the Brain of Male and Female Japanese Quail. P. Absif, J. van Gils2, F. Vandesande 2 and J. Balthazart ~ Lab. Gen. Comp. Biochemistry, Univ. Lidge (1) and Lab. Neuroendocrinology, Univ. Leuven (2), Belgium. Specific polyclonal antibodies were raised in rabbit against the fragments of the L H R H I and II molecules showing the highest degree of dissimilarity. They were used to analyze the distribution of immunoreactive (ir) perikarya and fibers in the brain of male and female Japanese quail (Coturnix japonica). Specificity of these antibodies was established by liquid and solid preabsorption and by cross-reactivity tests. LHRH I-Jr cells were found mainly in periventricular location throughout the preoptic-hypothalamic area, in a ventral position from the level of the tuberculum olfactorium to the mid-hypothalamic region, dorsal to the tractus occipitomesencephalicus, at the lateral edge of the tractus septomesencephalicus (TSM), in the lateral hypothalamus and in the lateral septum. L H R H II-ir perikarya were exclusively located in a single cell cluster around the roots of the third nerves. L H R H I and II-ir fibers were found mainly in medial position throughout the hypothalamus but also in the septal region and in the telencephalon. This pattern of innervation was specific for each peptide. No qualitative sex difference could be detected in the pattern of L H R H I or II innervation. The numbers of L H R H I or II-ir cells were similar in males and females, except for a small group of L H R H I-ir perikarya located at the lateral tip of the TSM in which neurons were about twice more numerous in males than females (p<0.05). These data confirm that LHRH I and II are present in separate neuronal networks and are presumably implicated in different functional systems.

P22.2 Immunocytochemicai and Electron-Microscopic Study of Pulmonary Neuroepithelial Endocrine Cells in a Bird, The Quail Coturnix Coturnix. D. Adriaensen, T. Gomi*, A. Kimura*, J,-P. Timmermans, M. H. A, De Groodt-Lasseel and D. W. Scheuermann Laboratory of Celt Biology and Histology, Department of Morphology, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium. *Department of Anatomy, School of Medicine, Toho University, 5-12-16. Omorinishi Ota-ku, Tokyo 143, Japan. Despite extensive knowledge of the neuroepithelial endocrine (NEE) system in the lungs of species of various vertebrate classes, data on avians are limited. This was clearly illustrated by recent comparative overviews of the chemical coding of the pulmonary NEE system (Adriaensen and Scheuermann 1992, Anat Rec, in press; Scheuermann et al. 1992, Eur J Morphol, in press). The present study aims to extend our information on the morphology and immunocytochemical characteristics of avian NEE cells. In the quail lung, solitary and grouped NEE cells can be found in the ciliated epithelium of the primary and

secondary bronchi and mostly lack a direct relation with the airway lumen. The NEE cells contain dense-cored secretory granules and are innervated by morphologically efferent, showing synaptic specializations, as well as afferent nerve endings. They appear to be immunoreactive for serotonin, and part of them for bombesin or somatostatin. Heretofore, no cleary identifiable efferent nerve terminals were reported to contact NEE cells in birds. In this study, at least three different bioactive substances are demonstrated in the avian NEE system.

P22.3 Localization of Vasoactive Substances in the Endothelial Cells of Human Term Umbilical Blood Vessels. IV. Q. Cai, P. Bodin, A. Loesch, A. Sexton and G. Burnstock Dept. of Anatomy and Developmental Biology, University College London, Gower Street, London WCIE 6BT, UK. The localization of neuropeptide Y (NPY), atrial natriuretic peptide (ANP), vasoactive intestinal peptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP) and vasopressin (VP) in the endothelial cells of term human umbilical blood vessels was studied using the pre-embedding peroxidase-antiperoxidase technique for electron microscopy and avidin-biotin peroxidase complex (ABC) immunostaining for endothelial cells cultured from umbilical vein. The results revealed that subpopulations of these immunoreactive positive endothelial cells were present in the umbilical vein and artery. The percentages of NPY- and ANP-immunoreactive umbilical vein cells in culture were 32.3% and 44.2%, respectively, out of the total 3013 cells examined; VIP- SP- and CGRP-positive cells were about 10%, while VP-positive cells were less than 10%. The vein contained more positive cells than the artery. The physiological significance of the presence of these potent vasoactive substances in the endothelial cells of the non-innervated umbilical vessels will be discussed in terms of the local regulation of blood flow.

P22.4 Distribution of Some Neuropeptides (VIP, SP, SS-14) in Whole-Mount Preparation of Caecum of Domestic Ducks. A. Costagliola, A. Vittoria and A. Cecio Department of Biological Structures, Functions and Technology, University "'Federico H". Naples, Italy. The distributive pattern of neuropeptides VIP, substance P and somatostatin-14 were studied in the caecum of adult ducks on whole-mount preparations. The reciprocal co-localization of the said neuropeptides was also examined. Whole-mount preparations of caecum from 6 cholchicine treated adult ducks were fixed in 4% Paraformaldehyde.

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Separated layers containing the myenteric or the submucosal plexuses were processed according to a double indirect immunofluorescent labelling technique. VIP-containing fibres and cell bodies were largely diffused in both plexuses studied, resulting more numerous in the submucous one. The neuropeptide VIP never resulted colocalized with substance P. Somatostatin-14 was scarce, and mainly contained within submucous neurons, isolated or grouped in small clusters. It was sometimes co-localized with substance P. This latter, finally, was often seen in nerve terminals surrounding negative or VIP-containing cell bodies, both in myenteric and submucous plexuses. The functional roles played by both the searched bioactive peptides and by their co-localization will be discussed.

with epoxy resin Epon, whereas the semi-thin tissue sections were prepared by LKB ultramicrotome. The parafollicular cells were demonstrated immunohistochemically with antiserum against the pig calcitonin (*) and with antiserum against somatostatin, using the avidin-biotin method. Calcitonin and somatostatin in the parafollicular cells were well stained in aIl three age categories of animals, in form of granula clearly distributed within their cytoplasm. It was also stated that some parafollicular cells simultaneously contain calcitonin and somatostatin. (*) Antiserum against pig calcitonin was kindly donated by Professor Cary Cooper, University of Texas, USA, and antiserum against somatostatin by Dr. Camille Vaillant, University of Liverpool, UK.

P22.5 Immunohistochemical Detection of Chromogranin in Rat Suhmandibular Gland.

P22.7 Immunohistochemical Localization of Chromogranins/Secretograuins in the Gut of a Lizard

M. Kunikata and M. Mori Oral Surgery, Asahi University School of Dentistry, Gifu, Japan. Chromogranin (CG) is a high molecular weight protein, costored and co-released with catecholamines from storage vesicles in adrenal gland and sympathetic nervous system, and also exist in several other endocrine glands. The function of CG is largely unknown. The autonomic innervation of submandibular gland (SMG), has tempted us to study the CG localization in rat. The distribution pattern of CG was studied in SMG of adult, postnatal (1 - 56 day), duct ligated and also in that of isoproterenol (IPR) treated rats. The techniques used were, immunohistochemistry, electron microscopy and radioimmunoassay. Adult rat SMG did not show any CG in aciner cells, where as in postnatal rats of 7 to 14 day showed a progressive increase in CG content with a highest on 14 day, subsequently, a decrease was observed with the lowest content on 42 day. IPR treated adult rat SMG showed positive reaction for CG. The initial phase of duct ligation, along with atrophied cells, did not show any CG in aciner cells, but subsequent proliferation of aciner cells took place with CG positivity. The injection of polyclonal antibody against chromogranin to the rats, from 7 till 21 day, showed a dramatic change in the cytomorphology of SMG cells. In normal rats, the aciner cell differentiation is almost completed by 21 day of postnatal life, In antibody treated rats, aciner cell differentiation remained indefinable till 21 day. The present study has clearly demonstrated that, CG may play a vital role in aciner cell differentiation mechanism.

P22.6

Immunohistochemical Demonstration of Calcitonin and Somatostatin in the Thyroid Gland Parafollieular Ceils of Pigs (Sus scrofa dora. ). A. Poga~nik ~, G. Majdi~, C. Vaillant 2 and S. V. Bavdek ~ Institute for A natomy, Histology and Embryology of the Veterinary Faculty, University of Ljubljana, Slovenia I and Department of Veterinary Preclinical Sciences, Veterinary Faculty, University of Liverpool, UK2. The thyroid gland structure of the three age categories (one day, three weeks, seven months) in pigs was studied. Tissue pieces were fixed with 4~ paraformaldehyde and embedded

(Lacerta sicnla ). L. D'Este, R. Buffa*, N. Carboni*, M. Pelagi**, A. G. Siccardi**, C. Casu and T. Renda Institute of Human Anatomy (IV), University "'La Sapienza" of Rome; *Third Chair of Morbid Anatomy, University of Milan; **Scientific Institute "'S. Raffaele'" of Milan (Italy). The distribution, the relationship with argyrophilia and the possible amine/peptide colocalizations of chromogranins/ secretogranins- (Cgs)like immunoreactive (-LI) endocrine cells in the alimentary tract of lizard Lacerta sicula have been investigated using novel monoclonal antibodies. CgA- and CgB-LI cells were everywhere numerous, except in the distal tracts of the small intestine. Almost all were positive to the Grimelius silver stain with parallel intensity. Some CgA- and CgB-LI cells didn't colocalize any investigated amine/peptide. CgA and/or CgB were found colocalized in almost all serotonin-, histamine-, substance Pand gastric polypeptide tyrosine tyrosine- (PYY-) LI ceils, but only in some somatostatin-LI cells and in no neurotensin-, gastrin/cholecistokinin-, pancreatic polypeptide- and intestinal PYY-LI cells. On the whole, the chromogranin/secretogranin content in the secretory granules appeared not homogeneous even within the same cell type.

P22.8 The Neural and Neuro-Endoerine Component of the Human Thymus. H.-J. Schuurman, E. Batanero, F.-E. de Leeuw, G. Jansen and D. van Wichen Department o f Pathology, University Hospital, P.O. Box 85.500, 3508 GA Utrecht, The Netherlands. Sections of formalin-fixed paraffin-embedded thymus (n = 17, between 26th week of gestation and 13 years) and of frozen thymus (n = 8, 1 8 9 5 89 years) were investigated in immunohistochemistry for pituitary hormones (FSH, LH, GH, ACTH, prolactin), somatostatin, chorionic gonadotropin, neurofilaments (68, 160 and 200 kD), neuron-specific protein PGP9.5, chromogranin A, synaptophysin, and tyrosin hydroxylase. Noradrenergic nerve fibers (tyrosin hydroxylase) were observed in the medulla around epithelium surrounding Hassall's corpuscles. Fibers expressing neurofilament 160 kD

602 and PGP9.5 occurred in the medulla and cortex-medulla region. PGP9.5 was extensively present in the cortex following a dendritic labelling pattern resembling the epithelial network. Peptidergic nerve fibres occurred in the medulla along epithelial cells surrounding Hassall's corpuscles and around blood vessels in the medulla and cortex-medulla region. Labelling for FSH revealed extensive and long fibers in the outer cortex; in two-color immunohistochemistry these fibers were especially prominent in the epithelial network. Neuroendocrine hormones were located in epithelium, particularly cells in the cortex and more extensive in the medulla around Hassall's corpuscles. Chorionic gonadotropin was present on the surface of lymphocytes, that in two-color immunofluorescence appeared to be in the CD3-positive subset. Conclusion: The neural and neuro-endocrine systems have an extensive expression in the human thymus: (1) noradrenergic and peptidergic innervation, and (2) the presence of neuroendocrine hormones in mainly epithelial cells.

P22.9 Postnatal Maturation of Astrocytes in the Rat Spinal Cord and Effect of Neonatal X-Irradiation. Z. ~irnonovd and E. Sykov~ Laboratory of Cellular Neurophysiology, Institute of Experimental Medicine, CAS, Prague, Czechoslovakia. G F A P is the major protein of intermediate filaments in mature astrocytes and in activated astrocytes during CNS injury. Antibodies against G F A P were used to follow postnatal maturation of astrocytes in the spinal cord of 2 to 21-day-old rats, and astrogliosis after X-irradiation of the lumbar region of the spinal cord in 1-day-old rats. In 2 to 6-day-old pups the membrane limitans and radial glia in the white matter, running from subpial zone inward, were densely stained and remained so even in adult rats. In the grey matter, small, but densely stained somata with very short processes were found in 2 to 6-day-old pups. Astrocytes on the 8th postnatal day become well stained throughout the entire spinal cord, they had large soma and short thick processes. On the 12th day the somata were less dense and processes were thinner and longer. In about 21-day-old pups the astrocytes closely resemble those in adult animals, i.e. they decrease in number, their soma are small and have thin and long processes. X-irradiation disrupts radial glia in the white matter and maturation of astrocytes in the gray matter. In 8 to 12-day-old rats, typical astrogliosis was found, with proliferative and hypertrophied astrocytes with densely stained large somata and thick, long processes. These finding correlate with the electrophysiological experiments which revealed that these immature and activated astrocytes are not able to ensure ionic and volume homeostasis in the rat spinal cord.

P22.10 Immunohistochemical Localization of NGF and NGF. Receptor in the Hypothalamus of the Adult Rat. L. M. Talamini and L. Aloe Institute of Neurobiology, CNR, Viale Marx 15, 00156 Rome, Italy. In the central nervous system of mammals, the nerve growth factor (NGF) has thus far been implicated mainly in the

P o s t e r sessions support and pathophysiology of cholinergic basal forebrain (CBF) neurons. In recent years, however, evidence has emerged indicating that N G F may affect non-cholinergic neurons as well. It was, for example, reported that N G F is present in the hypothalamus of mice and that its level here increases following aggressive behaviour. The aim of the present study was to extend these observations by using immunohistochemical techniques to localize NGF-positive cells and by evaluating the effect of anti-NGF antibody administration in the rat hypothalamus. The results show colocalization of NGF and NGF-receptor expression in the paraventricular, periventricular and supraoptic nuclei. To our knowledge this is the first evidence demonstrating that N G F and NGF-r are coexpressed in the hypothalamus. Our study furthermore demonstrates that NGF target cells in the CBF are affected by local injection of anti-NGF antibodies, whereas in the hypothalamus this does not seem to be the case. In conclusion, our findings support the hypothesis that N G F plays a role in the pathophysioiogy of CBF neurons, while prospecting a function of NGF in the hypothalamus. A function which is probably connected with neuroendocrine processes and which might be mediated by an autocrine mechanism.

P22.11 Immunohistochemical Study on Neuroblastoma and Metastatic Tumors. Z. Xiaofeng Anthropotomy Association of China, Dept of Histology and Embryology, Jiamusi, Medical College, Dexang Road, Jiamusi, P.R. of China. 5 cases of neuroblastoma coming from sympathetic ganglion and their metastatic tumors from bone, kidney, spleen, lung, bone marrow were studied by Ag staining, AgNOR counting, NF and F3 immunohistochemistry staining. It was found that the morphological and functional properties of tumor cells were different in original and metastatic tumors, the number of the neurofibril was also different in them. We thought that metastatic cells consisted of mainly small cells in which some differentiation was found. They were probably a subvariety mass from original tumor. The development and differentiation in these metastatic tumor cells were not similar. This article also discussed some questions about neuroblastoma results.

P22.12 Immunohistochemical Study of NGF Effect on Cultured Cerebellum Neurons. Z. Xiaofeng, L. Fenghua and J. Weizhe Anthropotomy Association of China, Dept of Histology and Embryology, Jiamusi, Medical College, Dexang Road, Jiamusi, P.R. of China. Primary cultured cerebellum neurons were obtained. A model was established in which neurons were more than 90%. Added N G F 5 microlitre per millilitre into medium, the neurons were stained respectively by Ag-staining, NSE and NF immunochemistry staining at 5d., 10d., 20d., 30d. It was

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found that NSE and NF immunohistochemistry staining degree of experimental groups was higher 1 - 2 grade than controls, the difference is more obvious at 10d., 20d. The neurofibril numbers of Ag staining were different in both

P23. CYTOCHEMISTRY

groups. The results indicated that NGF had an effect on the differentiation of the cultured cerebellum neurons. This article also discussed the properties of growth cone and endocrine phenomena of cultured cerebellum neurons.

O F T H E GOLGI A P P A R A T U S

P23.1

Uitrastructural Localization of UEA-I and AAA Binding Sites in the Human Fundic Glands. Implications in the Fncosylalion Processes. J. F. Madrid, M. T. Castells, M. Avil6s, J. A. MartfnezMenn~irguez and J. Ballesta Department of Cell Biology. Medical School University of Murcia. Murcia. Spain. The subcellular localization of a carbohydrate residue by different lectins with subtle differences of their binding specificites provides useful information on the topology of the glycosylation processes. Ulex europaeus (UEA-I) bind preferentially to terminal fucose residues a(1-2)-linked to Gal. Aleuria aurantia (AAA) specificity binds fucose residues a ( 1 - 6)-linked to the proximal GlcNAc moiety of N-linked glycoproteins. The intracellular distribution of Fuc residues on different cell types of human fundic glands has been studied by means of UEA-I and AAA lectins. Samples of histologically normal human fundic mucosa were obtained. H-blood group specimens were refused to avoid blood group interaction. For light microscopy, the samples were fixed in formalin, embedded in paraffin and the sections stained with UEA-I-peroxidase labelled and AAA-digoxigenin labelled. For electron microscopy, tissues were fixed in glutaraldehyde, and embedded in Lowicryl K4M. Sections were labelled with UEA-I gold and AAA-digoxigenin conjugates. External Fuc residues detected by UEA-I were observed in mucous granules of surface epithelium, clear areas of the secretory granules of mucous neck and transitional cells, apical membrane of chief cells, trans side of the Golgi complex of surface mucous, mucous neck and transitional ceils, and in the intracellular canaliculi of parietal cells. Core-linked Fuc residues (areas where only AAA-reactivity was observed) were located in dense areas of secretory granules of mucous neck and transitional cells, as well as zymogen granules of chief cells. Areas showing reactivity to UEA-I were always reactive to AAA; therefore, in these zones, differentiation between external and core-linked Fuc residues was not possible. In summary, external Fuc residues were detected in those areas containing O-linked glycoproteins while core-linked Fuc residues were observed in areas where the existence of N-linked glycoproteins have been previously reported. This work was supported by a grant from DGICYT PB 900310 (Spain).

AND LYSOSOMES

P23.2 Ultrastructurai Characterization of Terminal Oligosaccharide Sequences in the Glycoproteins of Human Laryngeal Glandular Cells. M. T. Castells, J. F. Madrid, J. A. Martfnez-Men~rguez, M. Avil6s and J. Ballesta Department of Cell Biology. Medical School, University of Murcia. Murcia. Spain.

The glycoproteins constitute the major component of human airways secretions. The occurrence of terminal residues of fucose and neuraminic acid in the human airways glycoproteins have been reported. These residues have been involved in protective and inter-cellular recognition functions. In the present study, lectin cytochemistry in combination with chemical and enzymatic deglycosylation treatments are used to characterize the terminal sequence of the oligosaccharides contained in human laryngeal glands. For light microscopy, samples of human larynx mucosa were fixed in 10070buffered formalin and embedded in paraffin. Peroxidase and digoxigenin conjugated lectins were applied (LFA, MAA, SNA, WGA, DSA, PNA, RCA-I, LTA, UEA-I and AAA). Desulphation and desialylation treatments were performed on some sections. For electron microscopy, samples fixed in 2~ glutaraldehyde in PBS were embedded in epon 812 or lowicryl K4M. One step (RCA-I gold, UEA-I-gold), two step (WGA/ ovomucoid-gold, DSA/ovomucoid-gold, LFA/fetuin-gold), and three step (PNA-, AAA-, LTA-, MAA-, SNA-digoxigenin conjugated/anti- digoxigenin/gold labelled antilg) lectin cytochemical techniques were applied. Neuraminidase digestion was also applied at the ultrastructural level. The glycoproteins contained in the secretory granules of mucous cells showed a variety of terminal oligosaccharide chains including GaI-(SO4)-/~t-3-GaINAc, Neu5Ac-Gal(SO4 or Fuc)/~I-4-GlcNAc, GaI-(SO4)-/31-4-GlcNAc, GIcNAc-SO4. Secretory granules of serous cells showed a different morphology and glycoconjugate cytochemistry. They mainly contained sialoglycoconjugates with terminal Neu5Ac-a2-3-Gal-pl-4GIcNAc and Neu5Ac-a2-6-Gal sequences and Gal-/~I-4GlcNAc, GlcNAc, GaI-(SO4)-/31-3-GalNAc oligosaccharide chains. Some of these sequences were fucosylated. This work was supported by a grant from DGICYT PE 90-0310 (Spain).

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P23.3 Study on Nematolysosome on Neurons in CNS by ACPase. X. Yu*, M. Sakai and K. Ogawa Dept. Anat., Fac. Med., Kyoto Univ., Japan *Dept. Histol. & Embryol., China Medical Univ., People's Republic of China. Since it has no reports about the presence of the nernatolysosomes in large pyramidal cells of cerebrum, Purkinje cells of cerebellum and motor neurons in anterior horn of spinal cord in CNS, we try to demonstrate the ultrastructural distribution of nematolysosomes in these cells by ACPase cytochemical technique using weak fixation of glutaraldehyde. Adults rats were perfused with 2~ formaldehyde and 0.25~ glutaraldehyde in 30 mM PIPES buffer for 10 min. The cerebrum, cerebellum and spinal cord were cut into about 1 - 2 mm block, the 50/am thick sections were made by Vibratome or Microslicer. The sections were incubated in Gomori's solution for 80min at room temperature. Ten mM NaF was added to incubation medium or the substrate was removed for the cytochemical control experiments. After the reaction, the sections were postfixed with 1070 0504 for 30 min. The sections were rinsed, dehydrated, embedded in Epon 812. The ultra-thin sections were stained with uranyl acetate and observed in a 1200 EX electron microscope. The nematolysosomes with positive ACPase activity were observed in the cytoplasm of the neurons of CNS. The motor neurons have more nematolysosomes which may extend into the processes. In these cells there were many microtubules and microfilaments. This may suggest the nematolysosomes is related to the ACPase transport in the neurons.

P23.4 Characterization of Autophagic Vacuoles Induced with Iodoacetate in Ehrlich Ascites Tumor Cells. H. Reunanen, J. Naarala and E.-L. Punnonen Department of Cell Biology, University of Jyvdskyl(t; P.O.B. 35, SF-40351 Jyvdskyld, Finland. Iodoacetate (2.5 mM) decreases cellular ATP-level and induces accumulation of early autophagic vacuoles in Ehrlich ascites tumor cells. The aim of this immunocytochemical study was to investigate whether these vacuoles contain cationindependent mannose 6-phosphate receptor (MPR), Cathepsin L or lysosomal membrane protein (LAMP-1). After isolation the cells were incubated for 60 min in phosphate buffer with or without 2.5 mM iodoacetate. The cells were fixed, treated with sucrose and frozen in liquid nitrogen. Thin sections of frozen ceils were cut at -90~ Immunolabelling was carried out using protein A-gold particles. Antiserums against MPR, cathepsin L and LAMP-1 were used as primary antibodies. The bulk of all the labels was found in large vacuoles, which were usually partly membrane-filled and partly electron translucent. These vacuoles probably correspond to the prelysosomal compartment (PLC). Early autophagic vacuoles were identified morphologically: they were surrounded by a membrane and contained cytoplasmlike material. They were usually unlabelled; only a weak labelling was observed in some vacuoles. Early autophagic vacuoles were sometimes situated very close to the PLCs. The results show that iodoacetate treatment induces accumulation

P o s t e r sessions of autophagic vacuoles which have lysosomal/prelysosomal markers.

not

yet received

P23.5 Ultrastructural Cytochemistry of Pulmonary Intravascular Macrophages (PIMs) of Sheep: Response to In Vivo Heparin Treatment. B. Singh, O. S. Atwal, P. K. Basrur and K. J. Minhas Dept. Biomedical Sc., Univ. Guelph, Guelph, ON NIG 2W1, Canada. The acid phosphatase staining has been used as a classical technique to identify lysosomes at the ultrastructural level. We utilized this method to study the cytochemical response of the PIMs of sheep to heparin. The PIM is a recently identified mononuclear phagocyte having a heparin sensitive globular surface coat (Am. J. Anat., 186: 2 8 5 - 299, 1989). Sheep were injected with heparin (50 IU/Kg Body Wt. IV) and sacrificed at 30 min, 48 hr and 120 hr post treatment. Lungs were fixed in situ by pouring the fixative (2.5~ glutaraldehyde and 2.0~ paraformaldehyde in 0.1 M Na-cacodylate buffer pH 7.4) intratracheaUy. Tissue was collected from different lobes of the right lung and processed for the acid phosphate localization. In the control sheep the PIMs had a diffused distribution of generally spherical lysosomes. After 30 min of heparin treatment most of the PIMs were devoid of any acid phosphatase staining and rarely a PIM showed a lysosome. However, a significant amount of the enzyme marker was observed around the PIMs in the lumen of alveolar capillaries. Majority of the PIMs were highly elongated and had ruffled plasma membrane. Following heparin administration no change in the cytochemical reactivity of type II ceils and the alveolar macrophages was seen. After 48 and 120 hr of heparin treatment the PIMs had lysosomes as seen in the control sheep. Our results suggest a high degree of sensitivity of the PIMs to heparin, a known vasoactive substance, as manifested by acid hydrolase secretion and physical elongation. (Supported by NSERC Canada, grant # 8 2 9 - 66).

P23.6 Cerium-Based Cytochemistry of Golgi Apparatus and Lysosomes in the Epithelial Cells of the Cyclic Rat Endometrium. L. Staneva-Dobrovski and H. Wilting Dept. of Anatomy, H.-Heine-University of Duesseldorf, Duesseldorf (FRG). In the present study we applied cerium-based methods for the ultrastructural localization of acid phosphatase (AcPase) and

thiamine pyrophosphatase (TPPase) activities to examine the cyclic variations in the cytochemistry of Golgi apparatus and lysosomes in the luminal and glandular epithelial cells in the rat endometrium during the four phases of the oestrous cycle. 40 gm Transversal sections of uterine horns of rats, sampled at each of the phases, were incubated, applying cytidine-5'monophosphate (AcPase) and thiamine pyrophosphate (TPPase), respectively, as the substrates and cerium chloride as the trapping agent. ACPase reaction product was localized primarily in lysosomes and extended (at metoestrus and dioestrus) to the l s t - 2 n d transmost cisternae (GERL-iike

cisternae) and to numerous Golgi-associated vesicles in both

Poster sessions epithelial cell types. TPPase activity was confined to the 1st-3rd trans eisternae. Concomitantly with the oestrogenstimulated secretion in the endometrial epithelium (prooestrus and oestrus) fine granular reaction product was also present in GERL-Iike eisternae and in the outermost eis eisternae and vesicules of the now complex and numerous Golgi apparatus. The established in our study AePase and TPPase modulation and redistribution in the endometrial epithelial cells during the oestrous cycle are in concord with previous observations of enzyme modulations in Golgi and GERL in various other secreting cell populations.

P23.7

Reconstruction of Golgi Apparatus Following withdrawal of Brefeldin A. S. Yamashina, H. Tamaki, Y.-I Kadoya and O. Katsumata Department of Anatomy, Kitasato University School of Medicine, 1-15-1, Kitasato, Sagamihara, 228 Japan. The Golgi apparatus in parotid acinar cells is a reticulated but continuous organella consisting of stacked cisterna and surrounding vesicles. Brefeldin A, a metabolite of soil fungus,

605 causes a rapid regression of Golgi apparatus to the rudimentary form. This process is reversible, and reconstruction starts immediately after withdrawal of the drug to recover the characteristic lamellated structure within 1 hr. A study was made of the mechanisms of morphogenesis and maintenance of the complicated structure of Golgi apparatus during reconstruction using various histochemical and cytochemicat markers of the Golgi, such as cis elements by osmium impregnation, and trans by TPPase and Golgi monoclonal antibody. Incubation at 20~ produced many transport vesicles that gathered in the vicinity of rudimentary Golgi, but there was no recovery of cisternal structure. At 30 ~C, the transport vesicle slowly fused to form cisterna of the Golgi, thus making it possible to follow process of lamellar formation morphologically. At this juncture, vesicles containing the cis marker became arranged to form cis elements, followed by the formation of stacked cisterna, in which the trans markers could be seen. Nocodazole, an antimicrotubular agent caused dispersion of Golgi elements, in which cis marker were distributed extensively, but the piling up of cisterna was delayed. These findings indicate microtubules to contribute the generation and maintenance of the normal lamellated structure of Golgi apparatus.

P24. E N Z Y M E H I S T O C H E M I S T R Y P24.1 Membrane Enzymes in Excretory Organs of Invertebrates and Vertebrates. B. Dare, A. Paraninfo, L. Labanca and P. Usai Dipartimento di Biologia Animale, Facolta "di Scienze MFN, Universita "di Torino, v. Accademia Albertina 17, I 10123 Torino, Italy.

In the course of several years we revealed enzymatic activities in Vertebrate and Invertebrate tissues embedded in polar resin (Glycol Methacrylate) at low temperature. In Vertebrate nephron (Mammalia and Amphibia) we constantly locate aspecific alkaline phosphatase (E.C. 3.1.3.1, alPase) activity in brush border membranes (BBM) of proximal convoluted tubules (pct). In these membranes (BBM) we often detect also activity of transpeptidases (Leucyl amine peptidase, E.C. 3.4.11.1, LAP; Gamma glutamyl transpeptidase, E.C. 2.3.2.2, GGT). It is interesting to note that in Xenopus laevis (Anura) and in Triturus carnifex (Urodela) the activity of these enzymes is not uniform along pct: there are patent gradients of the different activities; in literature, this is true in Mammalia too, for aiPase and GGT. Furthermore, we frequently observe aIPase activity in the membrane of vesicles underlying BBM and in pct cells basolateral membranes, sometimes associated with GGT activity. The colocalization of alPase, LAP and GGT activities in the pct membranes is certainly related with proteins, oligopeptides and amino acid reabsorption from ultrafiltrate. In addition, the association between these membrane enzymes is supported by the presence of alPase, LAP and GGT activities in Invertebrate nephridial organs (Macrotrachela quadricornifera, Rotifera; Unio mancus, U. elongatulus, Anodonta

cygnea, Bivalvia) and in Vertebrate and Invertebrate absorbing intestinal cells. Work supported by MURST grants (60%, 40%).

P24.2 Histochemical and Morphofunctional Studies on Urodela Integument. First Results on Alkaline Phosphatase. B. Dare, A. Paraninfo, G. Lodi, F. Andreone*, M. Operti and P. Usai Dipartimento di Biologia Animale, Facolta "di Scienze MFN, Universita "di Torino, v. Accademia Albertina 17, 10123 Torino; *Museo Regionale di Scienze Naturali, v. Giolitti 36, 10123 Torino, Italy. Aspecific alkaline phosphatase (E.C. 3.1.3. I; alPase) plays an important role in the processes of selective transport through membrane by a mechanism not yet explained. Using direct copulation of substituted naphtols (Burstone technique) we studied the alPase localization in the integument of eight Urodela species adapted to different habitat: Salamandra Salarnandra, S. lanzai and A m b y s t o m a mexicanurn (strictly terrestrial species); Triturus carnifex, T. alpestris and T. vulgaris (really amphibious life); Pachitriton brevipes and Pleurodeles waltl (mostly living in water). Moreover, Triturus alpestris and T. carnifex were studied in the course of annual cycle; T. alpestris was followed from larval phase, during metamorphosis and in the adult state. Data on transtegumental active sodium transport (estimated by means of the short circuit current, SCC) in Triturus alpestris and T. carnifex are related to histochemical results. In all the adult specimens the alPase activity is localized in the epidermis and in the endothelial cells of the dermal vessels. Concerning the integument organization, as well as the

606 localization and the intensity of the enzyme activity, intraspecific differences are evident, probably connected with the adaptation to the habitat. We are extending the study of the role of alPase taking into account some enzymes surely involved in active transport (Na/ K ATPase) and in respiratory gas exchange (Carbonic anhydrase). Work supported by MURST grants (60~ 40070).

P24.3 Carbonic Anhydrase and Lectin Labelling Distribution in the Quail Kidney. M. G. Gabrielli, G. Menghi and P. Palatroni Dept. M.C.A. Biology, Camerino University. Camerino (Mc), Italy.

Carbonic anhydrase distribution in the quail kidney was investigated by an immunocytochemical technique using an antibody against avian CA II. Comparison with previous histochemical findings (M. G. Gabrielli et al., Histochem. J. 22, 579, 1990), showing fair correlation on both distribution site and reaction intensity in the different tubules, strongly suggested that the soluble cytoplasmic isoenzyme may account for the bulk of renal enzyme activity. The highest CA II immunoreactivity in distal tubules further supported their involvement in urinary acidification and bicarbonate reabsorption. The mosaic-like pattern, established by CA and cytochemical stainings in collecting ducts, was detailed by HRP-conjugated lectins combined with sequential glycosidase degradation (G. Menghi et al., Histochemistry 90, 331, 1989). The approach, besides confirming the occurrence of two main cell types, namely principal and intercalated cells, proved to be a useful tool for investigating further heterogeneity within the two cell types in terms of carbohydrate moiety distribution.

P24.4 G6PDH Activity of Liver Cells in Normal, Ehrlich Carcinoma- and Colon Carcinoma-Bearing Animals. P. Griffini, V. Bertone, C. J. F. Van Noorden* and

I. Freitas Dept. of Animal Biology and CNR Center for Histochemistry, Univ. of Pavia; Italy. *Lab. Cell Biology and Histology, Univ. of Amsterdam, The Netherlands.

Liver plays essential metabolic, detoxifying and storage roles and is affected by neoplastic insult. Glucose-6-phosphate dehydrogenase (G6PDH) was studied histochemically in livers of normal mice and rats, mice with Ehrlich carcinoma on the hind leg, and rats with colon carcinoma metastatizing to the liver. G6PDH activity in normal animal hepatocytes was found to be present in a diffuse and a particulate form in discrete circular regions around pericentral veins and contiguous periportal zones respectively. These types of zonation agrees with the concept of Lamers et al. (Hepatology I0: 7 2 - 7 6 , 1989) of liver lobulus. In Ehrlich-tumor bearing mice, the liver parenchyma appeared to be affected although no metastases were detected. The zonation of G6PDH activity was progressively decreased and the amount of diffuse increased as the tumor became larger. In contrast, the zonation was not altered in livers bearing metastases from

Poster sessions colon carcinoma. Undifferentiated metastatic cells could be distinguished from differentiated cells since they showed a higher G6PDH activity and a different subcellular distribution of the coloured product. Kupffer cells, normally distributed in periportal tracts, and identified through their high G6PDH activity, were increased in number throughout the parenchyma in turnout-bearing animals. These observation suggest the plasticity of the distribution of G6PDH in liver as a response to increased tumor burden irrespective the localization of the tumor in the body.

P24.5 The Histochemical G6PDH Reaction But Not the LDH Reaction with Neotetrazolium is Suitable for Detection of Cancer Cells. P. Griffini, E. Vigorelli*, G. N. Jonges ~, I. Freitas and C.

J. F. Van Noorden* Dept. of Animal Biology and *Inst. of Gen. Pathology, Univ. Pavia; Italy. *Lab. Cell Biology and Histology; Univ. Amsterdam, The Netherlands.

The oxygen insensitivity of the histochemical reaction based on neotetrazolium (NT) reduction for glucose-6-phosphate dehydrogenase (G6PDH) activity was proposed for discriminating malignant and premalignant from normal cells. In the presence of incubation medium saturated with 02 the formazan generation was absent in normal but not in malignant cells. A competition for reductive equivalents between oxygen and Neotetrazolium, via superoxide dismutase (SOD), was suggested (Best et al. Cancer Res. 50: 5112-5118, 1990). Since SOD activity is decreased in tumor cells, Neotetrazolium reduction would not be inhibited in these ceils. We tested this hypothesis by demonstrating LDH activity instead of G6PDH activity in normal rat liver and metastases in liver from colon carcinoma. Enzyme reactions were determined cytophotometrically for both enzymes in the presence of pure 02 and N2. G6PDH acted as previously described. When an enzyme such as LDH is used that produces NADH instead of NADPH, this was not the case: normal liver tissue deviates. It appeared that the residual activity of G6PDH in 02 in normal liver tissue was 0070 whereas in colon metastasis it was 33 07o.However, the residual activity in 02 of LDH in normal female rat liver was 30070 and in normal male rat liver 75~ whereas the residual activity in metastasis was only 24070. These experiments clearly indicate that the oxygen sensitivity phenomenon is not solely an effect of competition for reducing equivalents between Neotetrazolium and oxygen via SOD, as NADPH generated by G6PDH and NADH by LDH have the same redox potential. Apparently, the system is more complex and other NADPHdependent cellular systems are involved as well.

P24.6 Histochemical Studies of Acetylcholinesterase and Cholinesterase in Various Developmental Stages of the Human and Rat Placenta*. T. Hahn Department of Anatomy, Free University of Berlin, Berlin, Germany.

The aim of this study is to clarify whether as suggested for other tissues cholinesterase (EC 3.1.1.8, ChE) may act also in

P o s t e r sessions the placenta as embryonic acetylcholinesterase (EC 3.1.1.7, ACHE) and where both enzymes are actually localized in this organ. Therefore, the placenta of rats and man was investigated during different developmental stages using routine and new histochemical methods (GEREBTZOFF 1953, KARNOVSKY & ROOTS 1964, TSUJI 1974, T A G O et al. 1986, ANDRJ~ & LOJDA 1986). In the human placenta AChE activity was found in the syncytiotrophoblast (STB), endothelial cells and media of fetal blood vessels. Stageindependently, weak ChE activity was demonstrated in the STB. In all developmental stages the STB, visceral and parietal yolk sac epithelial cells and Reichert's membrane of the rat placenta stained only for ACHE. These histochemical results show that generally ChE does not function as an embryonic AChE in the placenta, because the activity ratio of both hydrolases did not change during development. Furthermore, AChE and ChE appear in many structures not yet known before for the rat and human placenta and can no longer be considered as enzymes mainly present in the blood of this organ. *Supported by the German Research Foundation (Sfb 174). P24.7

Cerium as a Capturing and Amplifying Agent in Phosphatase and Oxidase Histochemistry. A Survey. K.-J. Halbhuber, H. Feuerstein and R. Bock Institutes of Anatomy, Friedrich Schiller University arenaand Saarland University Hamburg, Germany,

The appropriateness of cerium (CeIII) as a histochemical capturing agent can be attributed to the high speed of reaction with enzymatically liberated free orthophosphate and H202, extremely low solubility of both primary reaction products (PRP) cerium hydroxyphosphate (CeIIIHP) and cerium perhydroxide (CeIVPH) as well as the seldomly observed diffusion- and affinity artifacts. The latter occur frequently after application of lead as capturing ion. We developed new light microscopical techniques for cryostat and semithin sections based on several visualisation principles: Conversion of the 1) PRP into lead phosphate and lead sulfide, 2) CeIIIHP into CeIV (perhydroxy)HP, which is able similar to CeIVPH to oxidize DAB or pyrocatechol/pphenylenediamine into coloured compounds, 3 ) P R P into CeIII-oxalate, which is able to reduce OsO4 into osmiumblack. Moreover, different Gomori-based cerium methods based on a conversion of CaHP into CeIIIHP yielded satisfactory results. CeIII in complexed form can also be employed for amplification of PRP via reaction with CeIVperhydroxy compounds. The cerium-based methods represent alternatives in light and electron microscopical enzyme- and immunohistochemistry that are superior to the classical techniques.

P24.8 An Improved Method for the Detection of Minimal Residual Disease of Genetically Marked Leukemic Cells, P. Yo Hendrikx, A. C. M. Martens, C. O. Phorst-van Marrewijk, J. W. M. Visser and A. Hagenbeek TNO Institute of Applied Radiobiology and Immunology, Rijswijk, The Netherlands. In order to investigate the homing behaviour of leukemic cells

607 and the spatial distribution of leukemia regrowth immediately after therapy in the BN rat myelocytic leukemia model (BNML), the E. coli LacZ gene was introduced as a genetic marker into an in vitro growing subline of the BNML. A clone was selected which exhibits reliable, high expression of LacZ, in vitro as well as in vivo. A very sensitive suspension assay for these leukemic cells was developed. The fastest and most reliable method to screen large numbers of samples for the presence of a very small subpopulation of cells is by visual inspection under a microscope, using a low magnification. Using this method, the substrate X-gal, which yields a bright blue precipitate inside LacZ expressing cells, is the substrate of choice. To allow specific recognition of X-gal stained cells as genetically marked leukemic cells it is necessary to suppress mammalian type /~-galactosidase background activity. In a fluorimetric MUG-assay a number of substances described in the literature as ~-galactosidase-inhibitors were tested for their potential use as a specific inhibitor of mammalian type /3galactosidase. Taking these considerations into account, a new, highly sensitive method for the microscopic detection of LacZ-marked leukemia cells in suspensions was developed, in which cells are attached to poly-L-lysin coated flat bottom microwells, and stained with X-gal. Leukemic cell numbers are determined by visual inspection using an inverted microscope, where the leukemic cells appear as very intensely blue stained cells. At leukemic cell frequencies of up to ca. 50 cells/well entire wells are examined. At higher frequencies, leukemic cell numbers are determined by taking a number of "optical samples" from each well, using an eyepiece grid. The sensitivity of this assay is better than 1 per 5 • 106.

P24.9 Cytochrome c Oxidase Defects in Human Skeletal Muscle - - Correlation with Mitochondrial D N A Abnormalities. M. A. Johnson, L. A. Bindoff and D. M. Turnbull School of Neuroseiences, University of Newcastle upon Tyne, UK.

Cytochrome c oxidase (CCO) is terminal component of the respiratory chain and consists of 13 polypeptide subunits, 3 of which are coded on mitochondrial DNA (mtDNA) while the rest are coded on nuclear DNA. We measured the catalytic activity of CCO in single muscle fibres by micro-photometric enzyme assay (MEA) using diaminobenzidine as electron donor. Control data were derived from 18 adult and 20 juvenile subjects with no evidence of neuromuscular disease. In 32 patients with either Kearns-Sayre syndrome (KSS) or chronic progressive external ophthalmoplegia (CPEO), abnormal variability of CCO activity was seen within the muscle fibre population due to the presence of a mosaic of CCO-deficient fibres and fibres with normal catalytic activity. The proportion of CCO-deficient fibres was t I % - 9 5 % in individual patients. In many of these patients, mutations of mtDNA were demonstrated and the abnormal degree of variability of CCO activity appears to be associated with mtDNA heteroplasmy ie coexistence of mutant and wild-type DNA in the muscle fibre population. In contrast, in patients with Leigh's syndrome associated with CCO deficiency, the enzyme abnormality was expressed uniformly throughout the muscle fibre population. CCO activity was only 1 0 % - 30% of normal, with few fibres showing CCO activity within the

608 normal range. This finding is consistent with a primary defect of nuclear DNA in these patients, associated with retention of mtDNA homoplasmy.

P24.10 Expression of Enzymes in Microvillous Plasma Membranes in the Mammalian Epididymis. B. Miehe and R. Gossrau Departments of Anatomy, University of GreifswaM and Free University of Berlin, Germany.

In mammals the epithelium of the epididymis is equipped with microvilli and contributes to the maturation of sperms. The present study deals with the question whether this speciesindependent cell surface morphology and function are also reflected by a species-independent enzyme behaviour in the microvillous plasma membranes. Using catalytic histochemistry for the visualization of various hydrolases the epididymis of rats, mice, gerbils, guinea-pigs, marmosets, rabbits, cats, sheep and man was analysed. Species-independently non-specific alkaline phosphatase, nucleoside phosphatases, dipeptidyl peptidase IV and y-glutamyl transpeptidase were expressed in microvillous plasma membranes. Microsomal alanine aminopeptidase, a-glutamyl aminopeptidase and a-D-glucosidases were expressed species-dependently. All microvillous hydrolases were only expressed by the human epididymis. Furthermore, species-dependent regional expression differences were found. In conclusion, despite identical plasma membrane structure and function, microvillar enzymes behave differently in the mammalian epididymis. This shows macromolecular and therefore metabolic differences in the process of sperm maturation.

P24.11 Cytochemical Pattern of Neutral Maltase (EC: 3.2.1.20) in Human Granulocytic Cells from Chronic Myeloid Leukemia and Healthy Subjects: Functional Activities Associated to Maturation Stages. P. J. M. Philip Laboratoire Central d'H~matologie, CHU Nice Sophia-Antipolis, France.

Maltases are alpha glucosidases that split glucose residues from the non-reducing ends of alpha glycans of various lengths ranging from disaccharides to high molecular weight polysaccharides like starch. Biochemical studies have shown that Neutral Maltase Activity (NMA) is present in normal granulocytes from peripheral blood but in chronic or acute myeloid leukemia these cells have markedly decreased and no detectable activity respectively. In hematological diagnostic, enzyme cytochemistry techniques may be useful for the purpose of distinguishing different cells types of maturation stages. The objective of this study was to investigate cytochemically the presence and distribution of NMA in circulating polynuclear neutrophils from chronic myeloid leukemia (CML) and normal healthy subjects in order to: 1/analyze the variations of NMA observed between normal and neoplastic cells; 2/ research correlations between functional activities and maturation stages of granulocytes, Cytochemical detection of neutral maltase was realized using 5-Br-4-C1-3-indoxyl-alpha-D-glucoside as a substrate in an

Poster sessions indigogenic procedure. The product of the neutral maltase reaction was represented in neutrophils from peripheral blood smears by fine or coarse granule scattered throughout the cytoplasm. In all the circulating neutrophils from healthy subjects neutral maltase reactions were found to be strongly positive. Conversely, the products of neutral maltase in peripheral neutrophils from CML consisted of either less intensities or no reactions. These results suggest that circulating neutrophils from CML may be represented at least by two subpopulations showing different functional activities. Thus, negative NMA neutrophils could represent a subpopulation of immature cells whereas positive NMA neutrophils could belong to another one where terminal maturation stages of granulocytes are open. Thus, cytochemical pattern of neutral maltase could investigate the bio-functional activities associated to the maturation stages of granulocytes by quantitative image analysis system.

P24.12 Quantification of Cell Growth and Functional Activities From Cell and Tissue Cultures by Neutral Maltase Activity (EC: 3.2.1.20) and Wavelet Transform Analysis: A Model to Test Tissue and Cell Damages at the Implant Interfaces. P. 3". M. Philip, J. Giudicelli and A. Houri Laboratoires Central d'Hgmatologie, de Biochimie et de Recherches Neurosensorielles ORL/GBM, CHU -- Nice Sophia-Antipolis, France.

The alpha-glucosidases divided into two groups according to their optimum pH i.e. acid alpha-glucosidases (pH 4.5) and neutral alpha-glucosidase (pH 6.5), are present in many tissues and appears as ubiquitous markers reflecting functional activities. In implantology, cell damages implied in the human tissues during and after surgical processes may be associated with: 1/the nature of the mechanical or friction stresses; 2 / t h e thermic stresses following graft preservation, microwaves radiations such as ultrasonic waves, laser or hyperfrequencies; 3/the specific kinetics of cell growth known for each tissues; 4 / t h e tolerance of the toxicity of implants which induce in vivo cell stresses at the implantological interfaces. Thus, the question arise of how the physiological and physiopathological processes which occur at the implantological interfaces may be approached and quantified in vitro preliminary to the clinical applications. Here, we propose three models of cell and tissue cultures (osteoblasts, neuroblasts, lymphoid B-cells) where a biochemical marker (Neutral maltase) reflecting functional activities through its enzyme activity and a biophysical approach allowing to investigate micro and macro cell movements using wavelet transform analysis, were both characterized and quantified before and after physical damages in vitro induced. Cell cultures before and after physical stresses show biochemical and biophysical variations which appear correlated to degrees of cell damages. The nature of tissue and cell damages as well as the potentialities of physiological cell regeneration processes (cell growth, maturation index) occurring in vivo during and after surgical processes at the human implantological interfaces, may be thus predicted and in vitro quantified. The cell and tissue stresses induced by surgical processes as well as implantological biomaterials may thus be

Poster sessions modelized and controlled in order to determine the best procedures for any cIinical applications.

P24.13

Adaptation of the Liver and the Islet System to Different Forms of Physical Stress. K.-S. Pieper 1, P. Fehrmann 2 and C. Grofler 2 ~Abteilung A n a t o m i c - Universitdt Ulm; ~Institut fiir Anatomic der Medizinischen Akademie Dresden, Germany. Parallel investigations of the liver and islet system of Wistar rats who have undergone swimming training of six weeks indicate that differentiated adaptation of both systems occur - - with the level of adaptation being dependent on stress mode. By means of detecting phosphorylase (E.C.2.4.11.) and glycogensynthetase (E.C.2.4.1.11.), respectively, a distinct increase of phosphorylase and an activity decrease of glycogensynthetase was observed 24 hours after the intensively trained animals had been exposed to final stress. Extensively trained animals showed further increase of phosphorylase activity and only little synthetase activity. The glycogen content, which has been demonstrated with PAS-reaction and controlled with the help of distase preincubation for substrate peculiarity showed remarkable increase in group 2 (intensively trained animals) and was even more essential in group 3 (extensively trained animals). Particularly those animals extensively trained revealed striking adaptions of the endocrine pancreas. By using the detection of acid phosphatase (E.C. 3.1.3.2.) extreme islet enlargements as well as single transformations and small cell nests (islet shoots), which are histologically located in the acini region. Cellular reactions of the same kind could be observed in dogs following autogenic and heterotopic pancreatic segment transplantations. Findings suggest that extensive stress patterns may cause hypermetabolism and trigger a signal for an acino-insular transformation.

609 content of rat hepatocytes. We were therefore prompted to evaluate the effects of DG perfusion on" (a) the ultrastructure of hepatocyte organelles, (b) the hepatocyte content in cytosolic and membrane-bound enzymes used as zonal markers, and (c) the behaviour of specific mRNAs and secreted proteins. For this purpose, 10 male Sprague-Dawley rats were perfused by either the portaI vein or the vena cava superior with a 0.4~ DG solution. Liver samples were processed for electron microscopy (EM), cytochemistry, immunohistochemistry, and in situ hybridization. Upon EM, DG-altered hepatocytes were characterized by a barely visible plasma membrane and by a highly reduced cytosolic density. Except for mitochondria, which were swollen and irregular, the ultrastructure of hepatocyte organelles was preserved. Numerous ribosomes were attached to the endoplasmie reticulum. Golgi apparatus contained lipoproteins. Cytochemical examination of DG-altered hepatocytes showed the near complete extraction of the cytosolic enzyme lactatedehydrogenase and of the mitochondrial matrix enzyme glutamate-dehydrogenase. In contrast, the membrane-bound enzymes glucose-6-phosphatase, associated with the endoplasmic reticulum, fl-hydroxybutyrate-dehydrogenase, NADPH-reductase and succinate-dehydrogenase, associated with mitochondrial inner membranes, were still readily detected. Two secreted proteins, albumin and a~-glycoprotein, were detected in DG-altered hepatocytes by immunohistochemistry. Finally, reduced but significant levels of mRNAs for albumin and glutamine-synthetase were still present in DG-altered hepatocytes. In summary, despite their striking morphological alterations, DG-altered hepatocytes retain significant levels of membrane-bound enzymes, secreted proteins and specific mRNAs. Two conclusions about the possible pitfalls of DG use might therefore be drawn: (a) biochemical analyses of effluents released by DG-permeabilized hepatocytes must be interpreted with caution, (b) the apparent phenotype of cell suspensions enriched in either periportat or perivenous hepatocytes obtained by the DGcollagenase technique might be significantly altered by the presence of residual DG-altered hepatocytes from the opposite zone.

P24.14 The Effects of Digitonin on Rat Hepatocytes: A Morphological Assessment on Liver Tissue Using Electron Microscopy, Cytochemistry, Immunohistochemistry and In Situ Molecular Hybridization. L. Racine, J.-Y. Scoazec, A. Moreau, D. Bernuau and G. Feldmann Laboratoire de Biologie Cellulaire, INSERM U327, 16, rue Henri Huchard, Paris, France. Digitonin (DG) perfusion of rat liver by the portal vein or the vena cava superior selectively alters the hepatocytes of either zone of the hepatic lobule by permeabilizing their plasma membrane. This technique is used alone, to compare the biochemical characteristics of periportal and perivenous hepatocytes by analyzing the effluents released from the permeabilized ceils, or in combination with a subsequent perfusion of collagenase, to obtain cell suspensions enriched in hepatocytes from one zone of the lobule. Despite the wide use of DG as a tool the study of metabolic heterogeneity in the rat liver, little is known of its actual effects on the intercellular

P24.15 Human Urethra: Histochemical Parameters. M. T. Perra, A. Pinna*, P. Sirigu and F. Turno Department of Cytomorphology and Clinic of Urology*, University of Cagliari, Italy. Bioptic specimens, normal at histological examination, were obtained from five patients (ages 4 0 - 6 5 ) undergoing cystectomy and urethrectomy for carcinoma of the urinary bladder. Samples were processed in a conventional manner for paraffin embedment. Microtome sections were subjected to the standard procedures for the investigation of the glycoproteins. Other pieces were immediately frozen and, after a few hours, cryostat sections were cut. Groups of slides were then incubated for the localization of some androgen metabolic enzymes. Neutral/acid mucosubstances were detected mainly in the apical cells of the epithelium and in occasional groups of secretory cells interspersed within the epithelial lining of the urethra. 3fl-, 17fl-, 3a-hydroxysteroid dehydrogenase, G6PD and 6PGD reactivity were intense in all the urethral epithelium.

610 The presence of these enzymatic activities indicates that the human urethral epithelium normally is site of significant androgen activation.

P24.16 Enzyme Histochemistry of the Rat Decidua and Metrial Gland with Special Reference to Parturition. L Straatsburg and R. Gossrau Department of Anatomy, Free University of Berlin, Germany. There are many indications of involvement of the decidua in the onset of parturition. The search for cells participating in placental separation has lead us to investigate metabolic aspects of the rat decidua and metrial gland (MG). Cryostat sections of placenta were analyzed histochemically for activities of various enzymes, including proteases, nucleotidases and oxidases. Glycol methacrytate sections were used for the identification of various cell types. Results showed a heterogeneous enzyme activity pattern in cell populations of the basal decidua and MG. These cells are metabolically very active, because they expressed a variety of high activities of enzymes, which could possibly support the decidual degeneration at term. First, enzymes of the purine catabolic pathway show high activities, which may be related to nucleic acid breakdown in regressing granulated metrial gland-(GMG) and decidual cells. Second, proteases and other hydrolytic enzymes are also showing high activities and may be involved in degeneration processes in decidua cells or connective tissue elements. Third, the production of oxygen radicals and the release of acid hydrolases possibly contribute to lysis of pregnancy-related cells, including GMG cells, which

Poster sessions are believed to be involved in the special immunology of gestation.

P24.17 Histochemical Study of Tartrate-Resistant Acid Phosphatase (TRAP) and Fluoride-Resistant Acid Phosphatase (FRAP) in Medullary Bone Matrix and Osteoclasts of Laying Japanese Quail. T. Yamamoto and H. Nagai Department of Anatomy, Okayama University School of Dentistry, Okayama, Japan.

Tartrate-resistant acid phosphatase (TRAP) and Fluorideresistant acid phosphatase (FRAP) activities were histochemically examined in medullary bone matrix and osteoclasts of laying Japanese quail. In order to avoid nonspecific staining, reactivity of the enzyme was evaluated using both the azo-dye method and the lead-salt method, and nonembedded thick sections and resin-embedded thin sections. And the pH of the incubation medium was also varied from acidic range (pH 5.0 and 6.5) to alkaline range (pH 8.5). Tartaric acid ( 1 0 - 5 0 m M ) and NaF (2-10mM) were employed as inhibitors. Medullary bone osteoclasts contain both TRAP activity and FRAP activity, and no significant difference in intensity was detected between active and inactive osteoctasts. The entire matrix of medullary bone was positive for tartrate-resistant, fluoride-sensitive acid phosphatase activity. No reaction product was observed in sections incubated in substrate-free and pH 8.5 media. The results demonstrate the existence of FRAP in medullary bone osteoclasts, and that medullary bone matrix includes TRAP throughout the matrix.

P25. CELL G R O W T H A N D D I F F E R E N T I A T I O N P25.1 MIB 1 - 3, New Monocional Antibodies against the Proliferation-Associated Antigen Previously Defined by KI-67. M. H. G. Becket, G. Key, B. Baron, M. Duchrow, C. Schlfiter, H.-D. Flad and J. Gerdes Div. Molecular Immunology, Forschungsinstitut Borstel, D-2061 Borstel, Germany. The antigen defined by the monoclonal antibody Ki-67 is exclusively expressed in proliferating human cells, but is absent in quiescent cells. Numerous independent studies indicate that the Ki-67 labelling index is an immunocytochemical marker of unique diagnostic and prognostic relevance in the study of human neoplasms. Since Ki-67 is the only reagent available against this proliferation-associated antigen the aim of this study was to prepare new Ki-67 equivalent monoclonal antibodies. Using bacterially expressed parts of the Ki-67 antigen as immunogen, we were able to elicit several Ki-67-1ike antibodies. Three of them, designated MIB 1 - 3, were cloned and fully characterized. In immunohistochemistry and cytochemistry the reactivity of MIB 1 - 3 found to be identical to that of Ki-67. Furthermore, the specificity of MID 1 - 3 for the Ki-67 antigen was proved by Western blotting with native and recombinant parts of the Ki-67

protein. Using synthetic peptides, we could make it likely that the epitopes recognized by Ki-67, MIB 1 and MIB 3 reside within the same peptide sequence of 20 amino acids, whereas MIB 2 reacts with an epitope of the Ki-67 antigen clearly distinct from this latter structure. The reactivity of the new Ki-67 equivalent antibodies in formalin-fixed, routinely processed and paraffin-embedded tissues will be detailed by Dr. G. Cattoretti, Milano, during this conference. This study was supported in part by the Dr. Mildred Scheel Stiftung ffir Krebsforschung (W 49/90/Ge2).

P25.2 Involvement of Nuclear Protein Kinase C in PC12 Neural Differentiation. c . Pagliarini*, L. M. Neri *~, M. Marchisio*, L. Bertolaso*, M. Vitale*, E. Caramelh ~ and S. Capitani* *Inst. Anatomia Umana, Universita" di Ferrara; ~Ist. Citomorfologia CNR, Bologna; *Ist. Anatomia Umana, Universitd di Bologna; +Ist. Istotogia, Universita" di Bologna; Italy. We have studied the response of PKC to NGF treatment in PC12 cells by probing the subcellular distribution of the enzyme with a polyclonal antibody directed against all the

Poster sessions PKC isoform, by means of in situ immunocytochemistry, flow cytometry and western blotting. In untreated cells, cytoplasms are stained while nuclei appear negative. When differentiation occurs, the staining increases progressively and covers the entire cell, including the nuclear region and neurites. Western blot analysis shows different patterns between whole cells and nuclei with an increase of the nuclear staining after NGF treatment, also confirmed by flow cytometric analysis on isolated nuclei. These results suggest that the response of PC 12 cells to NGF includes nuclear changes mediated by PKC via the enzyme translocafion. In addition, our data show an overall increase of PKC associated to the fully differentiated neural phenotype.

P25.3 Transforming Growth Factors (TGFa and (~) Modulate GAG Accumulation in Embryonic Fibroblasts I n Vitro.

611 collagenases, is a potential regulator of cell proliferation; in particular, interferes with processes of bone formation and resorption. As glycosaminoglycans are extracellular matrix components involved in bone formation, in this study we investigated about the effects of IL-la in vitro production of such macromolecules. Human osteoblast-like cells utilized at the 5th subculture were treated in MEM + 0.5~ Fetal Calf Serum (FCS) with and without IL-la (at doses of 1 U/ml and 5 U/ml) for 48 h and pulsed for 24 h with 3H-glucosamine (5 uCi/ml- NEN; s.a. 29 Ci/mmol). Bone nature of cells was assayed by labelling them with antiosteonectin antibodies and by investigating the presence of receptors for parathormone. Glycosaminoglycans were precipitated separately in cells and media. Results show that IL-la induces a dose dependent decreasing of GAG accumulation in both intra- and extracellular compartment. Our data confirm that IL-la affects numerous metabolic activities of osteobtasts.

P. Carinci*, P. Locci**, C. Lilli**, L. Marinucci**,

R. Evangelisti* and E. Becchetti** *Ist. lstologia Embriologia Generale, Univ. Ferrara-**Dip. Medicina Sperimentale e Scienze Biochimiche, Univ. Perugia, Italy.

ExtraceUular matrix (ECM) of embryonic skin accumulates different amounts of glycosaminoglycans (GAG) according to developmental age and the interaction between the cells and ECM components influences tissue differentiation. Therefore the control of ECM composition has to be considered an important aspect for cell behaviours. Transforming growth factors, secreted by embryonic fibroblasts, are involved in cell proliferation, tissue differentiation and ECM synthesis and degradation. To clarify the rote of growth factors in the regulation of GAG synthesis we have examined the in vitro effect of TGFa and fl on fibroblasts from chick embryonic skin by determining the incorporation of ~H-glucosamine into total and individual GAG. 7 day-old fibroblasts are incubated for 24 h in medium 199 alone, or medium 199 plus TGFa or TGFfl (4 ng/ml): All cultures are labelled with 5 gCi/ml of 3H-glucosamine (40 Ci mmole-~). At the end of incubation GAG are precipitated from cells and media and then applied to a DE-52 cellulose anion exchange column. The addiction of TGFa or TGF/3 increases GAG accumulation both in the intra- and extracellular components and alters the proportions of GAG types secreted in the medium. TGFfl selectively stimulates the secretion of chondroitin sulphates and dermatan sulphate. TGFa the secretion of hyaluronate and dermatan sulphate. These results raise the possibility that TGFs modulate cell growth and differentiation through its regulatory action on ECM production.

P25.4 Interleukin 1-Alpha Modulates Glycosaminoglycan Synthesis of Human Osteoblast Like Cells In Vitro. M. Bodo*, E. Becchetti*, F. Pezzetti*, F. Carinci**, L.

Rossi*, R. Evangelisti** and P. Carinci** *Dip. Medicina Sperimentale e Scienze Biochimiche, Univ. Perugia; **Inst. Istologia Embriologia Generale, Univ. Ferrara, Italy.

Interleukin 1-alpha (IL-la), a growth factor produced by macrophages and other cells, shows numerous activities; it stimulates fibroblasts to produce prostaglandins and

P25.5 Monoclonal Antibodies Produced Against Recombinant Parts of the Ki-67 Molecule (MIB-1 to -3) Stain Proliferating Cells in Formalin Fixed, Paraffin-Embedded, Microwave Processed Tissues. G. Cattoretti, M. H. G. Becker, G. Key, M. Duchrow, C. Schlfiter, F. Rilke and J. Gerdes Dept. of Pathology, Istituto Nazionale Tumori, Milano, 20133 Italy and Forschungsinstitut Borstel, Div. Molecular Immunology, D-2061 Borstel, Germany.

The monoclonal antibody Ki-67 (Gerdes et al. 1983) stains a nuclear proliferation-associated protein of 345kD and 395kD and has been extensively used for diagnostic as well as prognostic purposes in human tumors. Recently, the Ki-67 gene has been cloned and monoclonal antibodies ( M I B - 1 to - 3 ) have been produced against recombinant parts of the antigen (see abstract of J. Gerdes et al). The M I B - 1 / - 3 MAbs, similarly to the original Ki-67, do not stain formalinor alcohol-fixed tissues when routinely processed for immunohistochemistry. However, deparaffinized sections from fixed specimens, irradiated in a microwave oven (Shi et al, 1991) in a 0.01 M citrate buffer pH 6 react with the M I B - t and M I B - 3 antibodies in the proliferative compartment of human lymph nodes, skin, thymus, gut, testis, etc. Fixation with Formalin 1007% Paraformaldehyde 4%, PLP, Zenker, Metacarnoy, but not Bouin's fluid allowed MIB-1/-3 staining, even after three days in formalin. M I B - 1 and - 3 staining survived bone marrow trephine EDTA decalcification and melanin bleaching. We succeeded in staining archivial material from our files, back to 1931. A new generation of true "Ki-67" antibodies is therefore available to be used on routinely processed histopathologic material.

P25.6 Thiol Groups Aikylation Inhibits T-cell Blasts Induction by Lectins. V. Cialdcu, C. M. Tzigaret, S. Murea and C. Cernescu Division of Cell Biology and Histology, Davilla University of Medicine, Bucharest 35, P.O. Box 35-10, Romania.

There is evidence showing that intracellular thiol groups are

612 involved in T-cell signalling. However, their role in the T-cell responsiveness to mitogenic stimuli is not welt defined. Human peripheral blood lymphocytes were separated on Ficoll/Hypaque density gradient medium. Lymphocytes (107 cells/ml) were pretreated with thiol-alkylating agents: iodoacetamide (IA) or p-chloromercuribenzoate (PCMB) (10-50/aM) for 15 min. Cells were then washed and incubated in RPMI-1640 medium containing phytohemagglutinin (PHA) (5/ag/ml) for 72 h. T-cell blasts induction was measured on a FACStar Plus flow cytometer (BectonDickinson). In addition, blasts were morphologically identified by optical microscopy. Both IA and PCMB inhibited in a dose-dependent manner T-cell blasts induction by PHA. In a different experiment, T-cell blasts were induced in the presence of thiol groups donors: thioglycolate (50 ~M) or 2-mercaptoethanol (50pM). Of these two compounds, thioglycolate was the most potent enhancer of PHA-induced blasts formation.

P25.7 A Confocal Scanning Laser Microscopy Study of Intermediate Filament Protein Expression in PreImplantation Mouse Embryos. E. Coonen, J. C. M. Dumoulin and F. C. S. Ramaekers Dept. of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands.

The intermediate filament protein expression patterns have been studied during early mouse embryo development. For this purpose pre-implantation embryos at different stages of development after in vitro fertilization were analyzed with antibodies to cytokeratins, vimentin and lamins. The levels of expression were quantitated and localization of the protein constituents was assessed by means of confocal scanning laser microscopy. Our studies show that, although the embryos grow in culture, vimentin could not be detected in a filamentous organization. With respect to the cytokeratins a diffuse fluorescent signal was detected in the 2-cell to 8-cell stages, while in the morula stage an increased level of cytokeratin expression with a transitional staining pattern, combining a filamentous and a diffuse occurrence, was seen. In the blastocyst stages cytokeratin filaments were seen in trophobtast cells but not in the inner cell mass. Analysis of cytokeratin subtypes showed that expression levels of cytokeratins 8 and 18 increased gradually up to a filamentous pattern in the blastocyst stage. Cytokeratins 7 and 19, although elevated in the latter stage, were not found as prominent as cytokeratins 8 and 18. A-type as well as B-type lamins could be detected as a faintly reactive nuclear lamina in all developmental stages and were present in both trophoblast and inner cell mass of blastocysts.

P25.8 DNA Polymerase Localisation of DNA Strand Breaks in the Nuclei of a Subpopulation of Cells in the Regenerating Skeletal Muscle of Dystrophic Mice. G. Coulton, B. Rogers, P. Strutt, M. Skynner and D. Watt Charing Cross & Westminster Medical School, London, W6 8RF, UK. Weintraub & Groudine (1976) showed that transcriptionally active genes were preferentially sensitive to nuclease digestion.

P o s t e r sessions Using DNA pol I for in situ 'nick' translation, dividing myoblasts do not incorporate biotin-dUTP whereas up to 24% of recently fused myotube nuclei are labelled. Hence differentiation depends upon transient induction and repair of single-stranded DNA breaks. We used in situ DNA polymerisation to study differentiation in regenerating mdx mouse muscle, (homologue of Duchenne Muscular Dystrophy). Mdx muscle contains surviving peripherally-nucleated fibres and lesions with necrotic and regenerating muscle fibres. After reaction in a medium containing DNA polymerase and digoxigenin-ll-dUTP, nuclei of a subpopulation of mononuclear cells in the inflammatory infiltrate of mdx lesions were intensely labelled, whereas nuclei of myotubes and muscle fibres were negative (C57BL/10 controls also negative). Labeiling was DNA polymerase-dependant, required a DNA template with singlestranded gaps but was not due to exogenous exonuclease activity as a range of DNA polymerases gave the same staining pattern. Labelling was protease insensitive, and was blocked by pre-incubation in a medium containing dideoxynucleotides. These results strongly indicate a role for DNA single-strand breakage in regenerative lesions of mdx mice. We must identify the cells involved and characterise the nature of DNA breaks within candidate genes such as, muscle creatine kinase. Clearly, in situ DNA polymerisation will prove to be beneficial for the investigation any cell differentiation system for example, during embryogenesis of carcinogenesis. Dawson B. A. & Lough J. (1988) Dev. Biol. 127, 362-367. Weintraub H. & Groudine M. (1976) Science 1 9 3 , 8 4 8 - 856.

P25.9 RAS-Transfected Caco-2 Cells: A Model for Endocrine Differentiation in the Large Intestine. A. de Brufne, J. de Vries, W. Dinjens ~, E. van der Linden, M. Pijls, J. ten Kate, F. Bosman' Departments of Pathology, University Hospital Maastricht and Erasmus University Rotterdam j, The Netherlands.

Colorectal adenocarcinomas with endocrine cells (about 30~ of all cases) appear to have a relatively poor prognosis. To study the biology of this subset of tumors, in vitro models reflecting the complete spectrum of intestinal epithelial differentiation are essential. However, endocrine differentiation in tumor cell lines is scarce and almost exclusively occurs in xenografts, as a result of differentiation induction by stromal components. We studied endocrine differentiation in the colonic adenocarcinoma cell line Caco-2, which thusfar is only known as a model for enterocytic differentiation. In vitro endocrine tumor cells were not encountered, whereas in vivo endocrine features could not be evaluated, because of poor tumor growth upon xenografting of Caco-2 cells in athymic mice. As increased RAS-oncogene expression has been claimed to correlate with both tumor progression and enhanced endocrine differentiation, Caco-2 cells were transfected with a point mutated H-RAS gene. The cell line Caco-2 E J6, generated from these experiments, could be successfully xenografted in nude mice, yielding a moderately well differentiated adenocarcinoma. In addition to enterocytic differentiation, xenografts also contained mucin producing and endocrine cells. This makes Caco-2 E J6 a suitable model to study endocrine differentiation in neoplastic intestinal epithelium.

Poster sessions P25.10

Expression of PCNA in Paraffin Sections from Human Colon Carcinoma Cells does not Correlate with BrdU Staining, o70 S P h a s e Cells, nor with Survival of Colon Carcinoma Patients. A . de Goeij, H. Ader and A. de Bruine Department o f Pathology, University of Limburg. P.O.B. 5800, 6202 A Z Maastricht, The Netherlands. The monoclonal antibody PC 10 was used to stain Proliferating Cell Nuclear Antigen (PCNA) in formalin fixed, paraffin embedded samples. Expression was studied in normal human colon mucosa, colon cancer cell line xenografts that were labelled in vivo with BrdU and colon carcinomas samples from patients. Normal human colon tissue was fixed ranging from 4 hours to 8 days. Nuclear staining was observed only in the lower half of the crypts, corresponding to the localisation of the proliferating cells in the normal colon mucosa. Fixation during three days and longer significantly reduced the percentage of PCNA-positive cells. Cells from the human colon carcinoma cell lines SW403, SW480 and LS174T were xenografted subcutaneously into immunodeficient mice. The animals received bromodeoxyuridin three hours before sacrifice. Parallel sections from the xenografts were immunostained for BrdU and PCNA. No significant correlation between the percentage of BrdU- and PCNA-positive cells was found. Routinely fixed, paraffin embedded colon cancer specimen from patients with a clinical 5 year-follow up were analysed for % of S phase cells with flow cytometry. No significant correlations were found with PCNA staining on adjacent sections. Also there was no correlation between the percentage of PCNA positive cells and the five year survival of these patients. We conclude that PCNA staining of colon carcinoma cells in paraffin sections does not allow quantitation of proliferative activity and has no prognostic value.

613 no reliable parameters to predict in vivo growth behaviour of human colorectal cancer cells in this model system.

P25.12

Cytogenetic Analysis and Proliferative Behavionr of SW480 After Transfection with the c-Ha-RAS Oncogene. J. de Vries, F. Kornips, M. Rousch, P. Marx, J. Geraedts, F. Bosman and J. ten Kate Departments of Pathology and Genetics, University of Limburg, The Netherlands. The c-Ha-Ras oncogene has been implicated in the induction of metastatic capacity. To study the effects of the c-Ha-Ras oncogene on cellular behaviour, the human colon tumour derived cell line, SW480, was transfected with this gene. The following questions were addressed: (1) do plasmids, with the VAL-12 pointmutated c-Ha-Ras oncogene integrate random or non-random into chromosomes?; (2) Is genotypic stabiliity altered after transfcction?; (3) Does Ras transfection alter the proliferative behaviour? Aberrant chromosomes, not observed in the parental SW480 cell line, were detected after transfection indicating increased genotypic instability. Four out of five integration sites in SW480 cells, transfected with the c-Ha-Ras oncogene, were identified in aberrant chromosomes. The rate of proliferation of SW480 after transfection was similar to that of the parental cell line. In conclusion, genotypic instability seems to be increased, but does not lead to alterations in proliferative behaviour. Integration of transfected c-Ha-Ras oncogene was not random. Further, alterations in cellular behaviour might not only be caused by the transfected gene but also by increased genotypic instability.

P25.13

P25.11 In Vitro Expression of L-CAM and u-PA of Human Colorectai Carcinoma Cells is not Related to their In Vivo Growth Behaviour. J. E. de Vries, A. P. de Bruine, W. N. M. Dinjens, E. P. M. van der Linden, H. W. Verspaget, F. T. Bosman and J. ten Kate Department of Pathology, University of Limburg, The Netherlands. A variety of human colon cancer derived cell lines (CaCo 2, S W l l l 6 , SW620, SW480, H716, LSI74T, 5583E, 5583S and HT-29) were cultured in vitro and xenotransplanted in the coecum wall of nude mice. Three modes of growth emerged. SWI 116, SW620 and SW480 developed primary tumors which were not locally invasive and did not metastasize. H716 and LS174T primary tumors were locally invasive but did not metastasize. 5583E, 5583S and HT29 primary tumors were locally invasive and also metastasized. L-CAM expression, suggested to inhibit invasive ability of cells, showed in vitro no correlation with in vivo growth behaviour. The expression of u-PA, suggested to enhance invasive ability of cells, showed in vitro no correlation with in vivo invasive capacity. We conclude that in vitro expression of L-CAM of u-PA, both believed to play a role in the invasive capacity of ceils, are

High Lysosomal Activity in Resting Quail Oocytes Disappears when Development Resumes, and Decreases During Provoked Atresia. K. D'Herde, B. de Prest and F. Roels Human Anatomy and Embryology, University of Ghent, 9000 Belgium. Acid phosphatase was localized for light microscopy with naphtyl-AS-TR-phosphate and p-rosaniline, which is a quantitative stain (Cornelis et al), and for light and electron microscopy with p-glycerophosphate and lead in the presence of NaC1 according to De Jong et al. Numerous lysosomes are present in prelampbrush oocytes (staging according to Callebaut) of 100~m diameter. On the other hand the surrounding follicle cells display very few lysosomes. The intraoocytal lysosomes contain partially digested "lining bodies"; the latter are secreted by follicle cells and taken up by the oocyte. In lampbrush oocytes of 1000 ~m acid PP positive lysosomes are rare. From this stage onward intermediary yolk is deposited in the oocyte. Lining bodies are known to contribute to protein yolk formation (Bellairs; Paulson and Rosenberg). Results suggest that the resting primary oocyte continuously is taking up pre-yolk constituents which are then destroyed. Protein yolk deposition is initiated after lysosomal activity is markedly reduced, in addition to enhanced secretion of lining bodies by the follicle ceils. Thus growth is regulated

614 in part by lysosomes. When atresia is provoked by starvation, enzyme activity decreases, suggesting that lysosomes are not active in this process. After glutaraldehyde fixation, lysosomes in some oocytes display latency, which is abolished by freezing. Bellairs, R. (1967). J. Embryol. exp. Morphol., 17: 267-281. Callebaut, M. (1973). J. Embryol. exp. Morphol., 29: 145 - 157. De Jong, A. H. S.; Hak, T. J.; Van Duyn, P; Daems, W. Th. (1979). Histochem. J., 11: 145-161. Paulson, J. L.; Rosenberg, M. D. (1972). J. Ultrastruct. Res., 40:25 - 43. Cornelis, A.; De Broe, M. E.; Roels, F. (1984). Anal. Quant. Cytol., 6" 2 7 9 - 283.

P25.14 Correlation Between Endogenous Membrane PKC Activity and Proliferation versus Differentiation of Normal and HPV16 Transfected Rat Myoloblast (L6 cells). M. M. Ennaji ~'2, M. Arella 2, H. Jouishomme ~, A. Merzouki 2 and J. Phipps' ~National Research Council of Canada, Ottawa, Ontario, Canada, KIA OR6. 2[nstitut Armand-Frappier, Laval, Quebec, Canada, H7N 4Z3. Protein phosphorylation is greatly involved in the regulation of cell growth and differentiation. In most cases proliferation and differentiation can be regarded as coupled processes. As cellular differentiation programs proceed, down-regulation of cell growth is generally observed. The possible role of membrane PKC in these two processes has been investigated by comparing the enzyme activity in normal and HPV16 transformed ceils. HPVI6 is known to be associated with epithelial cell proliferation during the course of productive infection. The HPV16 encodes three oncogenes: Es, E6 and E7 which are believed to be implicated in the deregulation of cell growth. Isolated clones, of rat myoloblast ( t 6 cells) co-transfected by electroporation with the full length HPV16 DNA and pSVtkneo3, were used in this study. Selection was achieved with selective marker G418 and dot blot and southern blot analysis. Differentiation stages have been assessed using flow cytometry with appropriate markers. Inducers of L 6 cells differentiation like Ca 2+ or DMSO 1% did not alter the HPV16 transformed L6 cells, these were blocked at the myoloblast stage. It was found that basal PKC activity in control L6 cells is low and decreases with the age of the cell culture. In contrast PKC activity in transformed cells was high and remained at high level throughout the experiment. Earlier reports in the literature suggest that CaM-kinase rather than PKC is involved in myoloblast differentiation. The results obtained with the transfected L6 cells support our earlier conclusion on matching control L6 cells that PKC may rather be linked to cell growth in this system. In HPV16 transfected myoloblasts, the long term increase in PKC activity above levels measured in resting ceils correlates well with proliferation.

Poster sessions

P25.15 Integrin Expression in Undifferentiated and Differentiated HT29D4 and CaCo2 Cells. C. C. Flohil, W. N. M. Dinjens, F. T. Bosman Dept. of Pathology, Erasmus University, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. For the study of differentiation in gut epithelium the HT29D4 and CaCo2 colon carcinoma celt lines are extensively used. Modulation of the culture conditions induces differentiation in these cells in the direction of columnar resorptive cells. We studied the expression of a2, a3, a6, /31 and /34 chains by immunohistochemistry on differentiated and undifferentiated cells. Furthermore integrin function was tested by adhesion experiments, using substrates coated with collagen type I and IV, laminin and fibronectin with or without prior incubation of the cells with anti-integrin antibodies. By immunohistochemistry a and 3 chains were visualised circumferentially on the surface of undifferentiated HT29D4 cells, but basolaterally on differentiated HT29D4 cells. CaCo2 and HT29D4 cells adhere to collagen type IV, laminin and fibronectin irrespective of their state of differentiation. In differentiated HT29D4 ceils laminin binding was reduced. Binding to collagen type IV and laminin is mediated by integrins VLA2, VLA6 and a6/34. Binding of differentiated and undifferentiated HT29D4 and differentiated CaCo2 cells to fibronectin seems not to be mediated by VLA or a6/34. These experiments indicate that: 1. in intestinal epithelial differentiation not the integrin expression as such but rather its topography on the cell surface changes, and 2. In these ceils a2, a6, 31 and f14 integrin chains play an important role in cellcell and cell-extracellular matrix adhesion.

P25.16 Study of Normal Human Lymphoid Subpopulations with Antitumor Activity. G. Gerzeli, E. Capelli*, R. Nano, M. Cuccia* and L. Lavezzi Dept. of Animal Biology, University of Pavia and Center for the Study of Histochemistry, C.N.R., Pavia; *Dept. of Genetics and Microbiology, University of Pavia, Italy. Activation, proliferation and differentiation of natural killer cells (NK) are regulated by different cytokines as IL-2, IL-3, IL-4 and IFN. These cells are constituted by lymphoid subpopulations with cytolitic non antigen specific activity and non MHC restricted, against neoplastic or virus-infected or allogenic cells. Cultured human peripheral blood lymphocytes in the presence of IL-2, induced the proliferation of cells with cytotoxic activity versus tumoral cells, named lymphokine activated killer cells (LAK). Cultures of peripheral lymphocytes maintained in IL-2 were performed and observed after 0, 12, 24, 48, 72, 96, 192 hours from the beginning of the treatment. PHA stimulated and non cells maintained in IL-2 enriched medium promoted the proliferation of activated cells for long period (probably killer?). In the cultures treated only with IL-2, this phenotype is observed early. Pure CD4 § and CD8 § lymphocytes cultures were obtained after incubation with magnetic beads coated with anti-CD4 and CD8 antibodies. After this selection, the cytotoxic and antitumor activity of these cells was evaluated towards human tumoral

Poster sessions lines in vitro. These pretiminar findings suggest a possible selective use of these cells for clinical applications as immunologic antitumor therapy.

P25.17

Expression of Insulin-Like Growth Factor I in the Submandibular Gland of the R a t . S. Iseki and O. Arnano Department of Anatomy, School of Medicine, Kanazawa University, Kanazawa, Japan. By use of irnrnunohistochemistry and in situ hybridization, we examined the expression and localization of insulin-like growth factor I ([GF-I) in the submandibular gland of rats. In adult gland, IGF-I-like irnnrnunoreactivity was demonstrated in the granular convoluted tubules (GCTs), striated and excretory ducts. The imrnunoreactivity in the GCT cells appeared to be localized in the secretory granules and disappeared upon the secretory stimulated by isoproterenol. By Northern blot analysis of adult rat poly A § RNAs using an oligonucleotide probe, the signal for IGF-I mRNA was even stronger in the subrnandibular gland than in the liver, but the pattern of the hybridized mRNA bands differed between the two organs. By in situ hybridization, the mRNA signal was exclusively localized in the GCTs and was not detectable in the other portions of the duct system. During the postnatal development of submandibular gland in rats, IGF-I mRNA occurred around 5 weeks after birth and increased progressively until t6 weeks, in coincidence with the development of GCTs. These results gave evidence that the GCT cell is the major site of the production of IGF-I in the rat subrnandibular gland and that IGF-I is probably secreted into saliva.

P25.18 Cell Proliferation Studies of Catecholamiue-Storing Cells in the Adrenal Medulla of the R a t . E. A . P. Janssen, IV. R. Ubink and A, A. J. Verhofstad Department of Pathology, University of Nijmegen, Nijmegen, The Netherlands. The parenchyma of the adrenal medulla consists of two types of highly differentiated endocrine cells, producing either noradrenaline or adrenaline (NA- and A-storing ceils). Cell proliferation in the adrenal medulla was examined by a recently described technique based on the incorporation of the thymidine analogue bromodeoxyuridine (BrdU) during the Sphase of the cell-cycle. BrdU was administered to adult rats by a single intraperitoneal injection or continuously using an implanted mini-osmotic pump. Eight days after single injection the number of labelled cells is low (BrdU labelling index = 0.09). But after 73 days of BrdU administration almost half of the cells are labelled (BrdU labelling index = 0.4). Apparently, cell division is needed to compensate for cell loss. In order to determine whether both NA- and A-storing cells are dividing we developed an immunocytochemical procedure which enables the simultaneous detection of BrdU in the nucleus as well as N A or A in the cytoplasm of the same cell. This technique will open new opportunities for a more detailed

615 study of cell kinetics in the adrenal medulla under normal as well as pathological conditions. Preliminary analysis indicates that eight weeks after birth cell division occurs in both NA- and A-storing cells.

P25.19 Populations of Peripheral Blood Leukocytes and Thymic Cells in Leukemia-Bearing Mice Treated with G-CSF. J. Kawiak ~, G. Hoser ~, B. Miks ~, Z. Pojda 2, A. Sobiczewska2, E. Machaj-', A. Wrembet 2 Dept. of Cytophysiology, Medical Centre of Postgraduate Education ~, Warszawa and Dept. of Radiology, Institute of Hygene and Epidemiology, Warszawa, Poland. It is well known, that G-CSF given to mice induces appearance of high number of leukocytes as well as relatively high number of hernatopoietic stem cells in the peripheral blood of the animal. There is, however not known influence of given GCSF on organs where the hernatopoietic cells differentiate e.g. central lymphatic organs. In our experiment mice were treated s.c. with G-CSF in doses known to induce leukocyte appearance in the blood, and populations of thymocytes were compared with appropriate controls. The populations of cells labelled with monoclonal antibodies in one-step imrnunocytochernical reactions were evaluated with flow cytornetry. Additional observations were performed on thyrnocytes of leukemia L1210-bearing animals. Bibliography Kawiak J, Kawalec M, Hoser G, Miks B, Skorski T, Pojda Z, Skurzak H. Changes of thymocyte subpopulations induced by activities diffusing from leukernai L1210 cells. Thymus 1991; 19: 185-192.

P25.20 Colocalization of Cholinesterase and Muscarinic Acetylcholine Receptors in Differentiating Embryonic Cells. M. Lammerding-K6ppel and U. Drews Institute of Anatomy, University of Tffbingen, FRG. In various species, our group observed the transitory expression of an embryonic muscarinic cholinergic system during distinct phases of development. Diverse constituents of the system (Cholinesterase - - ChE, choIine acetyltransferase CHAT, muscarinic receptors - - rn AChR) have been characterized primarily in chick embryo sections by means of histochemical ChE reaction and imrnunocytochemistry and in chick homogenates by biochemical and pharmacological techniques, as well. In all organs studied, the expression of the rnuscarinic system was correlated with rnorphogenetic movements. It disappeared when the organ structure had developed - - an observation which has been confirmed and extended by others. In the present study, we show the colocalization of ChE and rn AChR by combining the histochemical ChE staining according to Karnovsky - - Roots and the immunocytochemical detection of rn AChR by means of the rnonoclonal antibody M35. Using the double-staining procedure in cryostat sections and in single cell suspensions of chick embryo stage 24, we demonstrate that rn AChR and ChE are colocalized in the same ceils to a very large amount.

616 Moreover, in the differentiating neural tube, spinal nerves and myotomes, M35 reveals a staining pattern which matches quite well the AChE resp. BChE data earlier reported by LAYER et al. (1988). We suggest that not only ChE but also m AChR are involved in neuromuscular development. Furthermore, we present evidence for migrating neural crest cells expressing m AChR. In conclusion, all these results support the idea of an embryonic muscarinic system which is involved in embryonic tissue differentiation and cellular movements.

P25.21 Effects of Growth Factors on Proliferation of Luminal and Basal Cells in H u m a n Breast Explants in Serum Free Culture. N. P. Perusinghe, P. Monaghan, M. J. O'Hare and B. A. Gusterson Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Sutton, Surrey, England. Dissected breast TDLU structures (organoids) maintain the 3-dimensional relationships between cells in both epithelium and stroma. Unlike monolayer cultures this system permits interactions of the various cell types comprising breast tissue. Dissected breast organoids have been maintained in vitro for up to 10 days in serum-free medium without loss of fine structure of viability. Proliferation was quantified by incorporation of tritiated thymidine and light microscope autoradiography. Electron microscope autoradiography provided identification of the proliferating cell type. In response to 48 hr stimulation with insulin, hydrocortisone and cholera toxin a mean thymidine labelling index (TLI) of 15.7% was recorded from 3 experiments. The majority (98.5%) of labelled cells were of luminal cell type. The mean control TLI was 0.08%. Stimulation of explants with EGF and T G F a gave mean TLI's of 6.6% and 10.8% respectively, and an increased proportion of the labelled cells were of basal type after stimulation with EGF (11.5% basal cells) and TGFa (18.5% basal cells). The results indicate that this system allows the determination of both TLI, and proliferating cell types in human breast organoids in response to added hormones or growth factors.

P25.22 Effects of P M A Differentiative Agent on a H u m a n Colon Adecarcinoma Cell Line. S. Montagnani ~ G. Giordano-Lanza, P. Tagliaferri, P. Correale, R. Bianco, R. Vitelli and E. Di Vaia Second Medical School of Naples, ~ School of Reggio Calabria, Italy. We used a drug-resistance human colon adenocarcinoma cell subline (LOVO/DX) to study differentiative effects of PhorbolMyristateAcetate (PMA), an agent able to induce phenotypical changes on some tumoral cell lines. Our interest was pointed on some morphofunctional aspects of this human colon adenocarcinoma cell line which appear particularly affected by P M A treatment, like cell shape, expression of surface markers and adhesion proteins and appearance of some neuroendocrine-like characteristics. These changes appear correlate with alteration of functional parameters like abolition of drug-resistance. Immunocytochemistry demonstrated contemporary abolition of GP170 membrane expres-

Poster sessions sion and decrease of sensitivity to LAK-induced cytotoxicity. We used immunocytochemistry to evidentiate with monoclonal antibodies morphological changes in these cells and particularly interesting was the appearence of cytoplasmatic dendritic-like processes with secretory vescicles and wellstructured cytoskeleton, as evidenced by alpha-synaptophysine, S-100, G F A P and neurophylament positivity. In the same time, strongly reduced was the expression of epithelial markers like Epithelial Membrane Antigen, emidesmine and alpha-actinine which rearranged their distribution in P M A treated cells. We think our model can be useful to connect phenotypical with functional changes and this correlation may be important to associate chemio with immunotherapy in cancer patients.

P25.23 Immunohistochemical Localization of Inhibin Subunits in Bovine Oviduct During Oestrus Cycle. L. Passoni, S. Modina, F. Gandolfi, T. A. L. Brevini, F. Petraglia and A. Lauria Universit~ degfi Studi di Milano, Istituto di Anatomia degli Animali Domestici, Italy.

Various growth factors have been identified as morphogens in amphibian embryos. Recent data suggest that growth factors also play an active role in early embryonic development in mammals. Furthermore, it is known that in several species the oviduct is involved in early embryogenesis, through the secretion by nonciliated epithelial cells of proteins which then associate with the developing embryos. We have investigated whether inhibin/aetivin polipeptides, which are well characterized as mesoderm inducers in amphibians, are also present amongst bovine oviductal secretions. Proteins were separated by SDS-PAGE, blotted on to nitrocellulose and probed with antibodies specific for three subunits of inhibin/activin. Our results show that specific antibodies for the beta-A and beta-B subunit of inhibin/ activin recognized an antigen. No alpha subunits were detectable. Immunohistochemical techniques were used to localize these antigens in bovine oviducts during different phases of the oestrus cycle. Detection of antigen-antibody complexes was performed using an avidin-biotin-immunoperoxidase method. The results show that the beta-A subunit was present in the epithelial cells of both the ampullary and isthmic tract of the oviduct. Immunoreactive beta-B subunits were observed only in the isthmic tract. Alpha subunits were not detectable in any region of the oviduct, results consistent with the Western-blot analysis. No differences in signal intensity or intracellular distribution were observed between the periovulatory and luteal phase of the cycle. These results demonstrate that activin molecules are secreted by the oviduct epithelium, and indicate possible involvement of these molecules during early embryogenesis in cattle.

Poster sessions

P25.24 Light and Electron Microscopic Immunolabelling and Flow Cytometric Evaluation of a 33 kDa Stress Protein During the Cell Cycle of EUE Cells Grown in Hypertonic Medium. C. E. Pellicciari, G. Viale*, A. Fraschini, A. Giuliani**, A. Gaspari, L. De Grada*, A. M. FuhrmanConti* and M. G. Manfredi-Romanini Dept. BioL Anita. and CNR Ctr Studio Istochimica, Piazza Botta 10, Pavia, Italy; *Dept Biol. Genet. Sci. ivied, and **Dept. Biochem., Milano, Italy. Under hypertonic (HT) stress, EUE cells were found to enhance the expression of a 33 kDa protein (P~3). The aim of this work was to localize, by both light and electron microscopy, the distribution of P33 in EUE cells grown in isotonic or HT medium. The changes in P~3 expression at different times of growth in an HT medium were also monitored, to see whether they were related to the cell cycle phases. A polyclonal antibody against P33 was first produced, to be used for fluorescence-, peroxidase-antiperoxidase-, and gold-indirect immunodetection. P33 was found to be mostly located in the cytoplasm, without apparent association with subcellular organelles; a slight, but significant, labelling was also observed in the nucleus. Dual parameter flow cytometric measurements of fiuorescein-immunolabelled P33 and DNA content showed that P33 amount increased with increasing growth times in an HT medium, in all cell cycle phases. Thus there is no direct relationship between P33 increase and the exit of most of the cells from the cycle that occurs in an HT environment.

P25.25

Aspects of Ontogenesis of Neurosecretory Cells in Man. I. Radu Faculty of Medicine, Craiova, Romania.

We studied with light and electron microscopy, on sections of fixed embodded tissue from the human embryo and fetus - the appearance and the differentiation of neurosecretory cells. At the third month, the argyrophil cells were located close or in tile wall of branching ducts of the pancreatic parenchyma and after - - an in islets. At this date, we have also seen neurosecretory cells in the mucosa of stomach (corpus and antrum), duodenum, jejunum ileum, colon, appendix, pulmon, thyroid, parotid, etc. In the parotid of Macaca, Cercopithecus aetiops (monkey), we have revealed argyrophilic cells in the wall of the ducts. We studied correlations between neurosecretory cells - limitroph limphoid tissue and the relationship with autonomic nerve endings from the gut.

P25.26 Albumin Uptake by Preadipocytes in Different Stages of Adipogenesis Induced In Vitro; An Ultrastructural Study. M. Raicu, A. D. Petrescu and M. Simionescu Institute of Cellular Biology and Pathology, Bucharest, Romania. Specific binding of albumin (the main carrier of fatty acids) to

617 the adipocyte surface was previously reported (Brandes et al., 1982, Biochem. Biophys. Res. Commun. 105, 821). To investigate the structures involved in binding and uptake of albumin by preadipocytes in different stages of adipogenesis, precursor fat cells were isolated and cultivated in DMEM with 5~ fetal calf serum. Cell transformation was induced by adding 1.7 nM insulin to the culture medium. Cells in different stages of differentiation were incubated with albumin (alone or bearing oleic acid) conjugated with gold particles. In controls, polyetyleneglycol and lgG-gold complexes were used. After extensive washing, the cells were processed for standard electron microscopy. The results showed that; a) In adipogenesis induced in vitro, four cell types were simultaneously present: fibroblast-like cells (F); early adipocytes (EA); adipocytes (A); aged adipocytes (AA). b) In interaction with albumin-gold, the uptake was dependent on the cell stage, namely F
P25.27 Glycoconjugates and Regulatory Peptides in Bovine Abomasum During Development. B. Rodd and G. Lackovi~ Department of Biology, University of Zagreb, Zagreb, Croatia. Six lectins with different major sugar-binding affinities were investigated by indirect immunoperoxidase methods in paraffin sections of bovine abomasum during fetal and postnatal period. In addition gastrin and somatostatin were examined by PAP method in endocrine cells of same specimens. Ulex europaeus agglutinin (UEA I) which recognizes L-fucose residue and Dolichos biflorus agglutinin (DBA) which recognizes Nac-D-acetilgalactosamine proved to be the best markers of stomach glands during the process of differentiation. In early stages of development (10 to 14 wk of age) UEA I labels uniformly cell membranes of epithelium clusters and later on cytoplasmic compartments in separate regions of developing glands. With the progress of differentiation at 20 to 22 wk of age, DBA strongly labels primitive parietal cells at the bottom of crypts. Postnatally and in adults DBA selectively labels intra- and intercelluar canaliculi of parietal cells depending on the secretory state. Stereological analysis of gastrin producing cells which first appeared as single cells at 16 wk of gestation, showed a significant increase of frequency (Vv = O,13 _+ 0,001 mm ~ and volume of nuclei at 22 wk of age. This coincided with the beginning of parietal cell proliferation and general growth of glands. The frequency of G-cells in adult mucosa reached Vv = 0,28 _+ 0,001 mm ~ The multistep process of development of bovine stomach mucosa is apparently accompanied by structural changes in glycoconjugates and presumably by trophic action of gastrin.

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P25.28 Peritubular Myoid Cell Proliferation in Rat Testicular Atrophy Caused by Epinephrine. A Quantitative Immunohistochemical Study. L. Santamarfa, R. Martin and J. Codesal Dpt. Morphology, Sch. Medicine, Autonomous University. 28029-Madrid, Spain. It is known that repeated intra-scrotal injections of epinephrine (EP) causing testicular atrophic lesions with germ cell depletion. The aim of present work was to study the proliferative response of peritubular myoid cells (PMC) in EP-induced testicular atrophy. It was performed immunohistochemical detection of Bromodeoxyuridine (BrdU) incorporation, (that is a phase-S proliferating cells marker) and immunoreactive PMC nuclei to PCNA (proliferating cell nuclear antigen). Labelling Indexes (LI) to BrdU and PCNA, and counting of PMC per testis (NMT) were used to quantify cell proliferation. After 5 weeks of treatment by repeated EP injections in the left hemi-scrotum, both PMC LI's were significantly increased in comparison to controls, linear correlation (R = 0.98) was also founded between those indexes. The NMT was gradually raising and there was also linear correlation (R = 0.7) between LI (BrdU) and NMT. However, in the time course of experiment, a progressive decrease of LI (BrdU) for spermatogonia was detected in correlation to the germ cell depletion. It can be concluded that, in early stages of EP-induced testicular atrophy, the PMC's undergo a proliferating response. It is known that Sertoli cells inhibit in vitro PMC proliferation, this inhibition could be suppressed because the impairment of seminiferous epithelium caused by EP.

P25.29 Intranuclear Distribution of PCNA and Spatial Redistribution of DNA During S-Phase of the Cell Cycle. B. Schutte and F. C. S. Ramaekers Dept. of Molecular Cell Biology & Genetics, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands. During S-phase PCNA is observed in specific nuclear structures, which are similar to the actual sites of DNA replication. In a double labelling experiment using antibromodeoxyuridine (BrdU) and anti-PCNA immunofluorescence in combination with confocal scanning laser microscopy we were able to show that PCNA immunoreactivity does indeed colocalize with sites of BrdU incorporation, i.e. sites of DNA replication. In HT29 cells the PCNA and BrdU immunoreactivity was almost completely overlapping. Only a minority of the cells were exclusively PCNA or BrdU positive. Based on this observation we studied the spatial organisation and migration of newly synthesized DNA in the nucleus during S-phase. In a pulse chase experiment we were able to show that newly synthesized DNA is redistributed in larger patches, reassembling replication patterns of highly condensed late S-phase DNA as cells progress through Sphase, migrating away from the site of replication, i.e. PCNA-fluorescenee.

Poster sessions

P25.30 Cellular Differentiation and Migration of Mucous Neck Cells of Xenopus laevis Fundic Gland using Lectin Histochemistry and Bromodeoxyuridine Immunohistochemistry. T. Oinuma, J.-I. Kawano and T. Suganurna Department of Anatomy, Miyazaki Medical College, Miyazaki 88916, Japan. We studied the origin and differentiation of MNCs in fundic glands of Xenopus (X), laevis and its larvae using lectin histochemistry and bromodeoxyuridine (BrdU) immunohistochemistry. Griffonia simplicifolia agglutinin (GSA)-II specifically detected MNCs in fundic glands of adult X. laevis. During the morphogenetic period, GSA-II reactive cells randomly appeared in various portions of the immature fundic glands and then rapidly localized in the neck portion. At this time, two types of cells intermediate to MNCs and surface mucous cells (SMCs) and intermediate to MNCs and oxynticopeptic cells (OPCs) were detected with GSA-II. BrdUimmunohistochemistry revealed a proliferative cell zone between SMCs and MNCs. Labelling index (LI) of MNCs rapidly increased by week 5. LI of OPCs showed the delayedincrease by week 7, coincident with the decrease in LI of MNCs. In immature fundic glands of larvae, the labelled proliferating cells randomly distributed throughout the glands. During the metamorphosing period, LI of immature epithelial cells was the highest at stage 63. After the labelling at stage 63, there was no significant difference in LIs among the epithelial cells. In adults, stem cells might differentiate directly to SMCs in an upward direction. A majority of OPCs seemed to differentiate during a downward migration. Stem cells pass through a stage as MNCs, finally becoming OPCs. In metamorphosing larvae, stem cells might proliferate and differentiate to SMCs, MNCs and OPCs in situ. They may have migrated during stages 64 and 65. Histogenesis of adulttype gastric mucosa completed at stage 66.

P25.31 Expression of Insulin-Like Growth Factor 2 in Hepatitis B and Liver Cirrhosis - - an LM Immunohistochemical Study of 265 Cases. Q. Su, Y.-F. Liu and S. X. Zhang Department of Pathology Tangdu Hospital. The Fourth Military Medical University, Xi'an, P.R. China. With ABC immunohistochemical technique, we made an investigation on the role of insulin-like growth factor 2 (IGF2), a polypeptide highly expressed in both embryonic liver and hepatoma, in the process of hepatocarcinogenesis in 265 cases of hepatitis B and liver cirrhosis. It was demonstrated that the IGF-2-highly-expressing cells existed in chronic liver disorders, and even more significantly, the IGF-2-highlyexpressing cells remarkably increased both in sort and in number (P<0.005) with the progression of the pathologic changes, especially of the fibrosis and oval cell proliferation. It was found that only small polyonal liver cells (SPLC) could highly express IGF-2 in chronic persistent hepatitis; and additionally, some polypoid and multinuclear hepatocytes in active liver disorders and some of the regenerative nodules in some cirrhotic livers, especially those around hepatomas, could also express IGF-2 in large amount. It was suggested

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P o s t e r sessions that the overexpression o f IGF-2 be a crucial step in the process of hepatocarcinogenesis. The nature, function of SPLC and the mechanism of SPLC proliferation were discussed.

PCNA staining pattern may provide much information regarding the potency of cell proliferation.

P25.34 P25.32

Expression of Insulin-Like Growth Factor 2 in Hepatoma Cells in Culture - - An LM Immunocytoehemical Study. Q. Su, Y.-P. Han and Y.-F. Liu Department of Pathology Tangdu Hospital. The Fourth Military Medical University, Xi'an, P.R. China. With ABC immunocytochemical technique, we found that hepatoma celt line QGY 7703 and SMMC 7721 in culture could express insulin-like growth factor 2 (IGF-2), the IGF-2immunoreactivity was localized in cytoplasm. The spindleshaped cells and the cells in mitosis stained strong; Once the hepatomo cells contacted with each other to form a monolayer, the immunoreaction decreased remarkably and turned negative. These findings suggested that IGF-2, as a mitogen, be responsible for the proliferation of the hepatoma cells at least in culture, and inhibited by cell contaction. In addition, the cell lines was found to express A F P , which was localized in the similar subpoputation with IGF-2. This suggested that there may be some relations between the expressions of the 2 proteins.

P25~33

Differential Expression of PCNA in Human and Rat Tissues - - Analysis of Immunostaining with Image Analyzer. K. Suzuki, A. Henmi, R. Kato and A. Kawaoi Department of Pathology, Yamanashi Medical College, Yamanashi, Japan. Immunostaining of proliferating cell nuclear antigen (PCNA), which can be applied to conventional formalin-fixed, paraffin-embedded tissue sections, has extended the applications of cell kinetics studies. We used anti-PCNA monoclonal antibody (19A2) as the primary antibody and DAB as the substrate to immunostain various tissue sections, including human neoplasms and diisopropanolnitrosamine (DIPN)-induced rat thyroid tumors, estimated the relative PCNA staining intensity of each nuclei separately with an image analyzer, and then compared PCNA staining intensity distribution patterns. Normal intestinal mucosa and splenic lymph follicles stained in the same way were used as the standards of the PCNA staining pattern. In normal tissue, only a few nuclei showed strong staining, but some showed faint staining. The staining intensity varied widely in the specimens from neoplasms, and labelling index and average staining intensity were increased. In the lesions of diffuse hyperplasia seen in the early phase of DIPN-induced rat thyroid tumorigenesis, the population of weakly stained cells was increased, and most nuclei in the cancerous nodules which developed in the late stage showed strong PCNA expression. These results suggest that dull cellular expression of P C N A reflect the early or preliminary phase of the cell cycle before DNA synthesis, and support the previous biochemical findings that two forms o f PCNA exist. Differential assessment of

Association of Titin with Other Myofibrillar or Cytoskeletal Proteins During Human Myofibrillogenesis In Vitro. P. van der Ven", G. Schaart 2, P. Jap ~ and F. Ramaekers z ~Dept. of Cell Biology & Histology, University of Nijmegen, The Netherlands, 2Dept. of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands. Differentiating, cultured human skeletal muscle cells were used to study associations of titin with other sarcomeric constitutents during human myofibrillogenesis. Several stages of differentiating cells were stained with antibodies against titin, a-actin, sarcomeric myosin, nebulin, desmin, tubulin and vinculin. Titin primarily appeared in postmitotic mononuclear myoblasts in a random punctate fashion. Except for myosin, which was colocalized with titin in a minority of the cells, none of the other myofibrillar proteins studied, seemed to be associated with these spots. Subsequently the titin spots were found linked to longitudinally oriented filaments that contained a-actinin and a-actin. In somewhat further differentiated cells, titin was localized in a filamentous, stress fiber-like pattern together with a-actinin, a-actin and myosin. Nebulin was not found in these structures until an obvious periodicity of titin, myosin, a-actinin and a-actin was observed. A localization of desmin in a striated pattern was not observed before the myofibrils had aligned laterally. From these results we conclude that titin/myosin complexes can develop before association of titin with Z-disk components. Intermediate filaments, microtubuli and nebulin filaments do not seem to be essential for early stages of myofibrillogenesis.

P25.35

Histochemical Studies on the Testes of China Yak, Cattle and Plan (Ft). S. Wang, C. Chin, S. Zhao and X. Yu Depart. of Histology and Embryology, Xinjiang Medical College, Urumqi, China. 1. The testes of 2 . 5 - 3 years China Yak, Cattle and Pian(F~) were studied with RNA, PAS, AIP, AcP, ATPase, G-6-Pase, 5'-Nase, NSE, SDH and LDH staining methods. 2. The seminiferous tubules are thick in Yak, Cattle, thin in Plan, which diameter is 1 / 2 - 2 / 3 of the Yak ones. Many high columnar Sertoli cells, fewer spermatogonia, primary spermatocytes and occasionally pyknotie spermatid can be found, while no spermatozoon can be seen in the testes of Plans. Plans basement membranes, which PAS reaction showed intensive positive, were 1 . 5 - 2 times thicker than those of the Yak and Cattle. Plans' interstitial cells were smaller and blood vessels were fewer than the Yak and Cattle. 3. RNA granules were fewer and smaller in the interstitital cells and primary spermatocytes, rich in the residual bodies of the Yak and Cattle. 4. Glycogen granules were rich in the spermatogonia. 5. The reaction of A I P was strong in the interstitial cells and endothelium of small vessels. 6. The reaction of AcP and NSE was positive in the spermatogonia, Sertoli cells and interstitial cells. 7. The reaction of ATPase

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Poster sessions

was very strong in the intertitial cells, myoid cells and endothelium, positive in the spermatogonia, spermatocytes and Sertoli cells. 8. The reaction of 5'-Nase and G-6-Pase was strong in the spermatogonia, spermatocytes and interstitial cells. 9. The reaction of SDH was very strong in the endothelium, interstitial cells, moderate in the spermatogonia, and positive in other cells. 10. The reaction of LDH was strong in the spermatocytes and interstitial cells, moderate or positive in the spermotogonia and Sertoli cells. From above mentioned, the metabolism of the testes is identical, the male sterility of the Pian may be caused by the change of its genetic gene and the abnormal structures of the seminiferous tubules and interstitial tissue.

P25.36 Inhibition of bFGF synthesis and terminal differentiation of chondrocyte cultures grown as a pellet mass by exogenous bFGF. N. Yamarnoto and A. Masumoto Dept. of Functional Morphology, Kitasato Univ. School of Nursing, Sagamihara, Kanagawa 228, Japan. Chondrocytes undergo a series of changes including proliferation, formation of an abundant matrix, and terminal differentiation to hypertrophic cells prior to calcification of a cartilage matrix. The chondrocytes maintained as pellet mass in a centrifuge tube produced high levels of alkaline phosphatase (ALPase) and induced extensive calcification. In the present study, we examined the effects of exogenous bFGF on the synthesis of endogenous bFGF and the induction of ALPase using this culture system, and obtained the following results. 1) bFGF was localized in both proliferating and hypertrophic cells, but not recognized in maturing cell in rat femoral growth-plate cartilage by immunohistochemical staining. 2)bFGF was also localized in the chondrocytes grown as a pellet mass using immunohistochemistry, and mRNA for bFGF was detected in the cells by in situ

P26. CYTOCHEMISTRY

hybridization. 3)Addition of bFGF to the chondrocyte cultures suppressed the synthesis of bFGF, and abolished the increase in ALPase activity. These results suggest that an exogenous bFGF inhibits the synthesis of endogenous bFGF and then prevents the chondrocyte terminal differentiation.

P25.37 Histogenesis of the Human Stomach. W. Zhang and S. Y. He Central Laboratory, The First Medical College of PLA, Guangzhou 510515, People's Republic of China. We collected 70 human fetuses between 7 and 32 weeks of fertilization age and 3 neonates legally for studying their development of stomach by histochem- and immunohistochemistry under LM and EM. In 7-week old embryo, the superficial sheet of gastric mucosa was of stratified columnar epithelium. By 9-week, simple columnar epithelium had presented. At this stage the gastric pits and anlages of glands had formed. Occasionally, a few parietal cells in the bottom of the glands could be found. The muscularis mucosa had occurred at 14-week and the 4 layers of stomach formed. At 18-week, parietal cells were found in the neck of the gland. During the fetal period, the pyloric gland possessed parietal cells as well as fundic gland. In antrum, we observed the surface epithelium wasn't the same as that of fundus to a certain extent, and the muscularis mucosa developed earlier. The argyrophil cells of stomach were less at 12-week. They distributed in the base of the surface epithelium and gland, and might assume various forms. The argyrophil cell of antrum is more than those of fundus since 14 weeks. In 18 weeks, the argyrophil cells were the most numerous. Some cells possessed processes which extend to the basal membrane, parietal cells or the gland lumen, We also observed EC cell of fundus and G cell of antrum by immunohistochemistry, parietal cell, G and D cell under EM.

OF PEROXISOMES

P26.1 Identification and Characterization of Peroxisomes in the Digestive Gland of Marine Mussels*. M. P. Cajaraville ~ A. V61klb and H. D. Fahimi b ~Cell Biology~Morphological Sciences, Univ. Basque Country, Spain; ~Anatomy/Cell Biology, Univ. Heidelberg, FRG. Marine molluscs are sensitive indicators of environmental pollution and in this conjunction their microsomal and lysosomal enzymes have been extensively studied and used as pollution biomarkers. Because of paucity of information on molluscan peroxisomes, the present study was undertaken. Peroxisomes were identified by electron microscopy in all three main cell types of the digestive gland of the bivalve mollusc Mytilus gaUoprovincialis Link. They stained very weakly with the alkaline DAB reaction but showed distinct immunolabelling with the antibodies to mammalian catalase by the protein A-gold procedure. In addition, mussel digestive

gland peroxisomes were isolated by differential and Metrizamide-density gradient centrifugation and a 30-fold enrichment of catalase over the initial homogenate was obtained. The highly purified peroxisomes exhibited catalase as well as palmitoyl-Co A oxidase activities. By Western blotting, they cross-reacted with antibodies to acyl-Co A oxidase, catalase and urate oxidase from rat liver. These observations establish that peroxisomes in molluscan digestive gland contain some of the enzymes present in mammalian liver peroxisomes. Further studies of alterations of mulluscan peroxisomes by environmentally relevant xenobiotics are warranted. *Supported in part by grants from the Univ. Basque Country, DAAD and SFB 352.

Poster sessions

P26.2 The Liver and Kidney of Tumor-Bearing Mice: Catalase Activity and Peroxisomal Alterations. D. De Craemer, C. van den Branden, A. Vergeylen and F. Roels Human Anatomy & Embryology, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. It is well known that hepatic catalase activity is decreased in patients with malignant diseases and in tumor-bearing animals. Recently, it was shown that tumor necrosis factor reduces hepatic but not renal catalase activity in vivo. The mechanism of catalase depression is not elucidated. In addition, little is known about the peroxisomes, which contain the bulk of catalase. We investigated the catalase activity and evaluated the peroxisomal alterations in murine liver and kidney. Xenografts of two human pancreatic exocrine adenocarcinomata, differing in growth rate, were subcutaneously transplanted in four weeks old nude mice (Swiss nu/nu). Animals were sacrificed after three to six weeks, depending on the growth rate of the transplant. Samples of liver and kidney were homogenized for catalase activity assay or were stained with diaminobenzidine to visualize the peroxisomes. Results were compared to those obtained from control mice of the same age. A significant decrease in catalase activity in liver but not in kidney of tumor-bearing mice was observed. This is in agreement with the proposed role of tumor necrosis factor. Light microscopy revealed less peroxisomes in liver of mice with pancreatic xenografts; staining intensity of the peroxisomes was normal in most livers. In kidney, a normal number of well stained peroxisomes was observed. Ultrastructural morphometry of hepatic peroxisomes confirms the light microscopic findings. Our observations indicate that a decrease in hepatic catalase activity in tumor-bearing mice is due to a reduced peroxisomal number. In contrast, in livers of patients with cancer, the peroxisomal number is not decreased. Supported by grants from the Belgian FGWO and NFWO.

P26.3 Catalase Deficient, Giant Hepatic Peroxisomes Containing Four Other Enzymes in a Child with Bone Dysplasia. M. Espeel, J. Heikoop*, D. de Craemer, J. Smeitink**, R. Wanders*, T. Hashimoto***, B. T. Poll-The** and F. Roels**** Free University Brussels, Dept. Human Anatomy & Embryology, Brussels (Belgium); *University Hospital Amsterdam, Amsterdam (The Netherlands); **Wilhelmina Kinderziekenhuis, Utrecht (The Netherlands); ***Shinshu University Medical School, Nagano (Japan); ****University of Ghent, Ghent (Belgium). In the liver biopsy from an 8 89 year old girl with the biochemical characteristics of rhizomelie chondrodysplasia punctata (RCDP; phytanie acid storage, deficient plasmalogen biosynthesis and unprocessed 44 kDa 3-ketoaeyl-CoA thiolase) but normal limbs, eatalase containing peroxisomes were absent (Smeitink et al. 1992; J. Inher. Metab. Dis., in press). Catalase, the peroxisomal marker enzyme, was localized only in the cytoplasm as demonstrated by DAB- and immunocytochemistry. In the parenchymal cells rare (numerical density 3~ of control

621 values), extremely large (mean corrected d-circle = 1.44 ~m; controls: 0.64 gm), and irregularly shaped vesicles were found in close association with cisternae of the endoplasmie reticulum. They contained a reticular matrix, enclosed by a single limiting membrane. All three peroxisomal fl-oxidation enzymes, and alanine-glyoxylate aminotransferase (AGT), were immunolocalized in these organelles. In cultured skin fibroblasts from the patient normal catalase containing peroxisomes were found. The findings in the liver represent an intermediary situation between "classical" RCDP, having catalase containing peroxisomes in most cells (De Craemer et al. 1991. Virchow Arch A 419,523 - 525), and the generalized peroxisomal disorders, having a cytosolic localization of catalase and AGT. The large size, rarity and irregular shape of the hepatic peroxisomes are reminiscent of peroxisomal "ghosts". In one RCDP patient, we found similar organelles in the liver cells that lack catalase containing peroxisomes. Supported by N.F.W.O. Research Grant.

P26.4 Histochemistry of Peroxisomal Enzymes: A Useful Tool in the Diagnosis of Zellweger Syndrome. W. M. Frederiks ~, K. S. Bosch ~, M. Ankum ~, R. J. A. Wanders 2 rLaboratory of Cell Biology and Histology', eDepartment of Paediatrics, Academic Medical Centre, University of Amsterdam, The Netherlands. The localization of the activity of the peroxisomal enzymes catalase, D-amino acid oxidase and hydroxy acid oxidase was studied at the light microscopical level in livers and kidneys from control subjects and patients with an inherited deficiency of peroxisomes (Zellweger syndrome). D-amino acid oxidase and hydroxy acid oxidase activities were demonstrated in unfixed cryostat sections with the cerium-diaminobenzidinecobalt-hydrogen peroxide procedure, in which cerium ions capture hydrogen peroxide, the product of both enzymes, whereas in a second step decomposition of cerium perhydroxide gives rise to the formation of a diaminobenzidine polymer eomplexed with cobalt ions. Catalase activity could not be detected when demonstrated with diaminobenzidine in glutaraldehyde fixed cryostat sections in livers and kidneys of control humans, which may be a consequence of the very low enzyme activity. D-amino acid oxidase activity was found in peroxiomes of liver parenchymaI cells and proximal tubular cells in the kidney of control humans. Hydroxy acid oxidase activity with glycolate as substrate (isoenzyme A) was found in peroxisomes of liver parenchymal cells, whereas isoenzyme B (normally present in rat kidney) could not be detected in peroxisomes of proximal tubular cells with hydroxybutyrate of lactate as substrate. The activities of D-amino acid oxidase and hydroxy acid oxidase could not be demonstrated in livers of Zellweger patients, as was the case also for D-amino acid oxidase activity in kidneys of these patients. It is concluded that the cerium-diaminobenzidine-cobalthydrogen peroxide procedure enables the detection of peroxisomai enzyme activities in human tissues which can be applied in diagnostic procedures with regard to peroxisomal disorders.

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P25.5 Species-Dependent and Species-Independent Sites of H202-Generation in Mammalian Tissues as detected with Cerium Ions. R. Gossrau and G. Nakos Department of Anatomy, Free University of Berlin, Germany. Using cerium ions, PVA media and a DAB-H202-Co visualization medium H202-producing sites can reliably be detected in all kinds of tissues. This study deals with the question whether H202-generation is a general phenomenon, i.e. shows no species-dependence or behaves speciesdependently for H20~-generating D- and L-amino acid oxidases (AAOX), a-hydroxy acid oxidase, monoamine oxidase (MAOX), benzylamine oxidase (BAOX), diamine oxidase and the xanthine oxidase-superoxide dismutase (XOX-SOD) system in rat, mouse, gerbil, guinea-pig and marmoset tissues. With very few exceptions, e.g. D-AAOX in proximal renal tubular cells, MAOX in the same cell type and hepatocytes, BAOX in smooth muscle cells and XOX-SOD in enterocytes most of the other cells express these H202generating oxidases species-dependently, e.g. in nerve cells, capillary endothelial ceils as well as lung, uterine or epidydimal epithelial cells, enterocytes or endocrine cells. Furthermore, species-dependent intraorgan, intercellular and intracellular differences existed indicating that there is no uniformity of H202-generating cellular and subcellular sites in mammals.

P26.6 Immunolocalisation of Peroxisomal Proteins in Patients with Peroxisomai Disorders. J. L. Hughes ~, D. I. Crane 3 and A. Poulos 2 1Department of Histopathology and 2Department of Chemical Pathology, Adelaide Children's Hospital, North Adelaide, South Australia, 3Division of Science and Technology, Griffith University, Queensland, Australia. Peroxisomal disorders are a group of inherited diseases affecting children which result in severe clinical consequences including abnormalities of the liver, kidney, brain, adrenal and bone. The disorders are characterised biochemically by a deficiency of one or more peroxisomal enzymes, and an accumulation of substrates in the patient's tissues and plasma. The peroxisomes in the liver of these patients display marked ultrastructural changes ranging from a complete absence to extremely enlarged, flocculent organdies. An on-grid immunogold technqiue was used to localise the peroxisomal matrix protein catalase; the/3-oxidation enzymes, acyl-CoA oxidase, bifunctional protein, and 3-ketoacyl-CoA thiolase; and a 68 kDa peroxisomal integral membrane protein. Liver tissue was fixed in a mixture of paraformaldehyde and glutaraldehyde and embedded in Lowicryl resin at low temperature. Liver from 6 control patients, and patients with Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, rhizomelic chondrodysplasia punctata, pseudo-Zellweger syndrome, and several unclassified peroxisomal disorders was investigated. The results of the immunolabelling show that in cases where the peroxisomes are enlarged the labelling density for catalase is generally decreased. In those disorders which display small, ultrastrucrurally abnormal peroxisomes labelling for the matrix proteins

Poster sessions is often absent, although the 68 kDa membrane protein is still present. In some cases labelling for catalase and/3-oxidation enzyme was present within the matrix of these peroxisomes but excluding an electron-dense core.

P26.7 Quantitative Analysis of Hepatic Peroxisomes in Marmoset (CallRhrixjacchus) Following Chronic Ciprofibrate Treatment. S. L. Old, J. A. Rees, A. J. Spencer, M. J. Graham, S. A. Wilson and F. W. Bonner Toxicology Department, Sterling Winthrop Pharmaceutical Research Division, Willowburn A re., A In wick, Northumberland, UK. Hypolipidaemic drugs of the phenoxyisobutyrate series such as ciprofibrate (CP) induce marked peroxisome proliferation, changes in peroxisomal structure and enzyme composition, and, in chronic studies, lipofuscinosis and formation of hepatic carcinomas in rodents. The aim of this study was to investigate the effects of chronic treatment with CP on peroxisomes and lipofuscin deposition in a non-rodent species, the marmoset. Male marmosets were treated with 0, 2, 10 and 20 mg/kg CP by daily oral gavage for 3 years. Samples of liver were processed for biochemical assay (peroxisomal/3oxidation and carnitine acetyl transferase). Samples of the median liver lobe from all animals were thinly cut, fixed for 1 hour in modified Karnovsky's fixative (2.5~ glutaraldehyde, 2~ paraformaldehyde) and incubated for visualisation of peroxisomes using a 3-3 diaminobenzidine technique. Samples were then processed conventionally for electron microscopy examination. Uttrathin sections were examined using a Philips CM10 electron microscope. For each section, 10 non-overlapping electron micrographs were taken by a systematic sampling technique. The micrographs were photographed directly using a macrostand linked to a "Magiscan" image anayser. Quantitative image analysis examination was carried out "blind". Total area and number of both peroxisomes and lipofuscin per unit area of hepatocyte were calculated using a user-defined, partially interactive program. Statistical analysis of the results (analysis of variance) showed no significant difference or trend with treatment in any of the parameters measured. These findings were in accord with the biochemical data, which found only slight increases in peroxisomal enzymes with treatment (2fold, as opposed to 20-fold in rodents). These findings are in marked contrast to the effect of CP in rodents and support the theory that primates are relatively insensitive to the peroxisomal effects of this class of compound.

P26.8 Peroxisomai Ghosts in Human Liver. F. Roels, M. Espeel, S. Yokota and P. Ramos Universiteit Gent, Belgium; Vrije Universiteit Brussel, Belgium; Yamanashi Medical School, Japan; Universidade Novo, Lisboa, Portugal. Peroxisomal membrane ghosts were originally described by Santos in cultured fibroblasts from patients without peroxisomes (i.e. with ZeUweger syndrome), and considered to be empty vesicles, reflecting the impairment of import of

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623

matrix constituents. Later, 44 kDa thiolase, catalase and acylCoA oxidase were found in the same membrane fractions (G~irtner; Van Roermund; Roels). We review data on liver from 16 patients. Infantile Refsum disease: abnormal organelles deficient in catalase and four other peroxisomal enzymes were numerous in some parenchymal cells but rare in others; their volume density was 37%, and surface density 42% of normal; they displayed an electron-transparant zone. In 3 other IRD children, no peroxisomal structures were seen (Roels and Poll-The). Neonatal adrenoleukodystrophy-like girl (Auborg n~ enlarged vesicles with a pale matrix; they were not observed in 4 other NALD(-Iike) cases. In one, four peroxisomal enzyme proteins and catalase activity were correctly localized, notwithstanding storage of VLCFA and bile acid intermediates (Espeel et al, 1991). Bone dysplasia and mental retardation with phytanic acid storage and

P27.

RECEPTORS

AND

plasmalogen deficiency (Espeel et al, this Congress): large vesicles with minimal catalase (DAB or immunogold) but immunoreactive for oxidase, bifunctional enzyme, thiolase and AGAT. In a similar patient, cousin of the former, catalase particles were reported (Pike et al, 1990). Zellwegers cerebrohepato-renal syndrome: in 4 cases immunolabeUing for 70 kDa membrane protein was negative. In a 5th case very large, irregular and pale vesicles were deficient in catalase activity. Conclusion: in liver, prevalence and composition of ghostlike vesicles vary among comparable patients, and point toward molecular mechanisms other than a generalized import deficiency. Espeel et al, 1991, Virchow Archiv A 419: 301; Roels F, Peroxisomes - - a personal account. VUBPress, 1991; Roels et al, J Inher Metab Dis, 1991, 14: 853.

LIGANDS

P27.1

Raised Cytosolic Calcium and Cell Rounding in ATP Induced Cells as Visualized with the Argon Laser Confocal Microscope. K. H. Sit, B. H. Bay and K. P. Wong* Departmenl of Anatomy and *Department of Biochemistry, National University of Singapore, Singapore 0511. Extracellular ATP, an established phosphoinositide (PI) signal transducer induces raised cytosolic calcium and Na +/H + antiporter mediated which is sensitive to quinidine at 1 mM (a N a + / H + antiporter blocker) and staurosporine at 10 mM (a protein kinase inhibitor) (Sit et al. Japan J. Physiol. (in press)). To evaluate [Ca2§ mobilization by ATP at a concentration that causes cell rounding, Chang liver cells are loaded with fluo-3 acetoxymethyl ester (cell permeant ester form) for 30 minutes, and incubated for 0, 1, 2, 5 minutes with 0.7 mM ATP in Na+-HEPES. The reaction is stopped by fixation in 5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3. Fluorescence imaging done in the Bio-Rad MRC600 confocal system as scaled by the 12 BAND pseudocolour lookup table called up from the software, show an upsurge of cytosolic Ca 2+ at two minutes. The cell profile area and perimeter of the controls and 0.7 mM ATP rounded cells as measured by microspectrophotometric digitization (Sit and Wong, Tissue & Cell 22: 7 8 5 - 8 0 2 , 1989; et al. 1992, Lithium (in press) 1992) show a significant difference in the cell area, perimeter and cell roundness.

P27,2

Autoradiographic Localization of Binding Sites for Vasoactive Peptides on Neurones and Astrocytes of Cultured Rat Central Nervous System. E. Hdsli and L. H6sli Department of Physiology, University of Basel, Vesalgasse1, CH-4051 Basel~Switzerland. There is good evidence that vasoactive peptides act as neurotransmitters/neuromodulators in the mammalian central nervous system (CNS). By means of autoradiography, we have investigated the cellular localization of binding sites

for ~zSI-vasoactive intestinal peptide (VIP), 3H-angiotensin II (Ang II), ~25I-endothelin (ET) and J25I-arginine vasopressin (AVP) in explant cultures which were prepared from cerebral cortex, cerebellum, brain stem and spinal cord of fetal and newborn rats. Binding sites for the peptides were found on the soma and processes of a great number of neurones. In addition to neurones, also many astrocytes revaled binding of ~25I-VIP, 3H-Ang II, t2~I-ET and t25I-AVP. The number of labelled cells and the intensity of labelling varied considerably between the different CNS areas studied, providing evidence for a heterogeneity of both neurones and astrocytes in various brain regions. Binding of the radio-ligands to both cell types as inhibited or markedly reduced by adding excess unlabelled peptides to the incubation medium suggesting specific binding. Our autoradiographic studies that not only neurones but also glial cells possess binding sites for these peptides are supported by electrophysiological investigations demonstrating that VIP hyperpolarizes the glial membrane whereas Ang II, ET and AVP cause a depolarization of astrocytes. These findings provide strong evidence that astrocytes express functional receptors for vasoactive peptides.

P27.3 Erythropoietin-Receptors on Myelodysplastic Bone Marrow Precursor Cells. S. R. Joosten-Achjanie and H. C. Schouten Department of lnternal Medicine, University Hospital Maastricht, The Netherlands. The myelodysplastic syndromes (MDS) are characterized by various cytopenias in the peripheral blood. Clinical studies suggest that recombinant human erythropoietin (rh-Epo) is not able to relieve the anemia due to MDS. We studied the percentage Epo-receptor positive cells in seven morphologically normal bone marrow aspirates and nine MDS bone marrow samples, from eight different patients [RA (n = 3), RARS (n = 2), RAEB (n = 2), CMML (n = 1) according to the FAB criteria], using biotin-labelled bioactive rh-Epo. From four patients the marrows were obtained while on rhEpo treatment, and from one patient during and after the therapy. The results were compared with the percentage

624 erythroid cells in the May Grtinwald Giemsa (MGG) stained bone marrow smears. Epo-receptor positive ceils were found in 7/7 normal marrows (49.7 _+ 16.8~ (mean _+ SD), and in 9/9 MDS marrows (53.1 + 18.2 (mean _+ SD). The correlations between Epo-receptor containing ceils and morphologicaly erythroid cells were similar in normal marrows (r = 0.793, 0.02
P27.4 GPIIb-IIIa and Bound Fibrinogen (FG) are Arranged in Clusters on Spread Platelets: Immunogold Electron Microscopical Investigation. H. Kuhn, J.-C. Brun and B. Steiner Pharma Division, Preclinical Research, F. Hoffman-La Roche Ltd, CH-4002 Basel, Switzerland.

The GPIIb-IIIa is present on the surface of unactivated platelets. Following platelet activation, GPIIb-IIIa changes its conformation and becomes the receptor for FG. We have prepared two monoclonal antibodies (MAbs): p 1 - 62 binds to the inactive and active receptor form of GPIIb-IIIa, whereas the MAb pl - 55 only recognizes the functionally active form. For the identification of bound FG, we used the MAb E3F8F5 (Serotec). The immunogold technique was applied for the localization of both forms of GPIIb-IIIa and of bound FG on spread platelets at ultrastructural level. In double labelling studies, different sizes of the gold-particles, either for the two forms of GPIIb-IIIa or for FG and GPIIb-IIIa, were used. Gel-filtered human platelets were allowed to adhere for 3 min on collodium/carbon-coated Ni-grids. After mild fixation, the platelets were incubated with the primary MAb. The bound MAbs were detected with gold-labelled goat anti-mouse IgG antibody. Active and nonactive GPIIb-IIIa as well as bound FG were distributed over the whole surface of the spread platelets and arranged in clusters. The active form of GPIIbIIIa represented 25 - 35~ of total GPIIb-IIIa. Mixed clusters of active and nonactive GPIIb-IIIa were frequently observed. We conclude that GPIIb-IIIa exists in two molecular forms on fully spread platelets. Both these forms, as well as bound FG were often arranged in clusters, distributed on the whole surface of the platelets.

P27.5 Replica-Gold Cytochemistry on the Distribution Pattern and Dynamics of Mistletoe Lectin I Binding Sites in Hela-Tumor Cells.

Poster sessions Prague, 36, 181 - 188, 1990). P t / C replicas, prepared from the outer surface of fixed and CP dried HeLa-tumor cells show an irregular threedimensional distribution pattern of gold markers after the incubation of MLI-gold and MLI B chaingold at O~ In contrast, only few markers could be found after the incubation of MLI A-chain gold. No ultrastructural alterations were detectable in replicas prepared from the extracellular or protoplasmic surfaces of freeze-fractures plasma membrane. Warming up the cells to 37~ for 3 - 5 min. induces a strong aggregation of markers into long bizarre ribbons and large aggregates of 2 0 - 5 0 markers. Microvilli and large areas of the cell surface are now not langer labelled. Ultrastructural alterations in replicas from the extracellular and protoplasmic fracture faces are demonstrable, and with that internalization into the cell starts via membrane bound structures, visible in replicas from intracellular fracture faces. Supported by Gemeinntitzige Hertie Stiftung, Frankfurt/ Main.

P27.6 Epidermal Growth Factor Receptor on Human Breast Cells Detected by Scanning Electron Microscopy. P. Monaghan, M. J. O'Hare and C. Clarke Institute of Cancer Research, Haddow Laboratories, 15 CotswoM Road, Sutton, Surrey, SM2 5NG, England.

Cells from normal human breast epithelium have been prepared as a single cell suspension, and flow sorted to provide pure populations of epithelial and myoepithetial ceils. The single cell suspension was also analysed for expression of EGFR. The sorted cells were immunolabelled in vitro with anti-EGFR antibody detected by FITC-labelled second antibodies for light microscopy and colloidal gold labelled antibodies for scanning electron microscopy (SEM). For SEM, the EGFR was detected in a field emission microscope using a YAG crystal backscattered electron detector. Individual myoepithelial cells expressed EGFR over their exposed surfaces, but when growing in the centre of confluent islands of cells the myoepithelial cells showed low levels of expression of EGFR on their exposed membranes. EGFR was, however, detected on the basal membranes of cells growing at the periphery of confluent islands. EGFR localisation on epithelial cells demonstrated lower levels of receptor expression than on myoepithelial ceils, but similar behaviour of the receptor when comparing isolated cells with cells growing in confluent islands. These results demonstrate the value of multiple analytical approaches to the localisation of antigens, and that the SEM is a valuable tool in immunodetection of cell membrane molecules.

K. Mannweiler ~, H. WalzeP, L. Jonas 3, H. Hohenberg ~,

P27.7

J. Brock ~ and St. Pfeiffef

Melatonin Receptors are Associated with the Visual Pathways in the Japanese Quail.

IHeinrich-Pette-lnst. f. Exp. Virol. u. ImmunoL a.d. Univ. Hamburg, W-2000 Hamburg 20, 21nst. Med. Biochem., 3Inst. Pathol. Med. Fac., Rostock, 0-25 Rostock, FRG.

EM studies of preembedded gold (MLI Aul5) labelled L1210 leukemia cells revealed at ultrathin sections that MLI-like recin - - a heterobifunctional glycoprotein with cytotoxic activity on these ceils - - is internalized via coated pits and long membrane bound structures (Walzel, H. et al. Folia Biolog.

G. C. Panzica, B. Cozzi*, N. Aste, C. Viglietti-Panzica, F. Fraschini ~ and B. Stankov ~ Dept. Human Anatomy & Physiol., Torino, *Inst. of Anatomy of Domestic Animals, and ~ of Chemotherapy, Milano (Italy).

The localization, the kinetic and pharmacological characteristics of melatonin receptors (MR) were determined in

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discrete areas of the adult quail brain by autoradiography and in vitro binding, using 2-[125I]-iodomelatonin. Binding was widely distributed throughout the entire brain. In particular, MR were predominantly associated with the visual pathways. High density MR were found in the major targets of direct retinal input, such as the optic tectum, the nucleus of the optic basal rout, the ventrolateral geniculate nucleus. A relatively wide area was labelled near the decussatio supraoptica, corresponding to the location of the visual suprachiasmatic nucleus, that was identified as a retino-recipient hypothalamic region in other avian species. Several areas not directly connected to the retina, but representing terminals in the visual pathways (nucleus rotundus, ectostriatum, thalamohyperstriatal pathway), expressed strong binding. Furthermore, 2-[125I]-iodomelatonin binding was also found in the oculomotoris nucleus, and its associate Edinger-Westphal nucleus, which are involved in the eye movements. The quail, as many avian species, is strongly photoperiodic and profoundly influenced by the light-dark cycle. The distribution of MR in the quail brain clearly indicates a direct action of this indole on the regulation the avian visual system at different levels.

with anti-Trf-receptor FITC-conjugated monoclonal antibodies (Becton Dickinson) on human tumor cell lines A431, HeLa, PA-1, HEp-2, MG-63, IMR-32, KB, HT-1080, COLO 320 HSR, CaOv, K562, U937, SEw (Ewing's sarcoma). With regards to their reactivity with antibodies cell lines were subdivided into three groups: highly-expressed Trf receptors (U937, K562, A431, KB, HEp-2), poorly-expressed (HT-1080, SEw, COLO 320 HSR, HeLa, IMR-32) and without Trf receptor (Mg-63, PA-1, CaOv). According to the Trf receptor expression the cell lines were ranged as follows: K562>U937>A431>KB>HEp-2>COLO 320 HSR>SEw> IMR-32>HT-1080>HeLa. In addition, it was found that level of Trf receptor expression significantly correlated with the level of DNA synthesis induced by exogenous Trf as determined by ~H-thymidine incorporation assay. All epithelial origin cell lines posses functionally active Trf receptors but were different in the level of its expression. Our data suggests that Trf/Trf receptor system may be involved in neoplastic transformation of epithelial cells. Therefore Trf receptor may be a suitable target for immunotoxins which are actively investigated now.

P27.8 Modulation of Transferrin (Trf) Receptor Expression by TNFa: Flow Cytometric Detection.

P27.10

A. A. Phylchenkov, I. I. Slukvin* and Y. I. Kudryavets Institute of Experimental Pathology, Oncology & Radiobiology and *Kiev Institute of Pediatrics, Obstetrics, & Gynecology, Kiev, The Ukraine.

T. Renda, L. Negri*, C. Casu and P. Melchiorri* Institutes of Anatomy and *Pharmacology, University "'La Sapienza", Rome (Italy).

Modulation of Trf receptor expression on K562 (eritroleukemia) and U937 (monocyte-like lymphoma) cell lines by recombinant TNFa were studied using flow cytometric analysis with anti-Trf-receptor FITC conjugated monoclonal antibodies (Becton Dickinson). The level of Trf receptor was not changed after treatment K562 cells with various concentration of TNFa (10, 100 and 1000 U/ml) during 1 6 - 18 hours. In contrast, Trf receptor expression decreased after incubation of U937 cells with TNFa. Moreover, U937 and K562 cells were different in their sensitivity to cytotoxic effect of TNFa: K562 cells variability after treatment with 10,100 and 1000 U/ml were 100%, 87% and 80% whereas only 25%, 20% and 5% of U937 cells were viable, respectively. It was found also that TNFa is a negative regulator of K562 and U937 cells proliferation induced by transferrin, as determined by ~H-thymidine incorporation assay. The mechanism(s) of cytotoxicity of TNFa requires further study but we speculate now that this effect of TNFa can be achieved via modulation of Trf receptor expression. Thanks to flow cytometric analysis it was possible to demonstrate the modulation of surface receptor expression by different cytokins and growth factors.

Autoradiographic Study of (D-Ala2)-Deltorphin-I Binding Sites in the Rat Brain.

(D-Ala2)-Deltorphin I is a heptapeptide isolated (1) from methanolic extracts of the South American frog Phyllomedusa bicolor. Its amino acid primary sequence is: TYR-D-ALAPHE-ASP-VAL-VAL-GLY. NHv It displays a very high affinity and selectivity to the delta binding sites of opiod receptors. This activity is strictly linked both to the " D " configuration of the amino acid in position two and to the C-terminal tetrapeptide. In order to support these findings by morphological data, we performed autoradiography on rat brain sections. Coronal, sagittal and horizontal 20 um-thick cryostatic sections were incubated with tritiated heptapeptide and superimposed with (3H)-Hyperfilm (Amersham) for twelve weeks. Olfactory region and neostriatum displayed highest binging, followed by II-III and V-VI cortex layers, nucleus accumbens, amigdala, nucleus interpeduncularis, pontine nuclei and hyppocampus. (1) V. Erspamer et al., Proc. Natl. Acad. Sci. U.S.A. 86: 5188-5192, 1989.

P27.11

The Changes of Receptor for Con A on the Surface of Human Sperm during Fertilization.

P27.9 Flow Cytometric Analysis of Surface Transferrin (Trf) Receptors in Certain Human Tumor Cell Lines.

J. Sha, Z. Zhou, Z. Hu and S. Zhang Dept. of Histology, Nanjing Medical College, 2]0029 Nanjing, P. R. of China.

A. A. Phylchenkov, I. I. Slukvin* and Y. I. Kudryavets Institute of Experimental Pathology, Oncology & Radiobiology and *Kiev Institute of Pediatrics, Obstetrics & Gynecology, Kiev, The Ukraine.

Con A conjugated to colloidal gold was used to study the distribution of surface receptor for Con A on human sperm and to monitor the possible changes of its distribution during fertilization. The observation showed that 1. before fertilizing, the receptor for Con A on the surface of ejaculated

Trf receptor expression were studied using flow cytometry

626 sperm was totally blocked by coating substances supplied by the accessory glands. 2. the surface both the inner and outer acrosomal membrane were labelled with colloidal gold all the times. 3. during acrosome reaction, it expose the receptor for Con A that the changes of structure of the plasma membrans in the postacrosomal region. It is suggested that the receptor for Con A has an important role in fertilization.

P27.12

Immunohistochemical Evidence that the Majority of Placental Insulin Receptors is Located on Endothelial Cells. M. Hartmann, A. Blaschitz, G. Siwetz, G. Dohr and G. Desoye Depts of Histology and Embryology, and Obstetrics and Gynaecology, University of Graz, Austria. It has been known for a long time that the human placenta is a rich source of insulin receptors (IR). The only histologic study to date found non-uniformly grouped IR on the surface of the syncytiotrophoblasts (Nelson et al, Diabetes 27, 530, 1978). However, no data have been reported about any possible expression of IR on other cell types in the placenta. Therefore, we were prompted to re-investigate the localization of IR on cryosections of human non-pathologic full term placentae by indirect immunofluorescence. Two monoclonal anti-IRantibodies against the extracellular (Clone B6, Immunotech, MarseiUe: Clone MA20, Amersham) and one anti IR-mab (Clone 29B4, Oncogene Sci, USA) against the internal domain of the receptor were used. In the villi some discontinued areas of the microvillous membrane of the syncytiotrophoblast were very weakly stained. In contrast, the endothelial cells (EC) of the capillaries in the terminal villi were strongly decorated, both on the cellular surface and in the cytoplasm. The same staining pattern was observed with the EC of the vessels in intermediate and stem villi and in the chorionic plate. However, the EC of arteries and veins in the umbilical cord did not react with either antibody, whereas they showed strong staining with a monoclonal anti-van WiUebrand-factor antibody (clone 4F9, Immunotech, Marseille) which served as a positive control. The data indicate: 1) The syncytiotrophoblasts of human term placentae express, if any, a very low level of surface IR, 2) the majority of IR in human term placentae is located in EC of the micro-circulation, and 3) macrovascular EC do not contain an amount of IR detectable by our immunohistochemical techniques. Our data might support the concept of two functionally different IR in human term placentae. Furthermore, the well-known differences between EC of small and large vessels with regard to antigen expression was also observed in the present study.

P27.13 The Mechanism Underlying IGF-1 NonResponsiveness during Experimentally Induced Arthritis: IGF-1 Receptor Expression In Sitn. P. J. Verschure, L. A. B. Joosten and W. B. van den Berg Dept o f Rheumatology, University Hospital Nijmegen, The Netherlands. IGF-I is the main anabolic factor for proteoglycan [PG] synthesis in articular cartilage. Chondrocytes of the intact

Poster sessions murine patella respond to physiological concentrations of IGF-I by enhancement of the PG synthesis. However, during experimentally induced arthritis, chondrocytes exhibit a markedly inhibited PG synthesis and cannot be stimulated by IGF-I. This non-responsiveness does not implicate a general inhibition of the chondrocyte metabolism. The mechanism underlying this phenomenon could be the appearance of suppressive factors overruling the IGF-I stimulation. Alternatively, there might be a lack of anabolic signaling caused by receptor malfunction or receptor expression defect. We immunohistochemically examined the IGF-1 receptor expression in situ of normal murine articular cartilage compared to arthritic cartilage by light microscopy [LM] and confocal scanning laser microscopy [CSLM]. CSLM offers two advantages above conventional microscopy i.e., large reduction of out-of-focus signal detection and optical sectioning of the specimen. 6/am cryostat sections of the mouse articular cartilage were stained with IGF-1 receptor monoclonal antibody [aiR-3] or with biotinylated IGF-1 and as secondary signal peroxidase- or fluorescence-conjugated antibody. For CSLM 1/am optical sections were scanned and the fluorescence intensity signal from the chondrocyte membrane and chondrocyte inner part of control versus arthritic cartilage were quantitatively compared. LM immunohistochemical staining of the IGF-1 receptor with biotinylated IGF-I showed intracellular as well as membrane staining of the patellar articular cartilage chondrocytes, whereas the cartilage matrix remained negative. aiR-3 demonstrated a more intense granular staining. No striking differences in staining pattern of the receptor on normal versus arthritic cartilage could be observed. CSLM confrmed the LM findings; no reduction in IGF-1 receptor expression on control versus arthritic cartilage could be qualitatively viewed or quantitatively measured. This indicates that IGF-1 nonresponsiveness is not related to highly diminished receptor expression. Studies to investigate a potentiated disturbance in signal transduction measured by specific phosphorylation patterns, are in progress.

P27.14 Immunohistochemicai Demonstration of Growth Hormone Receptors (GH-R) in Stromal Cells from Long-Term Human Bone Marrow Cultures (LTBMC). D. T. Lincoln, W. Breipohl and A. Wegener Department of Pathology, Royal Brisbane Hospital, Herston, Australia and The Institute of Experimental Ohthalmology, University of Bonn, Germany. Studies on antigen-defined subpopulations of bone marrow stromal ceils have shown that the adherent fibroblastoid cells obtained from LTBMC are different from those of nonhaemopoietic organs (Sullivan et al., 1989, Lab. Invest., 60, 667) and leukocyte common antigen has been detected on fbroblast of marrow but not of skin, whilst Thy-1 antigen is present on fibroblast from skin, but not from marrow (Ploemacher et al., 1984, Blood Cells, 10, 341). Growth Hormone (GH) is mitogenic for the major cellular stromal elements consistig of fibroblast, adipocytes, macrophages, endothelial cells and acts directly on erythroblast and erythroleukemia cells (Merchav et al., 1988, Br. J. Haematol., 70, 267). A local action of GH requires the presence of GH-R,

Poster sessions which hitherto have not been demonstrated in human cells from LTBMC. Using a panel of well defined monoclonal antibodies (MAb's) reactive against the rat, rabbit and human GH-R (Lincoln et al., 1990, Acta histochem., Suppl. 40, 47), this study found immunohistochemical evidence on the presence and localisation of binding sites of GH in the cell membrane and extra-nuclear Golgi area of LTBMC derived stromal cells. GH-R immunoreactivity was present in small proliferating progenitor cells, large reticular fibroblast, adipocytes and endothelial cells. Only MAb known to be reactive against human tissue resulted in strong immunoreactivity. Hyperthermia treatment and addition of MAb against GH-R to the culture medium resulted in reduction of GH-R expression and number of cells respectively, while addition of 10% haemopoietic growth factor containing conditioned medium increased initial colony formation.

627 Omission of hycrocortisone and/or horse serum, supplementary addition of interleukin-3 or cytokines translated into differences in cell number, size and altered the adhesive properties of stromal cell layers. In addition to GH-R expression, enzyme- and immunohistochemical reactions were used to define the composition of the stromal marrow cells. The expression of GH-R, not only on small proliferating but also on the well differentiated cells, indicates a role for GH on non-progenitor cells. GH-R immunoreactivity on differentiating and/or differentiated cells suggests that GH is also necessary for, or has a trophic function in differentiation. We propose that direct GH action is necessary not only for differentiation of progenitor cells as implied by the dual effector hypothesis (Green et al., 1985, Different. 29, 195), but also for their subsequent clonal expansion, differentiation and maintainence.

P28. C E L L U L A R T R A N S P O R T M E C H A N I S M S P28,1 Application of Immunoelectronmicroscopy for the Study of lntracellular Processing of Progastrin Using Transformed Rat Cultured Cells and Different Antibodies. Y. Itoh, R. Shinoda, R. Y. Osamura, K. Watanabe and T. Takeuchi Department of Pathology Tokai University School of Medicine and Institute of Endocrinology Gunma University, Isehara Kanagawa 259-11, Japan. Gastrin is known to be processed by proteolytic enzyme and amidated at C-terminus. This immunoelectron microscopic studies are aimed at to elucidate the site of processing by using two different antibodies recognizing progastrin (Ab 8201) and processed form (Ab 8237) on rat cultured GH3 cells transformed by human gastrin cDNA. GH3 cells are the rat pituitary tumor cells containing secretory granules (SG). Immunoetectron microscopy pre-embedding method was performed on the cells fixed by 4~ paraformaldehyde. As results, Ab 8201 showed the presence of progastrin in perinuclear space (PNS), rough endoplasmic reticulum (RER). Ab 8237 showed of processed gastrin fragment in not only RER, PNS but also in SG. These results suggest that immunoelectron microscopy is useful in elucidation of the sites of processing, i.e., SG and probably PNS and RER.

P28.2 The Relationship Between Lysosomophagy and Receptor-Mediated Endocytosis (RME) of Colloidal G o l d Labelled Concanavalin A (ConA-Au). Y.-Z Piao, W. Hu, L.-P. Liu, S.-Q. Luo and L. Ren Central Laboratory, The First Medical College of PLA, Guangzhou, 510515, People's Republic o f China. We studied the lysosomophagy in macrophages (Md) after the RME of Con A. The Con A was labelled with colloidal gold as probe, then was injected into the peritoneal cavity of mouse. At intervals ranging from 5 - 12 rain after injection, the peritoneal Mr5 were isolated and observed. For control, the animals were injected i.p. with pure colloidal gold solution

(Au). When the M~ of control group were incubated with Au in vivo for 5 - 120 rain, only a few Au particles were seen on the cell surface or in the vesicles of the cell. The M~ were incubated with Con A-Au in vivo. After 5 rain, a large number of Con A-Au particles were endocytosed by M~ into vesicles. After 10 min, the Con A-Au particles were seen in the endosomes in which the ligand dissociated receptors. After 20 min, the number of lysosomes increased and lysosomes were heavily labelled. These excessive lysosomes were degraded by the other lysosomes (lysosomophagy) and this process has been named "lysosomal wrapping mechanism" (LWM). Some Au particles could be seen in the wrapping and wrapped lysosomes, which involved in the process of lysosomophagy. The facts indicate that lysosomphagy occurs after RME, and in the process of lyaosomophagy the secondary lysosomes play an important role. After 30 rain, the lysosomophagic vacuoles gradually decreased and after 6 0 - 120 min, LWM was not observed. We infer that factors which can induce excessive lysosomes in Md will lead to lysosomophagy.

P28.3 The Effects of High Temperature on ReceptorMediated Endocytosis (RME) in Cultured Cells. L. Ren, W. Chang, Y.-J. Piao, S.-Q. Luo and H.-C. Xiao Department of Pathology, Air Force Medical College, Jilin, 132011, People's Republic of China. It was the purpose of this study that, in high temperature, the ultrastructural changes of the binding, interatization and intracellular routing of colloid gold labelled Con A (Con AAu) and the surface receptors of rat peritoneal macrophages (Mt) cultured were observed with transmission electron microscope. The rat peritoneal M~ were collected and incubated with Con A-au in different temperature (39~176 fixed in different intervals of time (5 m i n - 1 2 0 min) and prepared for electron microscopical observation as routine. For control, the animals were divided into two groups which were injected with Con A-Au (group A) and pure colloidal gold solution (group B), respectively. Many Con A-Au of group A binded to the receptors on the

628 cell membrane, a few of the ligands entered into the cells only after 5 min incubation in vivo. A large number of Con A-Au particles were endocytosed by M~ through coated vesicles, uncoated vesicles and small tubules 10 rain later. Con A-Au mainly presented in the endosomes after 20 min. Few colloid gold particles were endocytosed by Mt~ in group B even in 120 rain. There weren't distinguish between experimental groups in 3 9 - 4 0 ~ and group A. The changes of RME were unmarked at 41~ RME was inhibited slightly in 43~ M~ pseudopods decreased. RME was inhibited markedly at 45~ the non-pseudopods and spherical Mt~ were appeared. Few Con A-Au were endocytosed, it was similar to group B besides the cells injured. The lysosomophagy could happen below 41~

P28.4 Comparative Analysis of Intracellular Traffic of Mannose-6-Phosphate Receptor in Ras-Transformed and Control HBL100 Cells. S. A. Rudchenko, N. Sdiqui and A.-C. Roche Centre de Biophysique Moldculaire, CNRS, 1, rue Haute, 45071 Orlgans, France.

The transfection of HBL100 cells by the E J/T24 Harvey-ras oncogene induces their tumorigenic conversion which leads to the modification of cell morphology in culture as well as capability to induce tumors in athymic mice [Lebeau J. et al. (1991) Oncogene, 6, 1125- 1132]. Using confocal microscopy and flow cytometry we compared the mannose-6-phosphate (Man6P) receptor-mediated uptake and the intracellular traffic of fluorescein-labelled Man6P-substituted bovine serum albumin (F-Man6P-BSA) in ras-transformed and control HBL100 cells. The specific uptake of F-Man6P-BSA was more effective in ras-HBLtO0 cells and the main part of the ligand was partly colocalized with Texas-red-labelled wheat germ agglutinin which labels preferentially the Golgi apparatus. With control HBL100 cells, Texas-red-labelled wheat germ agglutinin gave roughly the same labelling when cells were in exponential growth, but a very faint labelling when cells were confluent: F-Man6P-BSA appeared as spots remaining in vesicles such as endosomes or lysosomes.

P28.5 A Distribution of Anion Sites on the Cell Membrane and Liposome-Cell Interaction. M. J. Velinova Department of Anatomy, Histology and Embryology, Medical University of Sofia, boul. G. Sofiiski No 1 1431 Sofia and Christo L Schoilew Central Veterinary Research Institute Sofia 1606 boul. P, Slaveikov 15 A Bulgaria.

Liposomes with different phospholipid composition and three cellular models were used: alveolar macrophages, lymphocyte and tumor cells obtained from lymphoid leukemia L 1210. Aiming to characterize the distribution of negative charged sites on the membranes of cellular models we used Polyethyleneimine mono and bimetal complexes as an electronically dense markers. A relation between the distribution of the anion sites on the surface of the cellular membranes and the penetration of the liposomes was found. In the areas of firmly adsorbed liposomes the electronically

Poster sessions dense marker was not observed. Liposomes in contact with the nuclear membrane as well as liposomes in the nuclei of lymphocytes and tumor cells can be seen. The liposomes' penetrations in lymphocyte is mainly by fusion with the plasma membrane with a consequent disintegration and pooring of the liposomal content in the cytoplasm and the nucleus of the cell. For the macrophages the dominant mechanism of penetration was endocytosis, but in single cases fusion with the plasma membrane was noticed. In the tumor cells all the mechanisms mentioned are taking place.

P28.6 Cytochemicai Demonstration of Acid Phosphatase Activity in Alveolar Macrophages and Tumor Cells Incubated with Liposomes. M. J. Velinova and W. A. Ovtscharoff Department of Anatomy, Histology and Embryology, Medical University of Sofia 1431 boul. G. Sofiiski No 1 Bulgaria and Christo L Schoitew Central Veterinary Research Institute, Sofia boul. P. Slaveikov 15 A 1606 Sofia Bulgaria.

In our study alveolar macrophages obtained by lavage from bronchoalveolar system of guinea pigs, tumor cells from lymphoid leukemia L 1210 and liposomes with different phospholipid compositions were used. After the incubation of the liposomes with the both types of studied cells, for the first time cytochemical method for demonstration of acid phosphatase was employed. A reaction product was observed in phagolysosomes, containing liposomes in a process of degradation. The same positive reaction was revealed in the Golgi complex. A fusion between phagosomes (with included liposomes) and lysosomes with a positive reaction for an acid phosphatase was observed. Keeping in mind these data we suggest that the liposomes entering via endocytosis or fusion could be digested by the lysosomes.

P28.7 Ultrastructural Aspect of Protein Absorption by Enterocytes. K. A. Zufarov and A. Y. Yuldashev Histology Department, I-st Med. Institute, Tashkent, Uzbekistan.

Proteins are transported by endocytosis from the intestinal lumen into enterocytes. This process is considered to be the selective concentrating mechanism being mediated by receptors and differing from other types of transport by its higher rate. Membranes of the endocytotic vesicles are fused with membranes of the Golgi complex structures within the enterocytes. Process of the exogenous proteins endocytosis is followed by enhanced formation of primary lysosomes in which the proteins are then depleted partly. It takes about three hours for the protein absorption, its transporting to the Golgi vesicles and outloading into the intercellular space. During six hours the absorbed proteins are transported from the dialated intercellular spaces to interstitia and then to lumina of the lymhatic vessels of the villi stroma. Thus, protein absorption by villi enterocytes is complex and many-staged process and is provided by cyclic functioning of the cytoplasmic structures.