In the present report we examine the composition and organization of the heterochromatic fraction of the genome, making use of cytochemical techniques, in a few species of primates of the genus Eulemur, a homogeneous group of lemuroidea from Madagascar. Among the species examined, Eulemur coronatus shows large blocks of heterochromatin distributed in nearly all the large metacentric chromosomes, in the large and small acrocentrics and in the microchromosomes. According to our measurements, such heterochromatin represents about 35% of the genome of the species, probably originated through amplification processes after the splitting off of the species from the common trunk. It must be recalled that in the genus, Robertsonian fusion represented the major mechanism acting in chromosome evolution. E. coronatus heterochromatin shows peculiar FRET (fluorescence resonance energy transfer) characteristics both before and after dehistonisation protracted to the destruction of the nucleosomal structure. Its characteristic FRET pattern differs greatly from that of E. macaco, supposed to have a longer history of evolution. Furthermore, quantitative histochemical analysis and imaging measurements highlighted a peculiar structure of the E. coronatus heterochromatin. Finally, the comparison of the data obtained with in situ hybridization, with probes like (TTAGGG) n and with probes obtained after genomic digestion with some restriction enzymes (Bam HI, Hae III, Pvu II) on E. coronatus, macaco, rubriventer and fulvus chromosomes clearly showed that E. coronatus heterochromatin has a more heterogeneous composition than the other species. Overall the data obtained lead us to reconsider heterochromatin in terms of its potential evolutionary plasticity.

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Different lectins conjugated either to horseradish peroxides (HRP) or fluorescein isothiocyanate (FITC) were used to detect sugar residues of the epithelial glycoprotein in the gastric mucosa of the one humped camel (Camelus dromedaries). All mucus secreting cells were rich in oligosaccharides with terminal galactose (Gal), N-acetylgalactosamine (GaINAc), N-acetylglucosamine (GIcNAc), and neuraminic acid residues while α-L-fucose (a-L-Fuc.) could not be localized by FITC- conjugated UEA-I (Ulex europeus agglutinin-l). Surface mucous cells (SMC) and foveolar mucous cells (FMC) were devoid of α-D-glucose (α-D-Glc.) and α-D-mannose (aD-Man.), although glandular mucous cells (GMC) were rich in these sugars. Sialic acid with mono or di-0-acetyl substitution at carbons 7, 8, 9 of their polyhydroxyl side chain was the predominant form of the neuraminic acid.

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In situ carbohydrate binding of the secretory granules in 3 cell types from rabbit submandibular glands (acini, granular tubules and intercalary ducts) with labeled lectins has been used to provide markers for the secretory changes that can be induced in the cells by prolonged parasympathetic or sympathetic nerve stimulation. This work has confirmed that parasympathetic stimulation causes exocytosis from acini and granular tubules and sympathetic stimulation causes a less extensive degranulation of acini without any apparent change to granular tubules. These studies have also demonstrated that granules in the intercalary ducts are more clearly revealed by lectin bindings than by more conventional histological methods used previously. This has shown that neither parasympathetic nor sympathetic impulses induce discernible exocytosis of granules from intercalary ducts, so it is a question as to whether they are secreted more gradually during spontaneous secretion from the gland, as part of an ongoing process, independent of neural drive. The lectin histochemistry also revealed that, after parasympathetic stimulation, there was an accentuation of Golgi-like staining in acinar cells, which suggests that replenishment of the secretory glycoproteins had already begun by means of protein synthesis and glycosylation. Despite some loss of acinar secretory material with sympathetic stimulation, no corresponding accentuation of Golgi-like staining was seen at that time.

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The atrial specific granule has been described as an intracellular Ca store (ICaS). It is reasonably proposed that other polypeptidesecretory granules could also play the function of ICaS. In the present work, high Ca concentration in the dense granule (dG) and α granule (αG) of platelets was confirmed by using x-ray microanalysis in both ultrathin cryosections and ethanolic phosphotungstic acid staining sections, and Ca²⁺-ATPase activities were found on the membrane of both the granules. Furthermore, when platelets were activated by A 23187 1 mM, thrombin 2 U/ml or ADP 200μM, both the Ca content and number of the granules decreased, which means that the Ca²⁺ was released and secretion of the granules increased. It is suggested that the release Ca²⁺ raise the cytosolic Ca2+ level and, thus, the secretion of the granules is promoted. In addition, it was described that the ultrastructure of the platelet granules in ultrathin cryosections was somewhat different from that in routine sections.

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The expression of Novel Ca2+-binding pro teins S100A1, S100A2, S100A4, S100A6 and S100B; an intermediate filament protein, glial fibrillary acidic protein (GFAP); and an extracellular matrix glycoprotein, tenascin were evaluated in chondro-osseous tumors. The tumor specimens obtained from surgery and routinely processed for paraffin embedding were evaluated. The tumors were histologically diagnosed as chondroma (n=2), osteoma (n=2), chondrosarcoma (n=3), osteosarcoma (n=6) and Ewing's sarcoma (n=2). Chondrocytes or chondrometaplastic cells in both benign and malignant tumors as well as undifferentiated round, spindle and elongated cells in chondrosarcoma and osteosarcoma showed an intense immunoreactivity for S100B and occasionally for S100A6, S100A1 and S100A4 but not for S100A2. The intensity of immunostaining for osteosarcoma was less intense than that for chondrosarcoma. S100B reactive cells were also reactive for GFAP, although the intensity of staining was less intense for GFAP. Reaction products for tenascin was seen in the matrix of neoplastic cartilage in chondroma and chondrosarcoma, and uncalcified osseous matrix in osteoma and osteosar coma, and the immunoreactive areas of tenascin usually coincided with intensely reactive cells for S100B in neoplastic chondroid tissue. The results reasonably allowed to conclude that S100B may be a potential marker for the identification of chondroid cells in neoplastic lesions and the S100B containing cells may express GFAP and may be associated with an enhanced expression of tenascin in the tumor matrix, the functional significance of which is under investigation.

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Mast cells which show metachromasia with toluidine blue were observed in newt (Cynops pyrrhogaster) lung connective tissue. The mast cells showed strange granules in their cytoplasm. In this study, we examined the characteristics of the mast cells by using histochemical and cytochemical methods.
The mast cells were positive for PAS reaction and Alcian blue (pH 1.0). Electron microscopy showed that the mast cells stretched their cytoplasm into the collagen layer. The cytoplasm was filled with various types of granules, some homogeneous throughout, others heterogeneous, and were classified as follows: high and low electron density amorphous structure, straight lamella structure, and rough particle structure. These granules were irregular in size. They were stained by alkaline bismuth and dialyzed iron. The rough particle structure granules in particular showed stronger staining than others.
In order to determine which sulfated polysaccharide was contained in the granules, we performed enzyme treatment using two enzymes chondroitinase ABC (pH 8.0) and heparinase. Our results indicated that the granules contained heparin.

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In the present study we first investigated the changes in protein synthesis of respective cell types of mouse pulmonary alveoli from 16 days of fetal life to senility (postnatal 22 months) by using ³H-leucine light microscopic radioautography. The results revealed that the protein syntheses in type I epithelial cells, type II epithelial cells, interstitial cells and endothelial cells in the mouse lung changed due to aging. The protein synthesis of type I epithelial cells was the highest on postnatal day 1, increased again at 1 week after birth, and then decreased gradually to 22 months after birth with aging. The protein syntheses of type II epithelial cells, interstitial cells and endothelial cells reached the highest level on fetal day 16, declined progressively with age, and increased again at postnatal 1 week, and then decreased gradually from 2 weeks to senility. Our results suggest that the decreases in protein synthesis are correlated with the decremental changes in DNA and RNA synthesis in the lung with aging.

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In the present study, the histologic distribution of glucagon receptors in the mouse was studied by in vivo whole body radioautography. Male mice were injected intravenously with ¹²⁵l-labeled glucagon in the absence of and, in the presence of, excess unlabeled glucagon. Three, 6, 15 and 30 min after injection, the animals were perfused and subjected to radioautographic procedures. The results demonstrated that only the liver had a very high level of specific glucagon binding. Low level of specific glucagon binding was observed in the stomach, small intestine, heart, kidney cortex, adipose tissue and spleen. These quantitative results from in vivo whole body radioautography did not always correspond to those from the quantification of mRNA expression. This seems to support a suggestion that glucagon receptor expression is modulated at a step after mRNA formation. High level of non-specific glucagon binding was observed in the kidney, thyroid, sublingual and submandibular glands and seminal vesicle.

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The expression of human epithelial related antigen (hERA), a 40 kD transmembrane glycoprotein assigned to one of the small cell lung cancer antigen was evaluated in normal salivary glands and their tumors using immunohistochemical methods.
In normal salivary gland acini, hERA was seen in lateral borders and basal plasma membrane of serous cells of the parotid and submandibular glands and in the basal layer of mucous cells of the sublingual and minor oral glands, the luminally projecting cell membrane being consistently unreactive. In ductal segments, hERA was seen in basal infoldings and lateral borders of striated duct cells, excretory ducts or interlobular ducts and the ductal basal cells in particular, were devoid of hERA immunostaining.
Salivary pleomorphic adenoma showed luminal borders of tubulo-ductal structures immunoreactive for hERA whereas the nonluminal or the neoplastic myoepithelial cells were consistently unreactive. A similar pattern of immunoreactivity was observed in Warthin's tumor where lateral borders of luminally located tall columnar tumor cells showed reaction products whereas the basal cells were unreactive. Mucoepidermoid carcinoma showed hERA in epidermoid and intermediate tumor cells, but not in mucous secreting cells. In adenoid cystic carcinoma reaction product for hERA was seen in a limited number of lumen-lining cells and again the non-luminally located neoplastic myoepithelial cells were unreactive. The results of the present study suggest that hERA may be a useful marker to detect luminal cell components in salivary tumor and, apart from the neoplastic myoepithelial cells or their counterparts, these luminally located cells may have a potential implication in salivary gland tumorigenesis.

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The established human histiocytic lymphoma U-937 cell line is of monocytic origin. U-937 cells can be induced by tumor promoters, Vitamin D₃ and retinoic acid to differentiate into macrophage-like cells. U-937 cells express the gene for the cysteine proteinase inhibitor cystatin C. This was established by Northern blot hybridizations, reverse transcriptase-polymerase chain reaction and Western blots of partially purified cystatins from extracts of U-937 cells. Induction of macrophage-like differentiation by the tumor promoters phorbol-12-myristate-13-acetate or 12-O-tetradecanoylphorbol-13-acetate, or with Vitamin D₃, or retinoic acid reduced the steady-state level of cystatin C mRNA in U-937 cells, suggesting that the expression of the cystatin C gene varies with the differentiation state. U-937 cells secrete cystatin C. This secretion was not affected significantly by the calcium ionophore A23187, PMA, or dibutyryl-adenosine 3′: 5′-cyclic monophosphate. Since macrophages play pivotal roles in immune and inflammatory reactions involving intra- and extra-cellular proteolysis, cystatin C produced and secreted by these cells probably has a modulatory function on the action of cysteine proteinases produced or released locally.

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Membrane-bound forms of β1-4 galactosyltransferase (GalTase) are distributed in trans Golgi cisternae and plasma membrane, while a soluble form of the enzyme also exists in extracellular fluid. In epididymis, the soluble form has been found in luminal fluid and suggested to play a role on sperm maturation. In the present paper, regional distributions of the membrane-bound and soluble forms of GalTase were studied in rat epididymis. Immunohistochemical studies showed that a very high concentration of GalTase was localized in Golgi apparatuses of epididymal duct epithelium from initial segment to intermediate caput, although much lower amounts of GalTase were in efferent ducts, distal caput, corpus, and cauda. The same distribution pattern was also shown by biochemical assays of the microsomal GalTase activity. On the other hand, assays of the soluble GalTase activity in epididymal fluids revealed a relatively uniform distribution pattern. Even in initial segment and caput epididymis, only low levels of the activity were detected in the fluid.

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Effect of fixative concentration, fixation time, embedding media, condition by dehydration and embedding, and incubation time with primary antibody upon the labeling intensity, was investigated by quantitative immunoelectron microscopy. Rat liver tissue and erythrocytes processed under various conditions of preparation were embedded in LR White or Lowicryl K4M. As concentration of glutaraldehyde (GA) in fixative increased, labeling density decreased. In the tissue and cells fixed with fixative containing 4% paraformaldehyde and 0.25% GA for 5-10min, cell ultrastructure and antigenicity were well preserved. As fixation was prolonged, labeling density was lowered. In the materials embedded in acrylic resins, such as LR White and Lowicryl K4M, high labeling density was obtained, compared with that obtained from embedding in epoxy resins such as Epon 812, Durcupan and Quetol 651. Temperature difference between dehydration and embedding affected strongly the preservation of antigenicity. There was no difference in labeling densities between embedding at 23°C and -20°C, when tissue slices were dehydrated at 4°C. To saturate the reaction of primary antibody with antigen on thin sections incubation should be carried out over 8hr. From these results, we recommend the following points in immunoelectron microscopy; 1) perfusion fixation with 4% paraformaldehyde+0.25% GA for 5min to 10min, 2) minimized temperature difference between dehydration and embedding of tissue, 3) use of acrylic resins for embedding, and 4) over 8 hr-incubation with primary antibody.

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The cellular and spatial distribution of the presumptive retrograde transmitter nitric oxide, detected by NADPH-diaphorase histochemistry and of immunocytochemically labeled monoaminergic fiber systems was compared in regions of the medial prefrontal cortex of the precocious rodent Octodon degus. The staining patterns at two postnatal stages (PO and P14) were compared to those found in adult animals (P90). At birth, NADPH-diaphorase positive neurons were found in all cortical layers of the anterior cingulate, infralimbic and prelimbic cortex. During postnatal development the number of diaphorase-positive cells gradually decreased in layers II-VI and remained unchanged in layer I of these cortical regions. All NADPH-diaphorase positive neurons appeared to be spineless and pyramidal cells never contained NADPH-diaphorase. No colocalization of NADPH-diaphorase with either serotonin or TH was detectable. However, at all developmental stages the somata and proximal dendritic shafts of some of the NADPH-diaphorase containing bi-or multipolar neurons in layers V-VI were contacted by serotonin-and tyrosine hydroxylase-immunoreactive fibers. In a subpopulation of GABAergic interneurons in the deeper layers (V-VI) NADPH-diaphorase activity was colocalized with calbindin-D 28 k immunoreactivity.
The abundance of nitridergic systems in regions of the medial prefrontal cortex already at birth together with their close spatial relationship with monoaminergic afferent systems as well as local GABAergic units may indicate a yet to determine role of these transmitter systems in early learning.

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The activities of membrane-bound proteases: γ-glutamyltranspeptidase (γ-GTP), microsomal alanyl aminopeptidase (mAAP, formerly APM), glutamyl-aminopeptidase (EAP, formerly APA) and dipeptidyl peptidase IV (DPP-IV) -were examined in 11 cases with non-Hodgkin's malignant lymphomas (NHL), in 7 cases with Hodgkin's disease (HD) and 8 cases with reactive follicular hyperplasia (RFH) by means of enzyme histochemical methods. The results indicated that there was correlation between grade malignancy of NHL and γ-GTP activity: high grade NHL displayed strong γ-GTP activity, whereas low grade NHL showed weak activity. HD also showed various patterns of γ-GTP activity. Of 7 HD examined, 4 showed strong γ-GTP activity and 3 other were slightly positive. The lymphoid cells in RFH and neoplastic cells in NHL and HD failed to exhibit mAAP- and EAP activities. The activities of these enzymes were seen in stromal cells. Variable numbers of small mature lymphocytes in RFH and in malignant lymphomas showed dot-like DPP-IV activity. Thus, the study of γ-GTP activity may be a valuable diagnostic feature in the distinction between NHL of low and high grade malignancy and may prove to be a useful marker with which to predict drug- and irradiation resistance. Moreover, mAAP- and EAP pattern in the lymphoid cells may be useful in defining in lymph nodes the metastatic tumour cells with strong activities of these enzymes.

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In the present study, interactions between lutenizing hormone releasing hormone (LHRH) -like immunoreactive (LHRH-LI) nerve endings and processes of tanycytes and/or astrocytes in the median eminence (ME) particularly its external layer were investigated by double labeling immunoelectron microscopy employing antibodies against glial fibrillary acidic protein (GFAP) and LHRH. GFAP-like immunoreactivity was observed in some tanycytes throughout the ME and/or astrocytes in the subependymal layer of the ME. In the palisade layer of the ME, GFAP-like immunoreactive (GFAP-LI) elements were frequently seen to make intimate contact with LHRH-LI nerve endings. LHRH-LI nerve endings and processes of the tanycytes and/or astrocytes were also found to direct contact with the basement membrane of the pericapillary space. And they were partially engulfed each other in the vicinity of the perivascular region. The above findings strongly suggest that the processes of tanycytes and/or astrocytes with intimate communication of cerebrospinal fluid in the third ventricle may play a role in regulation of secretion of LHRH into the portal vessels.

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L-Arg as a precursor of nitric oxide (NO) can be used in experiments to observe the action of NO. The present work is aimed at exploring the effect of oral administration of L-Arg on the ischemia-reperfusion (IR) damage of endothelial cells (EC) and the clearance of reaction oxygen species in the IR carotid artery. Cerium-marked cytochemical technique was used to localize the H₂O₂ product in endothelium. The area of Ce-H₂O₂ denseprecipitate on the lumen surface of damaged endothelium per length of artery wall (nm2/nm) was measured. It was found that the dense precipitate was decreased to 102.1±27.6 in rats drinking tap water containing 2.5% L-arginine for 3 days, much less than in control rats drinking tap water only (203.7±31.8, p<0.01). Furthermore, the IR damage of EC was relieved markedly. Drinking tap water containing D-Arg or L-Lys had no effects either on the amount of dense precipitate or on the EC damage. N^G-nitro- L-arginine methyl ester, an inhibitor of NO synthase, decreased the effect of L-Arg markedly. These results indicate that L-Arg administration can reduce H₂O₂ production in the IR artery, and thus IR damage of EC is alleviated.

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Zinc ions have been demonstrated in the anterior pituitary of rat by autometallography (AMG) which involves in vivo exposure to sodium selenite leading to creation of zinc selenide crystal lattices that are later silver enhanced by AMG in the sections. In this study, autometallography is combined with immunohistochemistry in order to present the coexistence of zinc ions and hormones in endocrine cells of the rat pituitary. The results showed that the majority of zinc-enriched (ZEN) cells are somatotrophs. All the somatotrophs observed were loaded with AMG grains. Zinc ions were also found to be present in gonadotrophs, corticotrophs and thyrotrophs. A low density of AMG silver grains was found in about 60% of gonadotrophs, 8% of corticotrophs and less than 10% of thyrotrophs. Lactotrophs did not appear to contain zinc ions. The present observations may provide a clue to the physiological function of zinc ions in the rat pituitary.

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We studied the intracellular dynamics of alkaline phosphatase (ALPase) -containing granules in human neutrophils exposed to a phosphotyrosine phosphatase inhibitor, phenylarsine oxide (PAO). Unstimulated neutrophils showed ALPase activity in short rod-shaped intracellular compartments distributed dispersedly throughout the cytoplasm. Enzyme activity was observed in elongated tubular structures and vacuoles in cells stimulated with a protein kinase C activator, phorbol myristate acetate (PMA). When cells were exposed to PAO followed by stimulation with PMA, the enzyme activity was visualized in intracellular compartments, localized in the pericytoplasm, which were associated with the plasma membrane. A tyrosine kinase inhibitor, erbstatin, prevented this re-location of ALPase-containing granules. PAO also prevented the association of ALPase-containing granules with the limiting membrane of phagosomes containing IgG-coated latex beads. Biochemical studies demonstrated that the up-regulation of ALPase activity was not affected by exposure to PAO in cells stimulated with PMA, but was inhibited by erbstatin in cells exposed to PAO followed by stimulation with PMA. The present findings indicate that both tyrosine phosphorylation and phosphotyrosine dephosphorylation are involved in the intracellular dynamics of ALPase-containing granules, and demonstrate that the dynamical alterations of these granules are affected by different stimuli such as PMA or IgG-coated latex beads.

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3, 3′, 5, 5′-tetramethylbenzidine polyvinylpyr-rolidone platinum (TMB-PVP-PT) reaction was moderately sensitive to horseradish peroxidase (HRP), and gave an improved image of the tracer localization. When HRP-injected mouse liver and submandibular gland specimens were treated with this reagent, a blue reaction product in fine granular shape was formed only at the sites where the tracer was localized, while no endogenous peroxidase activity was detected at all. Some attempt to enhance the sensitivity of this reagent to peroxidase was also made. This reaction was suitable for making permanent microscopic preparations.

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To examine the relationship between immunostaining intensity and antigen amount, serially diluted cell lysates from the liver or skeletal muscle of rats were immobilized on nitrocellulose (NC) filters. The filters were stained with antibody against cytochrome P-450 (P-450) 1A, P-450 2B, P-450 reductase, albumin, monosialoganglioside 1 (GM1) or myosin by the indirect immunoperoxidase method under saturation conditions, and processed for image analysis. There was a linear relationship between immunostaining intensity owing to and the amount of P-450 2B, P-450 reductase or myosin immobilized on the filters. However, the relationship was nonlinear for P-450 1A, albumin and GM1. Subsequently, the relationship between immunostaining intensity in sections and section thickness was examined by microphotometry. The relationship was linear for P-450 2B or P-450 reductase, but nonlinear for P-450 1A, albumin, myosin or GM1. The results indicate that immunostaining intensity resulting from P-450 2B or P-450 reductase in sections is proportional to the antigen amount in sections, whereas that from P-450 1A, albumin, myosin or GM1 is not proportional to the amount. Therefore, accurate quantitative information on P-450 2B or P-450 reductase in section is obtained by measuring immunostaining intensity in sections, while measurement of the intensity owing to P-450 1A, albumin, myosin or GM1 in sections will give erroneous quantitative results.

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Urinary trypsin inhibitor (UTI) is an acidic protein with anti-proteolytic activity that constitutes the active domain of serum inter-α-trypsin inhibitor. UTI has been demonstrated not only in the adult urine but also in the amniotic fluid of late pregnancy. In order to examine the expression and localization of UTI in the rat embryo and placenta, we performed a combination of reverse transcription-polymerase chain reaction (RT-PCR), In situ hybridization and immunohistochemistry. UTI mRNA expression was detected by RT-PCR in the embryo on and after the day 12 of gestation (E12) but not in the placenta throughout the prenatal period. In situ hybridization revealed that UTI transcripts were localized in the liver but not in any other organs of embryo from E12 through E20. On the other hand, UTI immunoreactivity was detected not only in the liver from E12 but also in the kidney and intestine from E18. On electron microscopy, the immunoreactivity of liver was localized to the surface of hepatocytes and the lysozomes of Kupffer cells, whereas the en-dothelial cells and hematopoietic cells were free of the reaction. In contrast, the immunonoreactivities of kidney and intestine were localized exclusively to the apical lysosomes of proximal tubular epithelial cells and absorptive epithelial cells, respectively. These results indicated that in the rat embryo, as in the adult, the hepatocyte is the only site for production of UTI, that UTI is excreted into the urine and thereby into the amniotic fluid, and that a part of amniotic UTI is reabsorbed in the fetal intestine, suggesting a possible role of UTI in the protection of the fetus during the late pregnancy.

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The three classes of alcohol dehydrogenase (ADH) in the rat which have been described at the protein level were subjected to qualitative and quantitative analysis by histochemical and microelectrophoretic means. Enzyme activity was studied in the epidermis, sebaceous glands, hair roots, stratum papillare, stratum reticulare and panniculus carnosus of the rat skin. Total ADH activity was histochemically demonstrable in all these structures. After electrophoretic separation of microdissected samples by a newly developed ultrathin-layer gel electro phoresis technique, no class I ADH activity was found in any of the above-mentioned structures, whereas class III ADH activity was ubiquitously detectable, although only at a faint level. Class IV ADH however, showed high activity in the epidermis and the sebaceous glands. Maximum activity could be attributed to the fibroblasts of the stratum papillare. The metabolic role of class IV ADH in the skin is discussed with respect to retinoic acid synthesis and the degradation of lipid peroxidation products.

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The present study was designed to establish a method for measuring cultured neuroglial gap junctional intercellular communication (GJIC) of rat cerebral cortices by fluorescence recovery after photobleaching (FRAP) analysis technique. As a result of the managements, such as cell culture, fluorescence staining, fluorescence excitement, laser scanning and computer analysis et al., fluorescence inside all selected cells recovered in different degrees, i. e., fluorescence relative intensities in the 1st-5th cell raised 14%, 28%, 43%, 17.5% and 12.5% respectively after scanning 15.4 min, the mean value of recovery was 18.57±10.06% and the mean rate of fluorescence recovery was 1.223±0.785%/min for three samples (n=14 cells). The result showed that the FRAP is an ideal method for measuring GJIC.

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The expression of intercellular junctions in developing ciliary epithelium of the rabbit eye was studied utilizing monoclonal antibodies to two major proteins comprising these junctions, connexin43, for gap or communicating junctions, and ZO-1, for tight or occluding junctions. The immunoreactivity of cx43 appears early, before distinct recognition of the differentiating ciliary body. The appearance of immunoreactivity to ZO-1 was strongly positive only in the later phases of gestation. These findings imply 1) that the gap junctions tend to solicit a cooperativity between the two cell layers of the ciliary epithelium during early development ensuring communication between the outer (blood side) and inner (eye side) two layers of this uniquely constructed ciliary epithelium, and 2) that the sequential development of the tight junctions to create a secretory gradient occurs when the eye no longer receives its fetal internal vascular supply and now requires a nourishing aqueous humor.

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Caveolae have been hypothesized to be involved in Ca²⁺ signaling. By electron microscopy, caveolae were observed to be apposed to the endoplasmic reticulum (ER), which is a major intracellular Ca²⁺ pool. In the present study, we examined the relationship between caveolae and the ER when the distribution of the latter was changed by depolymerization of microtubules. Double immunofluorescence microscopy for detection of caveolin and the ER antigens, and time-lapse observation of green fluorescent protein (GFP) -tagged caveolin were employed. In normal human fibroblasts and PtK2 cells, the ER was seen as a network extending throughout the cytoplasm, and most caveolin occurred in patches along the cell edge. When microtubules were depolymerized by Colcemid or nocodazole, the ER became retracted from the cell periphery and aggregated around the nucleus; in the same cells, caveolin was not seen along the cell edge, but was aligned along the edge of the retracted ER. By time-lapse analysis, GFPcaveolin expressed in PtK2 cells was observed to move from the cell periphery toward the cell center in Colcemid-treated cells. The result shows that the apposition of caveolae and the ER is maintained even after the gross distributional change, and suggests a mechanical linkage between the two organelles.

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We prepared a monoclonal antibody (mAb), IgM class, to rabbit muscle aldolase (ALD) A₄. The mAb reacted with the ALD A subunit but not with the B and C subunits. Distribution of the ALD A subunit was studied employing immunohistochemistry, with the ABC procedure in the rabbit tissues. The ALD A subunit was localized in the l-band of the skeletal muscle fibers. In the liver, endothelial cells showed strong immunoreactivity while parenchymal cells were negative or yielded only a faint reaction. The collecting tubules were positively stained in the kidney. Strong reactivity was seen in the proximal tubules of the outer medulla but not in those of the cortex. In the adrenal gland, cells in the cortex exhibited strong immunostaining, while medullary cells were negative. In the testis, most of the seminiferous epithelium was moderately immunoreactive. Intense staining was present in interstitial cells. The ALD A subunit was localized in the interstitial cells and follicular cells of primary follicles in the ovary. In the cerebellum, the ALD A subunit was localized exclusively in nerve fibers of the white matter; Purkinje cells and nerve cells in the granular layer were negative.

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To elucidate the steroid-synthesis in the mammalian brain (i. e., neurosteroid), we immunohistochemically studied various kinds of steroidogenic cytochrome P450 enzymes in the rat brain. The primary antibodies used in this study were rabbit polyclonal antibodies to cholesterol side-chain cleavage (SCC), C21-hydroxylase (C21), 11β-hydroxylase (11β), 17α-hydroxylase/C17-20 lyase (17α) and aromatase (AROM). The immunoreactivities for the microsome enzymes, C21 and 17α were located in the neuronal cell-bodies and proximal parts of their fibers, while mitochondria enzymes, SCC and 11β were located in the cell-bodies and their fibers and terminals. These immunoreactivities were distributed in the limbic structures of prosencephalon including hippocampus and amygdaloid complex, the hypothalamus including preoptic area, the cerebellar cortex, and some of other regions. All the sets of these enzymes did not always coexist in the identical cells, however, in the hypothalamus and cerebellum these enzymes are thought to work one after another in adjacent cells, forming the “steroidogenic cellular circuits”. These findings strongly suggest that the steroidsynthesis occurs in the neurons; and the neurosteroids exist in the mammalian brain.

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Human immunodeficiency virus type 1 (HIV1) Rev has been reported to act by inducing the nucleocytoplasmic transport of unspliced and singly spliced RNAs that encode viral structural proteins. However, our initial experiments indicated the cytoplasmic expression of intron-containing mRNAs in Rev^-cells [12]. To determine whether or not the post-transcriptional induction of gene expression by Rev extends to the level of increasing the efficiency of translation, Rev⁺ and Rev^-cells were morphologically examined by means of in situ hybridization and immunofluorescence assays. Hybridization to an intron-specific probe revealed that the transfection of HeLa cells with a rev-defective HIV-1 expression plasmid caused the export of overexpressed, unspliced gag mRNAs, possibly through a default process of nuclear retention. However, subsequent immunofluorescence assaying demonstrated that mRNA exported by Rev, but not mRNA directed through the default process, was translated into the corresponding protein. The findings were extended to a group of singly spliced viral mRNAs that produce Env in the following biochemical analyses. These findings suggest that Rev is directly or indirectly involved in the translational regulation of HIV-1 structural gene mRNAs. We discuss the possibility that Rev could cause the intron-containing transcripts to follow the appropriate pathway to reach the translational machinery.

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Localization of facilitated-diffusion glucose transporter GLUT1 was examined in the rat mammary gland by immunofluorescence microscopy and immunoperoxidase electron microscopy. Secretory epithelial cells of acini were enriched with GLUT1, which was localized to the basolateral plasma membrane. Apical plasma membrane and cytoplasmic organelles including the Golgi apparatus were negative for GLUT1. In addition to acini, abundant GLUT1 was found in the perineurium of nerve fiber bundles, which is a site of blood-nerve barriers. GLUT1 in the basolateral membrane of secretory epithelial cells seems to serve as machinery to take up glucose into the cell for milk synthesis and secretion.

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The present investigation was undertaken with the aim of studying the relationship between apoptosis and autophagy through observation of the alternations in splenic lymphocytes after reversible sublethal cell injury. For this purpose, splenic lymphocytes of rats which were irradiated with γ-ray (total 800 rads), were studied by histochemistry and electron microscopy. After γ-ray irradiation, a large number of splenic lymphocytes developed apoptosis which was classified into two types, i. e. classical apoptosis or apoptosis and autophagic apoptosis. Ultra-structurally, classical apoptosis of lymphocyte assumed the aspects characterized by com-paction of nuclear chromatin and formation of cell fragments. 30-40 min after irradiation, classical apoptosis of lymphocytes was observed in spleen, and became most prominent 2-5hr after irradiation. 3-5hr later, most of apoptotic lymphocytes were phago-cytized by macrophages. Autophagic apoptosis was characterized by the formation of numerous autophagic vacuoles (autophagosomes). Early autophagosomes containing identifiable organelles e. g. mitochondria, ribosomes, smooth and rough endoplasmic reticulum were visible 2-24hr after γ-ray irradiation. The autophagosomes that contained multiplied organelles, nuclear fragments and portions of cytoplasm were formed through the enclosing of cisternae of endoplasmic reticulum around damaged organelles. In most of the autophagic apoptotic lymphocytes, their organelles were essentially increased in number. In addition, except that their nuclei were pyknotic, they did not shrink as other apoptotic cells. Thus, we considered the increase of organelles and formation of autophagic vacuoles in the apoptotic splenic lymphocytes was as one of the self-protective reactions against radiation damage. The results showed that part of the apoptotic cells exhibited marked enhancement of autophagic activity. After γ-ray irradiation, distinct organelle changes occurred in autophagic apoptotic lym-phocytes, and the autophagic apoptotic cells themselves perhaps took part in the elimination of apoptotic bodies. These indicated that the classical and the autophagic apoptosis are two types of cell death that assumed different morphological and functional aspects.

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The inhibitory effect of diethyl pyrocarbonate (DEPC) on ecto-ATPase activity was evaluated in the rat cardiac muscle, small intestine and kidney using correlated biochemical and histocytochemical methodologies. The activity of the enzyme decreased in nonlinear proportion in correlation with increase of concentrations of DEPC. Essentially all ecto-ATPase activity was abolished in response to 1.0 mM DEPC in biochemical assays. No reaction product was detected using both confocal and electron microscopy when the tissues were pre-incubated with 1.0 mM DEPC. The present results show that DEPC can be reliably applied to substantiate the specificity of the demonstration of ecto-ATPase activity both biochemically and histocytochemically.

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For clarification of the structural and cytochemical dynamics of the Golgi apparatus of the parotid acinar cell during mitotic division in vivo induced by repeated injections of isoproterenol, computer-generated three-dimensional reconstruction was conducted of serial section electron micrographs in conjunction with Golgispecific enzyme cytochemistry. Outlines of the cells, nuclei, thiamine pyrophosphatase (TPPase) -positive Golgi elements (trans Golgi) and TPPase-negative ones (middle/cis Golgi) could be clearly seen through the use of a commercially available computer software program.
In interphase acinar cells, the Golgi apparatus could be seen to spread out in the supranuclear region of the cytoplasm as a single continuous reticular structure with many branches and anastomosis. TPPase-positive trans elements were associated with the entire surface of the interphase Golgi stack. With the start of the mitotic phase, gradual disorganization of the stack into a loose structure followed by dispersion throughout the cytoplasm and marked disappearance of TPPase activity became evident. Up to the anaphase, the Golgi apparatus was completely disorganized as small TPPase-negative clusters comprised of tubulo-vesicular membranes were distributed throughout the cytoplasm. Recovery of the stack, single reticular continuity, supranuclear location and TPPase-reactivity of Golgi apparatus was apparent in daughter cells during the telophase. Distributional change of the Golgi apparatus of parotid cell during mitotic division was clearly confirmed by the present study. Dispersion and reassembly may be significant factors in the equal partitioning of the Golgi apparatus into daughter cells. TPPase activity would appear quite essential to the expression of the normal cellular secretory functions.

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The Mongolian gerbil (Meriones unguiculatus) is an animal model of epilepsy in which the epileptic behaviors develop along with chronological development. In this study, to clarify the mechanism of epileptogenesis in the gerbil, the developmental change of the behaviors elicited by posture change was followed in the previously established animals of a seizure-sensitive strain and a-resistant one. Posture change is a strong inducer of seizure in the adult animals of the sensitive strain. The results show that a bilateral and synchronized rhythmical movement of the pinnae was induced in the young animals of the sensitive strain, which seemed to correspond to an earlier stage, if not the first, in the establishment of epileptogenesis. In contrast, posture change did not induce either the adult-type seizure or the characteristic movement of the pinnae in the animals of the resistant strain. As the first step in clarifying the reason for this difference between these two strains, we studied the structure of the facial nerve which innervates the muscle of the pinnae, using consecutive paraffin sections of decalcified heads of both strains.
In both strains, the facial nerve left the brainstem, ran laterally along the vestibular nerve bundle and at the geniculate ganglion, it changed its direction posteriorly and laterally. When it left the skull, it changed its direction again and descended ventrally, extending a branch to the ear on its way. In the seizure-resistant gerbils, this branch entered the muscle in a similar way to the seizure-sensitive animals. Therefore, these two strains probably have difference (s) in other components although the possibility of a functional difference in the facial nerve remains.

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Prophylactic and curative effects of two tradi tional Chinese medicines, Rikkunshito (TJ-43) and Shigyakusan (TJ-35), against adriamycin (ADM) -induced cytotoxicity were studied in gastric epithelium and cardiac ventricular myocytes based on histochemical analysis of mitochondrial cytochrome oxidase (COX) activity. Oral administration of TJ-43 and TJ-35 prior to the injection of ADM preserved the COX activities both in gastric epithelium and cardiocytes to the normal levels. However, post-treatment with both drugs after ADM injection showed different enzyme activities between TJ-43-and TJ-35-treated groups. In both tissues, post-treatment of TJ-43 retained significant enzyme activity, whereas the enzyme activity of the TJ-35-treated groups was comparable or lower than that in ADM-injected controls without drug treatment. Although both TJ-43 and TJ-35 scavenge free radicals, no curative effect of TJ-35 against ADM-induced cytotoxicity was observed.

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Recently, interphase cytogenetics using fluorescent in situ hybridization (FISH) was performed on various kinds of solid tumor, and their inherent karyotypic heterogeneities were revealed. Concerning this heterogeneity, we evaluated both the exhibiting number of chromosome 17 and nuclear DNA content on an identical nucleus by means of computer-controlled auto-scanning stage in order to demonstrate the alteration in number of chromosome 17 among cytofluorometrically distinct subpopulations.
We investigated 8 lesions of surgically resected colorectal carcinomas, which were classified as aneuploid in quantitative DNA analysis and also exhibited an increase of 17-aneusomy nuclei. We used paraffin-embedded archival blocks. First, we prepared isolated cell specimens, and memorized the position of the cells on a glass slide using computer-controlled auto-scanning stage. Next, the specimens were stained with propidium iodide, and the fluorescent intensity was evaluated as nuclear DNA content in the order of cell position data. And lastly, FISH was performed with (peri) centromerespecific DNA probes for chromosome 17, and we enumerated the number of signals in a nucleus also according to cell position data. Then, we compared the distribution of number of chromosome 17 among cytofluorometrically distinct subpopulations. Three of 8 lesions showed a single GO+G1 peak, and the rest exhibited plural GO+G1 peaks in DNA profile. And 4 of 5 lesions, which showed plural GO+G1 peaks, presented a peak at the DNA value of (near) 2c. We could detect an alteration in the distribution of number of chromosome 17 between diploid peak and aneuploid peaks in 4 of 4 lesions which presented a peak at the DNA value of (near) 2c. However, we could ot find a difference in the distribution of number of chromosome 17 between GO+G1 peak and G2+M peak. These observations indicate that the distribution of number of chromosome 17 reflects an endoreduplication of genome content, yet, it does not alter in accordance with the phase of cell cycle. It is necessary to evaluate nuclear DNA content simultaneously in order to assess an essential cytogenetic change.

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The fusion of lysosomes to phagosomes was observed under high voltage electron microscopy, in 4μm thick rat retinal sections with the aid of acid phosphatase cytochemistry. The study of thick sections facilitates the observation of the moment of fusion in stereo view from two tilted pictures. From this study, the contents of the lysosome pored into the phagosome through the orifice, shortly after the collision of the two organelles. The hydrolytic enzymes such as acid phosphatase spread in a sheet under the limiting membrane of the phagosome to finally form a balloon of the reaction product. In some case the ballooning appeared to be doubled. The outer skin of the reaction product may be the result of a wrapping mechanism of phagolysosomes.

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