Activity of guanylate cyclase (GC) in the rat hippocampus was studied cytochemically and biochemically. Histochemical activation of the enzyme could be reliably demonstrated only with nitroprusside and nitroglycerin. For the histochemical demonstration of GC activity the cerium precipitation technique was used. The enzyme was localized in small and medium-sized neurons and astroglial cells. Intensely stained were postsynaptic densities and glial cell processes. Surrounding capillaries the reaction was restricted to microvascular glial cell processes and basement membrane.

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D-amino acid oxidase (DAAO) cytochemistry was undertaken in the ‘normal’ liver of fetal mice obtained from mothers carrier of muscular dysgenesis. Developing and differentiating hepatocytes revealed that DAAO is released from mitochondria and immediately enveloped by smooth membrane in the cytoplasm to form DAAO-containing peroxisomes. DAAO synthesized in the cytosol may be relocated in mitochondria before being released back into cytosol.

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Proliferating cell nuclear antigen (PCNA) was demonstrated in paraffin-embedded tissues by an indirect immunohistochemical method using both IgG₁ (19F4) and IgM (19A2) class monoclonal antibodies (MAbs). Various fixatives were compared, at different temperatures and different fixation times, and pretreatment procedures prior to staining were also studied. PCNA was demonstrated when tissues were fixed in aldehyde fixative for a short time; the staining was more intense when tissues were fixed at 4°C than when they were fixed at room temperature. Longer fixation times were associated with less intense staining, and none of the pretreatments tested prevented the loss of immunohistochemical reactivity in such cases. Sufficient PCNA staining was obtained in methanol-fixed tissues; the staining was reduced when tissues were fixed in ethanol. This investigation showed that the usual 10% formalin-fixed paraffinembedded tissues were applicable for PCNA immunohistochemistry when the fixation time was less than 24 hr, even at room temperature. Under these conditions, PCNA-positive nuclei were clearly demonstrated in physiologically growing and cancerous rat and human tissues.

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Using a locally bred strain of Wistar rats, the proteinases in submandibular glands have been assessed histochemically and biochemically by their amidolytic activities with oligopeptide substrates, with the purpose of testing for differences in male and female glands. Proteinase histochemistry with the chromogenic substrate D-Val-Leu-Arg-4-methoxy-2-naphthylamide (MNA) produced a strong reaction within secretory granules in the granular tubules and striated ducts and gave the clear impression that fewer granular tubules occur in female glands. This was confirmed morphometrically using DMAB staining of the granules in paraffin sections. The volume density in female glands was approximately 75% of that in male glands, which appears not to have been observed in previous work by others. For biochemical testing the fluorogenic substrates D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin (AFC) alone or with soya bean trypsin inhibitor and Z-Val-Lys-Lys-Arg-AFC with or without aprotinin were used. In relation to DNA, it was found that the female glands contained only about 56% of the overall proteinase activity of that present in male glands. Tissue kallikrein in female glands was found to be approximately 72% of that in the males when related to DNA and that for tonin was about 66%. Further testing of compositional differences was performed after separating the individual proteinases by isoelectric focussing. Their location was identified by applying a membrane overlay impregnated with the fluorogenic substrate D-Val-Leu-Arg-AFC. This enabled precise excision of individual bands in the gels, which were then eluted and assayed quan titatively. In this way tissue kallikrein in the female glands was found to be about 85% of that in male glands, whereas T-kininogenase and esterase A were only about half of that in the male. The results support the concept that male sex hormones exert a greater effect on the synthesis of T-kininogenase and esterase A than on tissue kallikrein.

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Correlations among the immunolocalizations of keratin, actin and type IV collagen were investigated in serial sections of methacarn-fixed, paraffin-embedded mastectomy specimens taken from infiltrating breast cancer patients. Stainings of the filaments in pretreated sections with actinase were often weaker and/or less apparent in cancer cells than in normal mammary gland cells. If at least 30% of living cancer cells were stained, the case was scored positive. Positivity for 312C8-1 specific for basal cell type keratin 14 was observed in 8 of 60 cases, while for NCL-5D3 specific for luminal cell type keratins, positivity was observed in 57 of 60 cases. Five cases positive for HHF35 specific for muscle actin were also positive for 312C8-1 and NCL-5D3. Type IV collagen in cancer foci was patchy and/or absent, but small cancer nests surrounded completely by continuous type IV collagen, termed “in situ carcinoma components” were found in 25 of 60 cases. In all the components, stainings with 312C8-1 and/or NCL5D3 were found in some cancer cells, and the former was almost always seen in a basal orientation within the components. In con- clusion, keratin 14-positive basal cells could be identified in paraffin sections, and these findings suggest that the basal cells are closely related to continuous type IV collagen and a progressive loss of the cells may promote metastasis through fragmented type IV collagen.

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In vivo experiments have shown that the magnitude of peroxisomal proliferation induced by xenobiotics varies markedly in different mammalian species. Hepatocytes from rats and guinea pigs In vivo also exhibit species dependent differences in the response to hypolipidaemic drugs. In order to investigate the mechanisms underlying this phenomenon and to rule out the influence of different culture conditions we established a technique for the co-cultivation of primary hepatocytes of rat and guinea pig. Hepatocytes from rats and guinea pigs were isolated by a modified collagenase perfusion technique. Cells were either seeded as homogeneous or mixed populations on standard culture dishes. For their species specific distinction the cells were fixed, stained with the alkaline DAB method for cytochemical demonstration of catalase and embedded in Epon 812. In contrast to rat hepatocytes those of guinea pigs possess not only a peroxisomal but also a cytosolic catalase resulting in a darker cytoplasmic label. Whereas in guinea pig hepatocytes peroxisomes are smaller, forming clusters, they are larger and more sparse in rat hepatocytes. These characteristics are sufficient for the distinction of the hepatocytes of rat and guinea pig in coculture permitting the separate study of their peroxisomes by automatic image analysis. The cocultivation of primary hepatocytes from different mammalian species provides a novel approach for the investigation of interspecies differences in drug metabolism and toxicity studies.

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Rat retina, which was intravitreously injected with 5-hydroxytryptamine (5-HT) or 5-hydroxytryp-tophane (5-HTP), was examined to identify indoleamine-accumulating cells by an immunohistochemical method in a flat-mount view and transverse sections. In the retina treated with 5-HT the immunostaining was observed in the cell processes, while in the retina treated with 5-HTP a large number of the immunoreactive cells with processes were noticeable. According to their morphological features and locations, they were divided into 5 types. Type 1 cells were large ovoid with a few processes and located in the inner lamina of the inner nuclear layer (INL). The majority of their processes ramified in the interface between the INL and inner plexiform layer (IPL). Type 2 and type 3 cells had large ovoid cell bodies, and the former was located in the ganglion cell layer (GCL) but the latter was placed in the IPL. Type 4 cells had small round cell bodies with a few fine processes and were placed in the inner lamina of the INL. Type 5 cells had the same features as type 4 cells but were displaced in the GCL.
These immunoreactive indoleamine-accumulating cells are categorized as amacrine cells from their shape and location. A morphological variety of indoleamine-accumulating cells in the rat retina indicates a wide range of involvement of amacrine cells in the signal transmission of visual information.

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Trimetaphosphatase (TMPase) was biochemically and cytochemically investigated using rat and guinea pig tissues. TMPase was partially purified, and its activity was visualized and measured by the method of Doty et al. (J. Histochem. Cytochem. 25: 1381, 1977), using a dot-blot apparatus. TMPase was salted out in fractions of 60-80% ammonium sulfate. TMPase activity was observed in early protein fractions of Sephadex G-100 column. The molecular weight and pI of the partially purified TMPase were 130KD and 6.1, respectively. Electrophoretically, TMPase and acid phosphatase (ACPase) activities were observed in different bands. The present results clearly demonstrated that TMPase and ACPase are two different proteins. Cytochemically, the TMPase activity was elucidated using an improved method which employs cerium salt as capture agent, and the results were compared with those of the lead-based method. The incubation medium of the cerium-based method contained 20 mM acetate buffer, pH 3.9, 2 mM cerium chloride, 1 mM trimetaphosphate, 5% sucrose, and 0.00015% Triton X-100. The localization of the TMPase activity differed from that of ACPase in all tissues employed. TMPase activity was observed mainly in tubular structures. Using the cerium-based method, nonspecific precipitates were considerably reduced as compared with the lead-based method.

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In the present work the characteristics of acid phosphatase (ACPase) positive thread-like structures (nematolysosomes) in NIH mouse hepatocytes were studied. The relationship between the thread-like structures and the spherical lysosomes was also investigated ultracytochemically. It was demonstrated that thread-like structures were branched. They connected with spherical lysosomes and linked up with each other to form a network in the cytoplasm. The thread-like structures and the spherical lysosomes could reasonably be considered as a whole. Thus, a concept was put forward: There is a “lysosome three-dimen sional network system”in the NIH mouse hepatocytes.

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To measure cytochrome P-450 (P-450) content in tissue sections, a dual wavelength microphotometry system linked with a personal computer was developed. A computer program to calculate P-450 content in sections was also established. With this system, accuracy and microphotometric efficiency of the measurement of P-450 content in sections were improved markedly. Furthermore, this system can analyze many data derived from numerous microphotometric spots in sections using the program during measurement. Therefore, the measurement of P-450 content in tissue sections can be done precisely and rapidly with this system.

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The localization of four biologically active peptides was studied by immunohistochemistry in the coeliac plexus and in the first ganglion of the frog sympathetic chain. Perineuronal preganglionic apparatuses reacting with anti-LH-RH were found in both ganglia. Counts of neurons provided with such an apparatus and of neurons devoid of it led to the conclusion that almost all small neurons (diameter<35μm) have a positive apparatus, while the larger ones (diameter>35μm) must be divided into two groups: about 25% are provided with an apparatus, and the others not. In the coeliac plexus the distribution of certain peptides is different from that of the paravertebral ganglion. The coeliac plexus contains more neurons provided with a pericellular reacting with anti-substance P than Paravertebral ganglia. Moreover, the coeliac plexus contains numerous SIF cell reactive with anti-met enkephalin and anti-neuropeptide Y, and some reactive with anti-LH-RH and anti-substance P, which were not encountered in other ganglia.

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The effects of carbonic anhydrase inhibition on the transepithelial potential (TEP) generated by the ciliary epithelium were invesigated using a specially prepared and isolated ciliary epithelial bilayer tissue from the rabbit eye.
The TEP was -733±34μV, aqueous side negative. The TEP is very largely bicarbonate dependent because, in bicarbonate free media, with a stabilized pH, the potential declines to zero. The carbonic anhydrase inhibitors (CAIs) acetazolamide, methazolamide, MK927, and ethoxzolamide decreased the TEP to 70-80% of baseline values when added on the blood (stromal) side (PE surface) of the preparation. When these inhibitors were added to the aqueous side (NPE surface), the decrease in the amplitude of the TEP was not much different. The magnitude of the decline corresponds very well to the percentage decrease in the rate of sodium entry into the posterior chamber after systemic carbonic anhydrase inhibition reported by others. These relatively lipophilic CAIs probably affect cytosolic carbonic anhydrase of the PE and/or NPE. Benzolamide, however, another carbonic anhydrase inhibitor that completely dissociates at neutral pH and hardly penetrates the cell, was considerably more effective from the aqueous side, suggesting that its effect is membranal, i. e., on a basolateral membranal carbonic anhydrase of the NPE. To test this hypothesis, quaternary ammonium sulfanilamide (QAS), a weak membrane impermeant CAI, was used. QAS caused a significant drop in the TEP but only when added from the aqueous (NPE) side. These results indicate that there is a membrane bound carbonic anhydrase associated with the basolateral membrane of the nonpigmented ciliary epithelial cells.

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Permeabilization procedures which allow the entry of immunocytochemical reagents may disrupt cells and tissues sufficiently to cause the loss of membrane associated structures. We show here that gap junctions may retain their integrity but become at least partially detached.

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Acid phosphatase has been used as a cytochemical marker for the identification of lysosomes in the seminiferous tubules of the domestic fowl with attention being focused mainly on the process of spermiogenesis. While small lysosomes were located in early spermatids, large autophagic vacuoles of diverse morphology were abundant in the elongating and late spermatids. In the Sertoli cells, lysosomes occurred as large irregular dense bodies. The residues of spermatid autophagy were released from the germinal epithelium in the form membrane whorls. The role of the spermatid autophagic vacuoles and the Sertoli cell lysosomes in the disposal of redundant spermatid cytoplasm is discussed.

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The effects of inorganic phosphate and divalent cations, Ca²⁺ and Mg²⁺, on autophagy were morphologically studied using perfused livers. High concentrations of phosphates in perfusate caused an increased number of macroautophagic vacuoles and translocation of lysosomes around a nucleus, like a glucagon solution. On the other hand, the depletion of phosphate in a glucagon solution showed no effect on the lysosomal elements or showed decreased numbers of microautophagic vacuoles. A divalent cation-free solution caused a similar decrease in the microautophagic vacuoles. There was a significant difference in the number of the macroor microautophagic vacuoles between either the high phosphate or the glucagon solution and either the phosphate-free or the divalent cation-free solution. The use of those inorganic ions in autopahgy was suggested. It has been proposed that inorganic phosphates along with Ca²⁺ are used for membrane fusion. The present study shows an increased number of autophagosomes in the high phosphate solution and high incidence of the autophagosomes in the phosphate-free or the divalent cation-free solution. Thus, these inoganic ions may be connected with the formation of autophagosomes and in the fusion of them with lysosomes.

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The effect of colchicine on the organization of lysosomal system in rat pancreatic exocrine cells was three-dimensionally examined by 2μm semithin section electron microscopy combined with acid phosphatase (ACPase) cytochemistry. In control rat pancreatic exocrine cells, ACPase activity was localized in trans Golgi cisternae, usual spherical lysosomes, and elongated lysosomes (nematolysosomes). The distribution of lysosomes was geographically characteristic. Most spherical lysosomes were located around the Golgi complex. The nematolysosomes elongated from near basolateral plasma membrane to the Golgi area. In colchicine-treated rats which were intraperitoneally injected with 5 mg/kg B.W. of colchicine 4 hr before fixation, the normal distribution of lysosomes was strikingly disorganized. The spherical lysosomes increased in number and size. The nematolysosomes appeared to be fragmented or shrunk, and change their form to spherical. The normal appearance of lysosomes was recovered at 12-24 hr after colchicine injection. The changes in distribution and shape of lysosomes by colchicine seemed to be correlated with the integrity of microtubules. The morphological association of microtubules with nematolysosomes was also revealed by electron microscopy. These results suggest that the microtubule is essential for nematolysosomes in pancreatic exocrine cells.

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The transitional epithelium of the rat urinary bladder was successfully dissociated to single cells and/or clumps of several attached cells, and the morphological features were well preserved in most of the cells. After tissue dissociation, therefore, three cell types, i. e., superficial, intermediate and basal cells, from the epithelium could be clearly and easily identified under the electron microscope. Ferritin-conjugated concanavalin A (Fer-ConA) and Ricinus communis agglutinin (Fer-RCA) binding sites on the surface of the dissociated cells were examined and compared by transmission electron microscopy.
In the dissociated superficial cell, Fer-ConA binding sites were essentially negative on the apical plasma membrane except for on its ridge regions, while they were numerous and continuous on the basolateral plasma membrane. Thus, between the apical and the basolateral portions of the plasma membrane, there were obvious regional differences in ConA binding pattern as well as differences in morphological features. On the plasma membrane of the dissociated intermediate cell, Fer-ConA binding sites were distributed irregularly; and on the entire surface of the basal cell, they displayed an even and continuous distribution. In other words, the particular regions of the plasma membrane of the cell types that contained the asymmetric unit membrane lacked Fer-ConA, while the remaining regions with the ordinary unit membrane had numerous sites of Fer-ConA labeling. The basal cell showed the highest density of FerConA binding sites on the plasma membrane among all the cell types.
Fer-RCA binding sites on the surface of the plasma membrane were numerous and continuous on the entire cell surface of all three epithelial cell types. In contrast to Fer-ConA, no differences were found in Fer-RCA binding pattern on the cell surfaces among three cell types except that the binding sites were somewhat more numerous in the basal cell.
The apical plasma membrane of the superficial cell in the transitional epithelium of the rat urinary bladder is thus a specialized membrane in terms of morphology and composition of membrane surface carbohydrates. The cellular polarity of the superficial cell can be readily appreciated by the ferritin-labeled lectin binding technique.

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To analyze in detail the dynamics of Golgi apparatus in exocrine cells, a monoclonal antibody against resident protein of the Golgi apparatus was prepared. A BALB/C mouse was immunized with fractions enriched in Golgi apparatus of guinea pig pancreas. The derived monoclonal antibody, GF-1, showed intense immunofluorescent staining of the Golgi area of rat pancreatic acinar cells by screening on cryosections. The antibody recognized a single polypeptide band on an immunoblot transferred from SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The polypeptide had a molecular weight of approximate ly 105-kDa and contained asparagine-linked carbohydrates. Light microscopic immunocytochemical study with rat pancreas, parotid gland, sublingual gland and gastric gland tissues indicated a staining pattern consistent with the Golgi apparatus but positive reaction was restricted to serous exocrine cells in these tissues. Immunoelectron microscopy with rat pancreatic and parotid acinar cells showed prominent staining of Golgi apparatus. The positive compartment of Golgi apparatus appeared to be its trans elements. It follows from these findings that monoclonal antibody GF-1 recognizes a glycopeptide of trans-Golgi membrane intimately related to specific functions of serous exocrine cells.

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The synaptic junction from three autopsy cases of adults with Down's syndrome and Alzhemer-type dementia was examined ultracytochemically, using the ethanolic phosphotungustic acid (E-PTA) method. A noteworthy finding was the presence of variable shaped vesicles which were not observed in the ordinary E-PTA-treatcd preparations. Many of these vesicles displayed a wide variety of abnormal shapes between the presynaptic dense projections. Such a change has escaped detection in the E-PTA-treated preparation so far. The presynaptic terminal was considered to be one of the central regions for observing pathological changes, associated with Alzheimer's disease. In addition, the possibility is raised that the qualitative analysis of synaptic junctions, using the E-PTA method, facilitates the acquisition of further information concerning synaptic changes.

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In preliminary experiments, we have applied the ultrathin frozen-sectioning technique to the immunoelectron microscopic labeling of DNA in procaryotic cells. In addition to two anti-DNA antibodies that immunolabeled total DNA, we used an anti-bromodeoxyuridine (BrdUrd) antibody that specifically detected the BrdUrd that was newly incorporated into the DNA of a thymine auxotroph that was deprived of thymine and then pulsed with BrdUrd. The immunolabeling of the incorporated BrdUrd was largely confined to the cell periphery, whereas the labeling of the bulk of the DNA was largely interior. These results are briefly discussed.

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When sections embedded in epoxy resin were conventionally stained with lead citrate and washed very briefly, about 1 sec, a very high staining intensity was attained. However, the staining intensity was quickly reduced by washing 10 min. When uranyl acetate-stained sections were washed for 1 sec, a much lower intensity was obtained. Nevertheless, a significant reduction in staining intensity occurred when washing was prolonged to 10 min. These observations indicated that not only in staining of frozen sections or sections embedded in hydrophilic resins but also those embedded in more hydrophobic resins, the staining intensity is closely related to the degree of washing, particularly in lead-staining.

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The morphological changes of autophagy in rat hepatic parenchymal cells treated with colchicine by intraperitoneal injection were observed ultrastructurally and cytochemically. Thirty min to 4 hr after injection, the double membrane limiting autophagic vacuoles (AV) increased and then the inner limiting membrane disappeared. Various organelles could be found in the AV. Many irregularly shaped lysosomes such as rod-, C-, tadpole- and ring-shaped lysosomes could be observed. The acid phosphatase (AcPase) activity was clearly positive in the matrix of AV and irregularly shaped lysosomes. All these lysosomes were situated in different stages of the lysosomal wrapping mechanism. Besides the mitochondria, endoplasmic reticula and ribosomes, several lysosomes were also segregated by these irregular lysosomes. This phenomenon was named lysosomophagy. The reason for this phenomenon seemed to be due to microtubule disassembling. The AV, the lysosomal wrapping mechanism (LWM), the lysosomphagy, and the exocytosis of residues are discussed.

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A calcium-binding protein, parvalbumin, was purified from the skeletal muscle of the Mongolian gerbil and a polyclonal antibody was raised against it in a rabbit. The antibody reacted with muscle parvalbumins from the gerbil and the rat and also with a protein in an extract of gerbil brain that had the same molecular weight as the muscle parvalbumins. Immunohistochemical localization revealed that this antibody bound to antigens in the neurons of the cerebral cortex, hippocampus and cerebellum of the human brain in a similar manner to its binding to parvalbumin-positive neurons in the corresponding areas of the rodents' brains. This antibody was also used in an immunohistochemical study of the central auditory system of the human brain. In the auditory pathway, the nervus vestibulocochlearis, striae acusticae, nuclei cochlearis ventralis et dorsalis, nucleus medialis olivaris superioris, lemniscus lateralis, nuclei lemniscus lateralis ventralis et dorsalis and colliculus inferior were found to contain immunopositive neurons and/or fibers. The nucleus lateralis olivaris superioris and nucleus medialis corporis trapezoidei in the human are difficult to localize. The antibody against parvalbumin stained two areas that seemed to correspond to the human homologs of these areas. Thus, in the human brain, the parvalbumin-specific antibody seems to be useful for the detection of the pathway of the central auditory system from the nervus vestibulocochlearis to the colliculus inferior. Such an antibody may also permit an examination of the correlation between the level of parvalbumin and the auditory functions within this pathway.

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Using the avidin-biotin-complex method, we examined nerve fibers with substance P (SP) - or calcitonin gene-related peptide-like immunoreactivity (CGRP-IR) in the labial gingival tissues of mandibular incisors affected by gingivitis in 210-day-old ODU plaque-susceptible rats (Sus rats) and in ODU plaque-resistant rats (Res rats) without gingivitis. In the Sus rats, large deposits of dental plaque were found at the labial side of the mandibular incisors, and gingivitis occurred in the areas in contact with the plaque. The labial gingivae of Sus rats lost their upper portion and became half the width and rounder when compared to the gingivae of Res rats. Immunohistochemical studies showed that varicosities and nerve fibers with SP- or CGRP-IR were present in the oral epithelium and connective tissue of the gingivae in both strains of rats. The number and density of the varicosities and fibers were similar in the oral epithelium of both kinds of rats. However, in the connective tissue affected by gingivitis (Sus rats), the fibers formed networks and showed a marked increase in number. Many of the fibers were located close to blood vessels. At the enamel side of the labial periosteum, a number of nerve fibers with SP- or CGRP-IR were located around the vessels and some fibers were close to the alveolar bone, but few were found along the oral epitheial side of the periosteum. The number and density of these nerve fibers along the enamel side of the labial periosteum in the rats with gingivitis were clearly increased compared with the rats without gingivitis. The presence of nerve fibers with SP- and CGRP-IR in the Sus rat gingivae may indicate an important role for neuropeptides in the progression of gingivitis.

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The present study deals with the effects of Urea, Nirma, DDT and Malathion in combination on 5′-nucleotidase activity in the liver, kidney intestine and gills of a fresh water teleost Heteropneustes fossilis at sublethal levels after short term and long term exposure. Marked alteration in 5′-nucleotidase activity were noted at most of the concentrations. Significant stimulation of enzyme activity was observed in the kindey. In the gills a notable synergistic effect was the presence of dense deposits of 5′-nucleotidase reaction product in the pillar cells of alternate gill lamina in the treated specimens. The most remarkable feature observed was that the enzyme leaves the area of operation by leaving the membrane system of its anchorage subsequent to the treatment of above mentioned contaminants and pollutants.

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We have done an acute toxicological experiment on the rat kidney by administration of SVATE-3 in clinical dose (high and low dose, respectively) for 3 weeks. By using morphological methods, light and electron microscopy, enzyme morphological methods, light and electron microscopy, enzyme histocytochemistry in light and electron microscope and biochemistry, we have observed the histological structure and ultrastucture of rat kidney as well as the activity and distribution of the index enzymes for the kidney cell membrane such as Mg-adenosine triphosphatase, 5′-nucleotidase, alkaline phosphatase and glucose-6-phosphatase for kidney cell organelle: endoplasmic reticulum. Futhermore, the biochemical quantitation has been given. The results show that there is no toxic damage to the histological structure and enzyme activity and distribution of rat kidney by administration of SVATE-3 in the low dose. But in high dose, the filter membrane of the kidney was damaged and the activity of Mg-ATPase were decreased. This suggests that we should pay more attention to SVATE-3′s toxicological threshold value when using it and get safe treatment.

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The precise establishment for the subcellular localization of guanylate cyclase would very much aid in clarifying a number of important involvements of cGMP metabolism on biological phenomena. Therefore, as reported, attempts were made to develop a cytochemical method for guanylate cyclase (GCase), using lead citrate as capture agent. Lead citrate acts as an inhibitor of GCase but 21.3% of its activity remains reserved in the tissue. The optimum pH examined on cytochemical sections was between 7.5 to 8.5, the manganese ion working as activator. In the rat retina given immersion fixation, the GCase activity was defined in the space of the disc membranes on the rod outer segment. However, after application of rapid freeze substitution fixation on the retina, the enzyme activity appeared at the cytoplasmic side of the disc membranes, showing good correlation with the biochemical estimations.

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This work was attempted to study cytochemically the localization of H⁺-ATPase on the lysosomal membrane in epithelial cells of renal proximal tubules. The method for demonstration of p-NPPase activity was adopted. In the incubation medium, p-nitrophenylphosphate (p-NPP) was used as the substrate, cerium chloride as the capture agent, and tricine-KOH as buffer to make a neutral optimum condition for the enzyme. Since p-NPP can be hydrolyzed by some ATPase, the enzyme specificity of the H⁺ATPase was identified in the present work. Levamisole, a potent inhibitor of alkaline phosphatase (ALPase), was added to the incubation medium to eliminate the effect of ALPase. Sodium fluoride (NaF), an inhibitor of acid phosphatase (ACPase), was added to elucidate the ACPase activity inside the matrix of lysosome. Ouabain in the medium had no effect and N-ethylmaleimide (NEM) inhibited the enzyme activity on the lysosomal membrane. The findings suggest that the enzyme activity on the membrane of lysosome in the proximal tubule cells was ouabain-insensitive and NEM-inhibitory proton-translocating H⁺-ATPase.

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The α-smooth muscle actin (aortic type) is one of the six isoforms of mammalian actin. It is difficult to distinguish them immunologically, because the amino acid sequences of the isoforms are well conserved. Here, we detected a-smooth muscle actin by means of in situ hybridization using an oligonucleotide probe complimentary to the 3′-untranslated region, which does not cross-hybridize with the remaining isoforms. We developed the following non-radiolabeled procedure, where labeled digoxigenin is intensively immunostained by a combination of PAP and ABC methods. In the both thick (100μm) and semi-thin (2μm) sections of rat parotid gland, actin mRNA was positively stained in myoepithelial cells which emitted flat and cytoplasmic prosesses over the basal surfaces of acini and intercalated ducts. These findings were also confirmed by immunohistochemistry using a monoclonal antibody which is slightly cross-reactive with the other actins. In the immunohistochemical study, however, the myoepithelial processes could be pursued to the distal end. It is probable that this discrepancy between the two methods is due to the different subcellular distribution of the actin protein and its mRNA.

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Cytochrome oxidase activity in extraocular muscles was examined by the diaminobenzidine method and analyzed with a computed image analyzer. Two types of mitochondria, described by Noda and Kuwahara, were found in muscles incubated for 30, 60 and 120 min. The reactivity and proportion of the two types of mitochondria showed time-dependent changes between 30 and 60 min of incubation. However, no changes were found between 60 and 120 min of incubation. It was suspected that potential cytochrome oxidase activity was present during 60 to 120 min of incubation. Quantitative analysis showed that type 1 mitochondria had more cytochrome oxidase activity than type 2 mitochondria. These findings suggest that the activity and proportion of the two types of mitochondria might be useful as an index of functional activity in extraocular muscles.

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We reported previously that fodrin is concentrated in the posterior portion of the human neutrophil stimulated with the chemotactic peptide (Fujimoto and Ogawa, J. Cell Sci., 96: 477-486, 1990). In the present study, the mechanically unroofed specimen of glass-adherent neutrophils was examined to define finer details of fodrin distribution. By immunofluorescence microscopy, F-actin and fodrin were positively labeled; after stimulation with the chemotactic peptide, fodrin was found more densely in the posterior half of the cell as in the whole cell observation. In ultrathin sections of the unroofed and immunogold-labeled specimen, two dimensional distribution of fodrin could be observed. Some gold particles were observed with filaments of about 5 nm in diameter, but others were on amorphous materials. In the stimulated neutrophil, labeling for fodrin was mainly found between straight filaments of about 10 nm in diameter which were densely distributed in the posterior portion of the cell. The results indicate that the redistribution of fodrin in the stimulated neutrophil is concurrent with other cytoskeletal changes.

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The nature of the outer surface of isolated lysosomes from rat livers was investigated by electron microscopic cytochemistry. After incubations with ferritin conjugated reagents, the outer surface of isolated lysosomal membrane revealed an intense binding for cationized ferritin (CF) and a moderate binding for ferritin conjugated wheat germ agglutinin (WGA-F), but not for anionized ferritin and ferritin conjugated Concanavalin A. Although CF particles were uniformly distributed on all lysosomal membranes, WGA-F particles showed a non-uniform distribution pattern, varying from lysosome to lysosome. These results suggest that the outer surface of the lysosomal membrane contains a net negative charge which is partially caused by N-acetylneuraminic acid judging from its characteristic of binding to WGA. The negative charges on the lysosomal membrane toward the cytoplasmic side may regulate the interaction of other organelle membrane with the lysosomal membrane.

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Received for publication October 8, 1991 and in revised form January 10, 1992 Resident peritoneal murine macrophages typically have acid phosphatase (AcPase) activity in lysosomes which are spherical in shape and have a perinuclear distribution. Upon stimulation with certain phorbol esters there is a rapid and dramatic modification of the AcPase-containing compartment into tubular structures which are often interconnected. The formation and maintenance of these tubular structures are dependent upon the presence of an intact microtubule system. Treatment of cells with the microtubule-depolymerizing agent nocodazole blocks the formation of the tubular structures and causes these tubular compartments to collapse toward the nucleus when nocodazole is administered after they are formed. We have employed whole mount electron microscopy and enzyme cytochemistry to document the association of the phorbol ester-induced AcPase-containing compartment and cytoplasmic microtubules. This methodological approach has the advantage of increased resolution when compared to optical microscopy and of an improved vantage point for analysis when compared to conventional thin section electron microscopy.

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Confluent monolayers of cultured bovine aortic endothelial cells (BAEC) at 4°C bound specifically, saturably, and with high affinity radioiodinated bovine serum albumin ([¹²⁵I] -BSA). Unlike bovine transferrin, fibrinogen, and immunoglobulin G, albumin significantly displaced [¹²⁵I] -BSA binding to the cell surface; 50% inhibition was reached at 15 nM of unlabeled albumin. Binding was reversible, and 45% of the surface associated radioactivity was lost after 2.5 min. The binding data calculated from Scatchard plots were consistent with a single saturable interaction of high affinity with a K_d of 1.6 pmoles, and a B_max of 45 fmoles/2.5 ×10₅ cells. The interaction was pH-dependent with an optimum at pH 6.0. Binding was diminished 40% by heparin, 25% by 1 M NaCl but was not markedly affected by cell surface treatment with hyaluronidase and chondroitinase-ABC. Ligand blotting experiments performed with [¹²⁵I] -BSA showed that extracts of BAEC expressed two albumin binding peptides of apparent molecular mass of 18 and 31 kDa. The albumin binding to BAEC surface was also demonstrated immunohistochemically.

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Microwave (MW) has the advantage of accelerating infiltration of fixatives into tissue blocks, but the disadvantage of causing the temperature of the fixatives and tissue blocks to rise. We tried to minimize the temperature elevation during MW irradiation by applying ultrasound (US) in combination with MW oven furnishing US generator. US was uneffective in reducing the rise of temperature, however, it was able to enhance the rapid tissue fixation. Electron microscopic examination showed that the fine structure of organs, such as liver, pancreas, submandibular gland, small intestine, and kidney, was well preserved. Characteristically, the matrix of mitochondria appeared electron-dense, and the marginal zone of zymogen granules in the pancreas was more electron-opaque than the core of the granule. The immunoreactivity of zymogen granules to anti-amylase antibody was strong. The deformation or disorder of cell structure due to the thermal effect and cavitation that can be caused by US was not seen. US can be useful for histologic and immunohistochemical study.

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The distribution and fine structure of [Met] -Enkephalin-Arg⁶-Gly⁷-Leu⁸-like immunoreactive (MEAGL-LI) neurons in the rat cerebellum were examined by light and electron microscopic immunohistochemistry. Immunoreactive neurons which were designated to be Golgi cells were found in the granular layer. These MEAGL-LI Golgi cells were distributed heterogeneously in the cerebellar cortex; the densest distribution was seen in the ventral and anterior vermis, moderate distribution in the pedunculofloccular lobe (PFL) and less in the dorsal and posterior parts of the cerebellar hemispheres. Immunoreactive Golgi cells showed well-developed organelles, such as rough-surfaced endoplasmic reticulum (r-ER) and Golgi complex as well as immunoreactive substances throughout the cytoplasm. On the surfaces of immunoreactive neurons non-immunoreactive axons made axo-somatic synapses. MEAGL-LI neuronal elements were distributed as components of the cerebellar glomeruli in the granular layer. Some of them were presynaptic axons which made axo-dendritic synapses with non-immunoreactive dendrites, and others were postsynaptic dendrites with which non-immunoreactive axons made axo-dendritic synapses. MEAGL-LI neuronal elements were also seen to make intimate contact with Purkinje cells in the Purkinje cell layer.

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We propose the use of fracture-flip combined with Triton X-100 extraction to visualize the cytoplasmic surface of plasma membranes. Unfixed human erythrocytes were freeze-fractured, carbon-cast, and thawed. The carbon casts, along with attached freeze-fractured erythrocytes, were treated with 2% Triton X- 100 to solubilize unfractured plasma membranes and to release haemoglobin. After repeated washing, the carbon-casts, along with attached protoplasmic and exoplasmic membrane halves, were picked on grids, flipped, and Pt-shadowed. Our method leads to extended views of the cytoplasmic surface revealing the fibrilar network that laminates the inner surface of the erythrocyte membrane. Spectrin immunogold labelling of fractured, carbon cast erythrocytes shows that the colloidal gold particles are associated with the fibrilar network at the cytoplasmic surface. Removal of membrane skeletal elements including spectrin by treatment with a low ionic strength buffer containing EDTA leads to loss of the network and reveals globular particles on the cytoplasmic surface of the membrane. These globular particles contained band 3, as shown by immunogold labelling. Our method can be extended to both the ultrastructural observation and the cytochemistry of the cytoplasmic surfaces of other biomembranes.

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A single intra peritoneal injection of colchicine to mice causes both functional and morphological changes on Golgi apparatus in pancreatic exocine cells. Structural changes included shrinkage of Golgi area in the secretory cell, whirling of the Golgi cisternae, decrease of the Golgi cisternae and increase of small vesicles around the remnants of Golgi cisternae. Cytochemical distribution of Golgi marker enzyme, nicotinamide adenine dinucleotide phosphatase (NADPase) which was found in the middle cisternae of Golgi stack in control, decreased the activity and expanded to the entire Golgi stack and in most small vesicles. Also by zinc-iodide-osmium (ZIO) stain which preferably stained cis-side of Golgi stack in control, the entire Golgi stacks were stained as well as these small vesicles. From these observation, by colchicine treatment, the NADPase and the osmium-reducing substance in the cis Golgi cisternae, first appeared to expand its distribution to all the Golgi components. Then, Golgi cisternae appeared to reduce the size either by vesiculation of the cisternae or by trimming the cisternae as cisternal whirling into autophagosomes.

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Distribution of cytokeratins No. 1-19 detected by MAB f 12-19, No. 1, 2, 9, 10, 11 detected by MABEE 21-06 and desmosome peptides 33-52 kDa of skin granular layer detected by MABs G 36-19 in Bowen's disease was studied by the immunoperoxidase and PAP methods. Monoclonal f 12-19 antibody uniformlystained all layers of normal and lesional epidermis in Bowen's disease.
MAB EE 21-06 in normal epidermis showed a reaction only in suprabasal cells, but in Bowen's disease the intensity of staining was considerably decreased. In normal epidermis, MAB G 36-19 stained granularlayer, while in Bowen's disease the reaction was reduced at the site of active proliferation. Thus the newl ysynthesized monoclonal antibodies allowed us to detect the disturbances in expression of keratinocyte differentiation markers in Bowen's disease.

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Two-dimensional membrane crystals were induced in purified preparations of membrane-bound Na, K-ATPase. The crystals were analyzed by cryo-electron microscopy in frozen-hydrated preparations using minimal dose conditions and elastic brightfield. The vanadate-induced crystals showed predominantly p1 or p21 symmetries and image analysis using correlation averaging demonstrated that the protomers in dimeric crystals were either similar or different in the projected structure. The observations suggest that the Na, K-ATPase protomes are gradually reorganized within the membrane crystals during the assembly process.

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PC12 rat pheochromocytoma cells were found to contain and release into the culture medium cysteine proteinase inhibitors, cystatins. Inhibitory activities in cell extracts and in spent culture media were assayed by titration of papain using a sensitive fluorescent method. Partial purification of the inhibitors using gel-exclusion and ion exchange chromatography and immunoblotting indicated that PC 12 cells contain high and low molecular weight cystatins. The low molecular weight (14 to 17 kDa) cystatins reacted with antibody against human cystatin C. Using the same antibody, cystatin-like immunoreactive material was localized in the cytoplasm of PC12 cells. Treatment of PC12 cells with sodium butyrate (6 mM), which is known to induce chromaffin cell-type differentiation, inhibited cell proliferation and led to a significant increase in cystatin level. The data suggest that cystatins in PC 12 cells may be involved in differentiation processes. The well-characterized PC12 cells, capable of neuronal or chromaffin cell differentiation, will provide a useful cell type to study the regulation of cystatin gene expression and the physiologic role (s) of cystatins.

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Histochemical reactions which proved to be useful for the detection of early and late changes of corneal hydration after the injury of the rabbit cornea (Na⁺-K⁺-dependent adenosintriphosphatase (ATP-ase), acid glycosidases, and reactions for glycosaminoglycans) were used for the evaluation of possible side effects on the rabbit cornea of compounds proposed as solvents or substances for the prolonged action of ophthalmic drugs. The following compounds were tested: Cellulose acetate phthalate (1%), propylene glycol (2%), polyvinylpyrrolidone (2, 5%), Tween 80 (1%), and polyethylene glycol 4000 (5%).
Single dropping of cellulose acetate phthalate, polyvinylpyrrolidone and propylene glycol on the corneal surface did not change the corneal hydration. After repeated dropping of these drugs decreased activity of Na⁺-K⁺-dependent ATPase in the corneal endothelium followed by subendothelial swelling was found. On the other hand single application of Tween 80 and polyethylene glycol 4000 caused substantial decrease of Na⁺-K⁺-dependent ATP-ase activity in the corneal endothelium connected with profound corneal swelling. If dropping was repeated, inflammatory cells with high activities of acid glycosidases infiltrated the corneal stroma. After some days the release of these enzymes into substantia propria took place. This was accompanied by decreased staining of glycosaminoglycans indicating their local degradation.
The histochemical approach was found to be more sensitive for the examination of the effect of drugs on corneal hydration than the biochemical examination.
It can be concluded that Tween 80 and polyethylene glycol 4000 cannot be recommended for the preparation of eye drops.

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Using an analytical color fluorescence electron microscope (ACFEM), we observed the cathodoluminescence (CL) emitted from lipid droplets (LDs) of cells in various stages of developing follicles and atretic follicles of the rat. The results showed that the CL at wavelength around 320nm (CL320), which had been proved to be derived from cholesterol ester, was distributed in the cells of theca layers as early as at the stage of preantral follicles; in the theca interna cells of all the follicles beyond this developing stage and atretic follicles; and in the granulosa cells of preovulatory and atretic follicles. This study demonstrates that the cholesterol ester is regularly distributed in follicular cells during the developing and regressing courses of follicles, and also cytochemically provides further detailed information about dynamic metabolism of steroids in follicular cells.

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Spectrophotometric study on kinetics of the reaction under the conditions mimicking its course in the tissue, carried out with α-glycerophosphate as substrate, rat spermatogenic mitochondria as a source of enzyme, tris/hydroxymethyl/aminomethane as copper complexing agent, and in pH 8.4, i. e. in such a range in which the final product, copper ferrocyanide, is soluble led to the following conclusions. Enzymatic oxidation of the substrate is immediately followed by ferricyanide reduction, whereas the final product preciptation starts with a few minutes latency. Dimethylsulfoxide (DMSO) in the final concentration of 5% does not alter the reaction course. However, at its concentration of 15% there is no latency in the final product formation that runs parallelly to the course of the reduction of ferricyanide but there is an inhibition of both the enzymatic reduction of ferricyanide and the formation of copper ferrocyanide as final reaction product. On fine structural level it was found that already at 5 per cent concentration of DMSO and at a shortened time of incubation the localization of the reaction for a-glycerophosphate: ferricyanide oxidoreductase in mitochondria and the other structures of rat spermatids was improved.

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The granular convoluted tubule segment (GCT) of the rodent submandibular gland is a unique structure and consists of granular cell, dark granular cell, pillar cell, and transitional cell. The granular cells contain secretory granules and synthesize biologically active peptides and related processing enzymes; EGF, NGF, kallikrein and renin, erythropoietin, atrial natriuretic peptides, large mobile protein as cystain andserin proteinase inhibitors, S-100 protein, glucagon and somatostatin. Granular cells may have amultiproduction system for growth factors or biologically active peptides for related processing proteinasesas well as proteinase inhibitors which are secreted by a-adrenergic stimulants. Pillar cells contain S-100protein, neuron specific enolase and other nerve related materials, and involve regulation of the nerve-related system as neurotransmitter-like or paraneuron-like properties. Transitional cells show combinedproperties of granular convoluted tubule and striated duct, and may participate in secretion and reabsoorption. Granular intercalated duct cells show similar category of granular cells in GCT segment.

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