The surface coats of non-ciliated bronchiolar cells and type II pneumocytes of six mammalian species, human, rabbit, rat, mouse, dog and sheep, were investigated by employing two lectins, Maclura pomifera agglutinin (MPA) and peanut agglutinin (PNA), and anti-Thomsen-Friedenreich (T) monoclonal antibody. Effects of neuraminidase, galactose oxidase (GO) and periodic acid (PA) on their affinities were also examined. MPA revealed strong affinity for the surface coat of non-ciliated bronchiolar cells and type II pneumocytes in most species examined. These epithelial cells largely maintained their reactivity for MPA after oxidation with GO and PA, and neuraminidase digestion did not alter the reactivity. PNA stained these epithelial cells in human, rat and rabbit, prior GO treatment enhanced or engendered the reactivity, and PA oxidation eliminated it. Anti-T antibody did not bind with any of these cells, but after prior neuraminidase digestion, it showed affinity for the surface coats of non-ciliated cells of dog, and of type II pneumocytes of human and rabbit. Interposed oxidation with GO and PA completely abolished anti-T reactivity. These findings show that non-ciliated cells and type II pneumocytes of different species have almost the same sugar structure on their apical membranes, and that staining with MPA, PNA and anti-T antibody are useful tools to identify non-ciliated cells as well as type II pneumocytes at the light microscopic level.

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The Golgi apparatus of Xenopus embryonic epidermal cells was examined by an inhibition experiment using brefeldin A (BFA), to know its target Golgi components since BFA caused rapid reorganization of the Golgi apparatus. Three different probes were used to identify the Golgi and Golgirelated components by fluorescence or electron microscopy: firstly anti-clathrin antibody for distinction of non-clathrin- and clathrincoated vesicles, secondly C₅-DMB-Ceramide as a trans-Golgi membrane marker, and thirdly anti-chondroitin 6-sulfate for identification of immature secretory granules. BFA caused rapid disorganization of the Golgi apparatus to form small vesicle clusters. Immature secretory granules did not contribute to the formation of the Golgi-derived vesicle clusters. The vesiculation of the stacked structures of the Golgi cisternae by BFA preceded the BFA-inhibition of non-clathrinand clathrin-coated vesicle formation. Thus, the results raise a question as to whether the BFA inhibition of coated vesicle formation and the vesiculation of the Golgi cisternae by BFA are different events.

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Localization and alteration of the levels of immunoreactive endotheoin-1 (ir-ET-1) products of canine basilar artery were examined in a one-hemorrhage model. Subarachnoid hemorrhage (SAH) was induced by a single injection of autologous arterial blood into the cisterna magna. In SAH dogs, the endothelial cells on day 2 showed minimal changes such as rounding nuclei or edema, and these injuries progressed on days 4 to 6 from moderate changes with subendothelial edema to severe damage characterized by denudation. In these dogs, ir-ET-1 products on endothelial cells were significantly enhanced and reached maximum levels on day 2 followed by gradual reduction on day 4 to 6. Although no significant changes appeared on endothlial cells in untreated dogs and control dogs on day 2 after the injection of saline, weak ir-ET-1 products were localized irregularly on those cells. However, ir-ET-1 levels on day 4 and 6 were significantly higher in SAH dogs than in untreated dogs. Cerebral arteries were covered with blood clots and a number of inflammatory cells had invaded the adventitia of basilar arteries, in addition to sorrounding the clots. The number of inflammatory cells and the quantity of ir-ET-1 products in the adventitia achieved maximum levels on day 2, and decreased on day 4 to 6. Almost all the inflammatory cells were positive for anti-macrophage antibody. Thus, the levels of ir-ET-1 products on endothelial cells were enhanced with minimal injury on day 2 and gradually decreased with the progression of the injury, while those in the adventitia correlated well with the number of macrophages migrating into the adventitia. These results suggest that ET-1 is derived mainly from macrophages migrating into adventitia as well as endothelial cells, and is an important factor for vasospasm after SAH, not only in the early phase but also in the late stages.

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To clarify the role of TGF-β1 in myocardial healing process after virus-induced myocarditis, we have examined the time course of TGF-β1 expression and its localization in myocardial healing process after Coxsackie B3-induced murine myocarditis. TGF-β1 immunoreactivity increased in parallel with its mRNA level and reached a peak at 10 days after Coxsackie B3 virus inoculation. Immunohistochemically, TGF-β1 was localized at pre-necrotic area at an early phase (5 days); it increased and extended to the area around the necrotic foci when necrosis became manifest (10 days); it decreased when macrophages and fibroblasts migrated to the necrotic foci (15 days); and then it reached a normal level and localized a little in perivascular and calcified regions (30 days). To our knowledge, this is the first study which demonstrates that TGF-β1 mRNA increases in viral-induced myocarditis at acute inflammatory phase. This result suggests that TGF-β1 promotes the migration of macrophages and fibroblasts to necrotic foci and induces fibrosis.

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The localizations of acid phosphatase and alkaline phosphatase activities were observed in Epon ultrathin sections of rat kidneys which had been either fixed with 2% glutaraldehyde or 4% paraformaldehyde, or fixed and then dehydrated by ethanol or acetone, and in ultrathin frozen sections of 2% glutaraldehyde fixed specimens. Prior to dehydration, acid phosphatase activity was positive in lysosomes, and alkaline phosphatase activity on the luminal side of brush border membrane. In the kidneys which had been fixed then dehydrated with ethanol, acid phosphatase activity was observed in the cytoplasm near lysosomes and on the plasma membrane, as well as in lysosomes, and alkaline phosphatase activity in the cytoplasm and on the cytosolic side of the brush border. These results seem to in dicate the displacement of enzyme proteins during a dehydration procedure. On the other hand, acid phosphatase activity was clearly localized in lysosomes, and alkaline phosphatase activity on the luminal side of the brush border membrane on the ultrathin frozen sections of rat kidneys which were fixed with 2% glutaraldehyde. The ultrathin frozen sections had not been exposed to organic solvents prior to demonstration of enzyme activities.
The present study shows that an ultrathin frozen section is the most suitable for demonstrating enzyme activities directly on an ultrathin section.

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Changes in patterns of metachromatic staining due to RNA were followed by a critical electrolyte concentration method in culture cells during mitosis, and compared with AgNOR staining results and with published data especially referring to nucleolar/nuclear proteins. A deep metachromatic response was found in association with the chromosomes, radiating along the spindle structure and then becoming greatly reduced in nuclei at late telophase/early cytokinesis. This response distribution was not exactly coincident with the reported relocation of nucleolar/nuclear proteins or with a non-nucleolar RNA-containing layer during mitosis. The sites of RNA metachromasy also differed from those of Ag-NOR positive response. The results indicate that RNA most probably of nucleolar origin participates in the complex architecture which is assumed to protect the chromosomes, to store rRNA maturation factors, and to contribute to distribution of nucleolar/nuclear proteins and particular RNA types to daughter cells.

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Trichosanthesjaponica agglutinin I (TJA-I) having a high affinity to the NeuAcα2-6Galβ1-4GIcNAc terminal sequence was used for an immunohistochemical study to analyze the expression of the NeuAcα2-6Gal structure in human colonic tissues. A TJA-I binding substance was detected in the mucosa of colonic carcinoma, but not in the normal and transitional mucosa. The TJA-I staining was correlated with the degree of histo logical differentiation of the carcinoma. TJA-I preferentially stained well-differentiated and moderately differentiated adenocarcinoma. In the carcinoma containing mucin lakes, the TJA-I staining reaction was either weak in intensity or negative. A comparative study using monoclonal antibody against sialosyl-Tn antigen (NeuAcα2-6-GaINAc) known to be expressed in colonic carcinoma tissues revealed that the areas exhibiting strong expressions of siaosyl-Tn antigen separated from the areas vividly reactive for the TJA-I staining. In addition, the expression of sialosyl-Tn antigen, unlike the TJA-I staining, was strong in the mucinous areas of the carcinoma. The results obtained in the present study suggest the possibility that a comparative staining method with TJA-I and monoclonal antibody against sialosyl-Tn antigen could be helpful to the diagnosis of human colonic carcinoma.

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Peroxisomes are present in virtually all eukaryotic cells and known to contain more than fifty enzymes. Their size, number and composition of enzymes vary considerably between different species and between different cell types within the same species. During the past two decades, there has been an explosion in the amount of information about their biogenesis, peroxisomal targeting signals, the role of these organelles in lipid metabolism, in the synthesis of cholesterol, bile acids and glycerolipids, and in the degradation of uric acid. The importance of peroxisomes in mammalian metabolism is underscored by the discovery of carcinogenic peroxisomes proliferators and by the occurrence of peroxisomal genetic diseases. Of considerable importance is the implication of induction of hepatic peroxisome proliferation and the peroxisomal fatty acid β-oxidation enzyme system in the development of hepatocellular carcinomas in rats and mice by structurally diverse peroxisome proliferators. The immunocytochemical and in situ hybridization procedures are extremely valuable in identifying qualitative and quantitative changes of specific enzymes in animals exposed to peroxisome proliferators. Peroxisome proliferators appear to exert their pleiotropic responses by activating a receptor known as peroxisome proliferatoractivated receptor. An understanding of the ontogeny of this receptor in relation to the expression of peroxisomal genes is an important consideration in assessing the implications of peroxisome proliferatorinduced toxicity and the role of peroxisomes in genetic diseases.

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The structure of hepatic peroxisomes has been studied by electron microscopy employing histochemical techniques. The recent progress of histochemical techniques, immunohistochemical and in situ hybridization, made it possible to visualize not only intralobular and intracellular but also the intracell organellar localization of various enzymes or proteins. These techniques were applied to normal and pathological liver tissues and the localization of catalase, acylCoA oxidase (AOX), bifunctional protein, 3ketoacyl-CoA thiolase, L-α-hydroxy acid oxidase (isozyme B), and glycolate oxidase (isozyme A), D-amino acid oxidase, peroxisome specific membrane polypeptides (70, 26, and 22 kDa PMPs) and allantoinase were visualized. The present study describes our recent findings on the structure of hepatic peroxisomes obtained by histochemical techniques, especially by the protein A-gold technique.

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The chain of enzymes necessary to convert uric acid to its metabolic products, urea and glyoxylic acid in vertebrates, is truncated through the successive loss of allantoicase, allantoinase and urate oxidase during evolution. In the rats, and most other animals that have urate oxidase activity, this enzyme is associated with crystalloid inclusion present within the peroxisomes in hepatocytes. We have previously shown that the absence of urate oxidase activity in man and hominoid primates is due to mutations in the gene encoding this protein. The presence of all three enzymes of uric acid catabolism in fish and amphibian liver enabled us to undertake their subcellular localization. Using specific antibodies against frog urate oxidase and allantoinase we have localized these two proteins by Immunohistochemical methods to frog liver and kidney. We demonstrated that urate oxidase is present in peroxisomes, allantoinase is localized to mitochondria and allantoicase in cytosol by protein A-gold immunocytochemical method. We have cloned the cDNA encoding allantoinase by immunoscreening a λgt11 bull-frog liver cDNA library and polymerase chain reactions. The cDNA is 2112-base pairs in length containing a 1449-bp open reading frame which correspond to a 483-residue protein. Structural analysis of the deduced protein suggested two potential transmembrane segments and also showed that it lacks a typical peroxisomal targeting signal. The protein sequence indicates the presence of a putative mitochondrial localization sequence in the amino terminus. Northern blotting revealed a single mRNA species in the liver and kidney of frog, which was confirmed by immunoblotting. The hepatic and renal specific expression of allantoinase coincides with the distribution of urate oxidase in the frog tissues. These studies clearly established that urate oxidase, allantoinase, and allantoicase the three enzymes involved in uric acid degradation are localized in different subcellular organelles in the frog liver and kidney.

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To elucidate the relation between Helicobacter pylori (H. pylori) and the surface mucous gel layer (SMGL), 32 cases (25 cases of gastric cancer and 7 of peptic ulcer) of surgically removed human stomach were examined by employing histochemical staining for mucins and H. pylori. This organism was demonstrated in 19 cases by histopathological and histochemical observation. All materials were fixed 2hr in cold Carnoy's solution to fix the SMGL and were embedded in paraffin. Serial paraffin sections were stained with galactose oxidase-cold thionin Schiff-paradoxical Concanavalin A staining (GOCTS-PCS). In addition, immunostaining with anti-H. pylori antibody was performed after the GOCTS-PCS sequence. H. pylori was observed not only on the apical surface of the surface mucous cells or between two lateral plasma membranes of adjacent surface mucous cells, but more abundantly in the SMGL. This organism was not found on the metaplastic cells showing intestinal properties, regenerated immature cells surrounding ulcerated lesions and neoplastic epithelial cells of carcinoma. In the SMGL, it tended to distribute in the layers of the surface mucous cell-type mucin, and, when it existed, marked disintegration of mucous layers and vacuolation was evident. These data suggest that disintegration of mucous in the SMGL is one of the factors which accelerate mucosal damage by this agent.

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Two monoclonal antibodies termed MSN-1 and MRG-1 were raised against endometrial cancer cell line SNG-ll and ovarian mucinous cystadenocarcinoma cell line RMUG-L, respectively, and were found to recognize carbohydrate antigens, Lewis b (Le^b) and bloodgroup A type 3 structures, respectively. Using these monoclonal antibodies, we examined phenotypic alterations of the corresponding carbohydrate antigens in gynecological malignancies including uterine cervical and endometrial cancers.
Additionally, since galactosyltransferase associated with tumor (GAT), which is derived from cellular galactosyltransferase (GaIT), is frequently elevated specifically in the sera of patients with ovarian cancer, the localization of GaIT and GAT was investigated in malignant ovarian tumors. Light-microscopically, MSN-1 gave a positive reaction in more than 90% of the cases of endometrial cancers. The suborganellar localizations of MSN-1-reactive antigen in the cancer cells were almost all in the cisternae of the Golgi, while the normal endometrial glandular cells showed a few positive signals in the trans-Golgi only. Using MRG-1 we investigated uterine cervical squamous epithelial lesions as well as their normal counterparts. Normal squamous epithelium showed a strong positive reaction exclusively along the cell surface. MRG-1-binding sites in squamous cell carcinoma were not only along the cell surface but also in the cytoplasmic space where they resided on vesicular structures, as observed at the electron microscopic level. To detect the galactosyltransferase, we employed two monoclonal antibodies, Mab8628 and Mab8513. Mab8628 strongly stained intracytoplasmic structures in ovarian cancer cells that coincided with the trans-Golgi, as observed with the electron microscope. When Mab8513, which can recognize GAT more specifically than Mab8628, was applied to endometrial cancer and normal cells, an increase in the positive signals was found in cancer cells. These histochemical analyses demonstrated that the localization and/or amount of complex carbohydrates as well as glycosyltransferase is altered between cancer cells and their normal counterparts.

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