Preneoplastic mammary hyperplastic alveolar nodules (HAN) and mammary tumors in four strains of mice (SHN/Mei, SLN/Mei, C3H/HeMei and GR/AMei) with different mammary tumor potentials were investigated histochemically by lectin from peanuts in comparison with the pattern of immunohistochemical prolactin (PRL) staining. Most HAN exhibited positive PNA and PRL staining in all strains. Meanwhile, the percentage of mammary tumors showing positive staining of all cells with PNA was lower in C3H/HeMei and higher in SHN/Mei than in the other strains. Little difference was found between strains in percentage of mammary tumors containing cells with positively immunoreactive PRL, although the tumors bearing PRL-positive cells always showed PNA-positive staining.
The cobalt acetylacetonate-diaminobenzidine (CAA-DAB) reaction for cytochrome oxidase has been examined. The medium consists of 2.5mM CAA, 2.5mM DAB·4HCl, 0.25mg/ml cytochrome c and 0.1mg/ml catalase dissolved in 0.1M cacodylate buffer of pH 7.4. Hepatocytes were stained deep blue in granular form due to mitochondrial reaction in rat liver specimens incubated in this medium at 37°C for 10min. The reaction was inhibited by KCN, and by 10min preheating at 80°C. It conformed to Michaelis-Menten's formula and was more sensitive than the color-unmodified DAB reaction processed in the complete medium minus CAA. The CAA-DAB reaction gave the reaction product whose absorption peak locates at 600nm. The microspectrophotometry, carried out by scanning the specimens at 600nm, gave the following results; 1) the adaptability of Beer's law was proved, 2) a decreasing gradient in cytochrome oxidase activity was found along the radial axis of the hepatic lobule from the periphery to the center, 3) using a standard step wedge as a reference, cytochrome oxidase activity was expressed in terms of DAB equivalent units.
Histochemical studies of human guanase have seldom been undertaken, in part because of technical difficulties which result in heavy background staining. We previously reported a modified procedure in which the methodological inadequacies had been overcome. In this report, the modified technique has been applied to determine the distribution of guanase in normal human tissues and in cases of chronic hepatitis, hepatocellular carcinoma and metastatic tumors in liver (adenocarcinoma of colon). Guanase was present within the cytoplasm of hepatocytes throughout the entire lobule. Portal components (bile ducts and veins), fibrous tissue and inflammatory cells were non-reactive, and the enzyme was absent from hepatocyte nuclei in the normal liver. However, in the case of non A non B hepatitis nuclei presenting guanase activity or otherwise were identified. Guanase activity was observed for primary liver tumor but not for metastatic tumor. Moreover, guanase activity was observed in proximal tubuli of kidney and mucosal epithelium of small intestine. No activity was found in other tissues. The present method can be regarded as a useful method, not only to examine the specificity of guanase for hepatic diseases, but also to elucidate the physiological significance of guanase in the kidney and small intestine.
Histochemical detections of sugar residues with the use of lectin bindings-Con A, PNA, RCA-1, DBA, SBA, UEA-1 and WGA, respectively, were described in Paget cells in both mammary and extramammary Paget's disease. Lectin binding in Paget cells was characterized by existence of stainings to PNA, DBA and SBA lectins, and these findings suggest that higher amounts of galactose, N-acetyl-galactosamine residues were contained. On the contrary, binding to either RCA-1, WGA or UEA-1 was very weak or negative. PAS positive stainability and Con A binding patterns showed parallel relations in Paget cells. Enzyme pre-treatment to Paget cells revealed the enhanced stainings to PNA, DBA and SBA lectins, respectively.
Ultrastructural H2O2 producing sites in guinea pig peritoneal (PM) and alveolar (AM) macrophages stimulated with soluble stimuli were studied using a cerium technique. Electron-dense deposits resulting from H2O2 and CeCl3 reactions were observed on the contacted surface of aggregated PM plasma membranes stimulated by formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). The AM stimulated by FMLP or PMA demonstrated no significant cell aggregation or H2O2 production. Although calcium ionophore A23187 did not induce any cell aggregation, deposit formation was apparent on the free surface of plasma membranes. These results indicate that cell-cell contact may have an important role in H2O2 production induced by FMLP and PMA, and that H2O2 production induced by A23187 may not depend on cell aggregation.
The present communication reports a modified technique for staining myoepithelial cells in normal and neoplastic human salivary gland tissues in 2-3μm thick sections from epoxy blocks conventionally prepared for transmission electron microscopy. The method employs tannic acid-phosphomolybdic acid-levanol fast cyanine 5RN procedure by Puchtler et al. after intense oxidation of plastic-free sections using 5% potassium permanganate for 20min followed by bleaching in 1% oxalic acid for 20min. As for the resin-removal procedure, treatment with saturated solution of potassium hydroxide in absolute ethanol for 5-10min yields an optimum result. Direct correlations of light and electron microscopic observations indicate that the cells positively stained with levanol fast cyanine 5RN correspond well to the myoepithelial and neoplastic modified myoepithelial cells containing bundles of microfilaments. This approach is useful in matching the corresponding areas at the light and electron microscopic levels in a single block of tissue for evaluation of myoepithelial participation in various salivary gland tumors.
Histologic and histochemical changes of epidermal growth factor (EGF), keratin-like immunoreactive materials (KLIM) and lectin bindings (PNA, SBA and DBA) were reported in duct-ligated submandibular glands (SMGs) of mice with varying treatments of testosterone and with castration or both. Testosterone treatment before duct ligation of SMGs was markedly influenced, and EGF staining in altered granular convoluted tubule (GCT) cells showed highly positive as in the normal GCT cells. Testosterone treatment after duct ligation as well as hormone treatments before sacrifice were also done. Testosterone effects on duct-ligated SMGs were evident in degranulated GCT cells and duct-like structures and in enhanced secretory granules of degenerated ductal epithelia. These changed GCT cell's findings were observed in EGF reaction, and lectin bindings for PNA and SBA. Keratins (KLIM) in duct-ligated SMGs appeared in duct-like structures, whereas they decreased in proportion to the number of secretory granules after hormone treatment. Testosterone effects on GCT cells and degranulated tubules appeared till the 7th day after injection, and were not evident at the 2nd and 3rd weeks, irrespective of different treatment methods: with pre-ligated testosterone and post-ligated groups and with castration.
Cytochemical analysis of sulfated glycosaminoglycans was carried out in cytoplasmic granules of mast cells from the bovine aorta and rat peritoneal cavity. The cytochemical staining reactions employed for this analysis were alcian blue pH 1.0, high iron diamine and aldehyde fuchsin with or without prior digestion procedures with glycosaminoglycan-degrading enzymes such as heparinase, chondroitinases ABC and AC and testicular hyaluronidase. The results obtained indicate that sulfated glycosaminoglycans contained in the mast cell granules were heparin and chondroitin sulfate D and/or E. The present results are consistent with the data of recent biochemical analysis of mast cell granules.
The distribution of 14C-fucose in mice was investigated by whole-body autoradiography. Following intraperitoneal injection of L-[1-14C] fucose, the mice were killed at various intervals. Autoradiography of the cryosections was performed before and after 6% perchloric acid treatment. At 30min, high radioactivity was found in the kidney cortex, small intestine, liver, urine and gall bladder. Radioactivity was relatively high in the blood, submaxillary gland and bone marrow. The brain did not show any significant radioactivity, but in the hypophysis and choroid plexus, it was significant. In contrast to the brain, the gray matter of the spinal cord exhibited substantial radioactivity, and the optic nerve had relatively high radioactivity. The radioactivities of these tissues retained a similar level at 1hr, but gradually decreased thereafter. To determine the distribution of fucose in the kidney cortex and the intestine, which showed the highest radioactivity in the whole-body auto-radiographs of perchloric acid treated sections 1hr after injection, L-[1-3H]-fucose was used and light microscope autoradiographs were made. In the kidney cortex, many silver grains were seen on the brush border of the epithelial cells and the basement membrane of the proximal convoluted tubules. In the small intestine, many grains of the epithelial cells were seen on the brush border and supranuclear region.
We demonstrated the localization of adenylate cyclase (ACLase) and guanylate cyclase (GCLase) activities in rat aortic endothelium and smooth muscle cells using a recently improved cytochemical method introduced by Fujimoto et al. (16). Cytochemical effects of some agents (isoproterenol, propranolol, acetylcholine, methylene blue) on cyclase activity were also examined with the direct incubation method in vivo employing the perfusate added with such agents utilizing the unique location of the aorta. On the endothelial cell, reaction products indicating ACLase and GCLase activities were localized on the cytoplasmic side of: a) the membrane covering the caveolae and vesicles of both luminal and abluminal surfaces, b) the abluminal plasma membrane underneath the mass of stress fiber-like structure, and c) the gap junctional membrane; and localized around the centriole. On the other hand, the basal activity of GCLase in the smooth muscle cell was intense in comparison with that of ACLase, and the reaction products of both cyclases' activities were localized on the cytoplasmic side of the membrane covering the caveolae, the gap junction, the sarcoplasmic reticulum and the rough endoplasmic reticulum, and on the dense bands, the myosin-like filaments and the centriole. ACLase activity in the endothelial cell and smooth muscle cell was activated by isoproterenol and partly inhibited by propranolol. GCLase activity was stimulated by acetylcholine and fairly reduced by methylene blue in both cell types. Here, we discuss the assessment of our method and the implications of cytochemical localization of cyclase activity in relation to the functional aspects in endothelium and smooth muscle cells.