To get a better understanding of the factors that are related to the rapidity of the postmortem changes, we studied the postmortem changes in various parts of the nephron in the rat kidney at room temperature histopathologically, electron microscopically, and enzyme histo- and cytochemically. We found that the rapidity of the postmortem changes in the tubular epithelia of the rat kidney, at room temperature, is related to the intensity of cellular K-NPPase activity, that is the partial phosphatase activity of Na, K-ATPase. The postmortem changes, characterized by cytoplasmic vacuolization and karyopyknosis, in the epithelial cells in the distal tubule and the thick ascending limb of the loop of Henle (TAL) with intense K-NPPase activity began earlier and progressed faster than those in the epithelia of the proximal tubules, macula densa and other parts of the nephron with low K-NPPase activity. These postmortem changes were delayed by flushing the kidney with PBS containing ouabain, ATP, verapamil, or EDTA. It is probable that the cells with intense ATPase activity tend to consume intracellular ATP faster than the cells with low ATPase activity after death, and the resulting lack of intracellular ATP and calcium ion influx cause various postmortem changes. On the other hand, the postmortem changes in the proximal tubules, with a large number of lysosomes, are slower and milder than those in the distal tubules and TAL at room temperature. Enzyme cytochemistry revealed hardly any signs of leakage of acid phosphatase, one of the lysosomal hydrolytic enzymes, up to at least 10hr after death, during which time, the postmortem changes in the proximal tubules are slight and those in the distal tubules and TAL are severe.
These results suggest that the rapidity of the postmortem changes in the rat kidney at room temperature depends mainly on the intensity of cellular Na, K-ATPase activity and that lysosomes do not participate in the cellular postmortem changes to a great extent until at least 10hr after death.

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In the first part of this study, we reported that the postmortem changes in rat kidneys at room temperature proceed most rapidly in the epithelial cells of the distal tubule and thick ascending limb of the loop of Henle, and that one of the factors which determine the rapidity of the postmortem changes at room temperature is the intensity of K-NPPase (Na, K-ATPase) activity. In the present study, the postmortem changes in rat kidneys maintained at 0°C for 20, 48, and 72hr after death were studied histopathologically, electron microscopically, and histo- and cytochemically to obtain further information on the mechanisms of postmortem changes. At 0°C, the postmortem changes in the distal tubules and thick ascending limb of the loop of Henle were delayed considerably, and only mild changes were observed electron microscopically even 72hr after death. Contrary to the results at room temperature, the postmortem changes in the proximal tubules were quicker than those in the distal tubules at 0°C, and the proximal tubules show obvious cytolysis 48hr or more after death. Electron microscopy revealed swelling of the lysosomes, and in enzyme cytochemistry, reaction products indicating ACPase activity increased in the cytoplasm around the lysosomes 48hr after death. These findings suggest that the lysosomal enzymes leak out from the lysosomes at least 48hr after death at 0°C. The lysosomal enzymes were considered to have activity even at 0°C, because ACPase activity could be detected cytochemically at 0°C in the lysosomes. The cytolysis observed in the proximal convoluted tubules at 0°C was not seen in the kidneys flushed with chlorpromazine, a membrane stabilizer, and was severe in the kidneys flushed with Triton X-100, a membrane labilizer. From these results, the postmortem cytolysis observed especially in the proximal convoluted tubules 48hr or more after death even at 0°C is considered to be autolysis caused by the lysosomal enzymes which leak out from the lysosomes. Together with the result of the previous part of this study, it is concluded that there are at least two biological factors which determine the rapidity of the postmortem changes in the cells of the rat kidney, the intensity of cellular Na, K-ATPase activity and the lability of the lysosomal membrane.

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A simple and new technique was developed to ensure intimate contact of film with whole-body sections mounted on glass slides. Mice were injected with ¹²⁵I-labeled insulin intravenousely. Whole-body frozen sections were prepared using adhesive tape along with a piece of dry paper and transferred to cold glass slides coated with egg albumin and glycerin. After freeze drying, a slide-mounted section was placed in a plastic bag, and brought into contact with the film. The bag was subsequently evacuated and sealed by a vacuum sealer. By this method, film maximum contact could be maintained with the section during exposure. Following development of the film, the section was stained and used for histological identification of tissues in the autoradiograph.

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The cell proliferative activity and capability of cytodifferentiation of cancerous and non-cancerous thyroid tissues were studied using ³H-thymidine (³H-TdR) autoradiography and immunohistochemistry for thyroglobulin. Cancerous lesions were induced by diisopropanolnitrosamine (DIPN) followed by methylthiouracil (MTU). Regenerative thyroid tissues were obtained through wounding. All the rats treated successively with DIPN and MTU were found to have carcinomas that invaded the surrounding tissue or blood vessels. By cumulative labeling with ³H-TdR, the maximal cell cycle times (Tc) of invasive and non-invasive parts of carcinomas were estimated to be 120hr and 180hr, respectively, whereas the Tc of regenerative and hyperplastic thyroid tissues were estimated as 113hr and 519hr, respectively. However, the duration of S phase (Ts) was greater in cancers (15-35hr) than in non-cancerous tissues (3-4hr), and the cancerous tissues showed lower expression of thyroglobulin than the non-cancerous thyroid tissues. These may reflect some injury in the DNA of cancer cells.

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Eosinophilic epithelial tumor cells of cystadenolymphomas consisted of two different cell types as determined by electron microscopy: light tumor cells, located on the apical side and containing mitochondria without cristae, and dark tumor cell, located on the basal side and containing mitochondria with large and tightly packed cristae and also lysosomes. As found immunohistochemically, keratins and other proteins were expressed differently in light (apical) and dark (basal) tumor cells. The dark tumor cells were positively stained by monoclonal antibody PKK1 (nos. 19, 18, 8; Carnoy's-fixed sections), K8.12 (nos. 16, 13; formalin-fixed sections), K4.62 (no. 19) and by monoclonals 115 F5 and 115 D8 against epithelial membrane antigens and by monoclonal KM231 against a human gastric cancer antigen. The immunohistochemical localization and pattern of staining is compared between dark tumor cells of cystadenolymphomas and ductal basal cells of the normal salivary gland.

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The increased collagen production in liver fibrosis is accompanied by many changes in the biosynthetic pathway, such as an increase in the transcription of procollagen mRNA and the activities of the post-translational enzymes. Prolyl 4-hydroxylase is a key enzyme in collagen biosynthesis, which stabilizes triplehelical conformation of procollagen. So, procollagen and prolyl 4-hydroxylase mRNAs are needed for mature collagen synthesis. In this study, the localization of type III procollagen and prolyl 4-hydroxylase mRNAs in fibrotic human liver was detected by in situ hybridization. Frozen and deparaffinized sections of human liver were examined using non-radioactive digoxigenin-labeled cDNA probes. Type III procollagen and prolyl 4-hydroxylase mRNAs were found to be localized not only in the cytoplasm of mesenchymal cells but also in hepatocytes. Furthermore, using serial sections, both mRNAs were found to be present in the same hepatocytes. These results suggest that hepatocytes produce triple-helical procollagen chains and promote liver fibrogenesis along with mesenchymal cells.

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The fine ultrastructural localization of acid phosphatase (AcPase) activity in mouse spleen ages; 1 day, 1 and 2 weeks, and 1, 2 and 10 months was carried out by cytochemistry, using cerium as the trapping agent. The AcPase enzyme activity was observed in the red pulp. The enzyme reaction product precipitates appeared dense and homogeneous, and were localized in the lysosomes of macrophages, phagocytic reticular cells and littoral cells. The presence of cerium in the reaction product precipitate was confirmed by X-ray microanalysis. The spectral line was present at La=4.84KeV and did not interfere with osmium emission line at Ma=1.914KeV. The results demonstrate variation in the degree of AcPase activity in different ages of the animal studied. The enzyme activity increased progressively from day one, recorded a peak on the first week and thereon, decreased gradually until the 10th month.

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The cellular distribution of argininosuccinate synthetase (ASS)-like immunoreactivity was studied in the intestinal tract and pancreas of the Japanese monkey by means of immunocytochemistry. In routinely immunostained sections, ASS-like immunoreactivity was localized in the nerve cell bodies of the myenteric and submucosal plexuses of the small and large intestines as well as in the pancreatic autonomic ganglia situated among the acinar cells. Dense nets of varicosed nerve fiber containing ASS-like immunoreactivity were observed in some smooth muscle layers of the intestines. In the pancreas, dense nets of ASS-like immunoreactive nerve fibers were observed around the pancreatic ducts and also near the exocrine and endocrine cells.

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Sphingolipid activator protein-1 (SAP-1), which stimulates the hydrolysis of several sphingolipids, including sulfatide, is increased in metachromatic leukodystrophy (MLD) and other lipidoses, but the reason for its increase in some lipidoses remains to be discovered. In a previous study, we found that intralysosomal sulfatide storage induced a compensatory increase in SAP-1 in cultured skin fibroblasts of MLD. In this study, the distribution of SAP-1 in the cerebrum of a normal subject and an MLD patient was examined. In the normal cerebral hemispheres, SAP-1 was found in almost all the cell types, especially in the neurons. Its subcellular localization was confirmed to be lysosomal by an electron microscopic study. In contrast, in the MLD cerebrum, SAP-1-positive cells were found throughout the white matter, especially in the corticomedullary junction. These SAP-1-positive cells in the corticomedullary junction were macrophagocyte-like cells, based on their structural characteristics and Mac-1-positive staining, and they had metachromatic granules, which reflect sulfatide storage in MLD. They were also stained with myelin basic protein (MBP) in spite of diffuse myelin loss in the white matter. The corticomedullary junction, known to be spared from demyelination, was actively infiltrated in the advanced stage by macrophagocyte-like cells, in which accumulation of sulfatide induced an increase in SAP-1 in the lysosomes. These results suggest that SAP-1 plays an important role in regulating intracellular lipid catabolism and help explain its increase in some lipidoses.

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Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the catecholamine (CA) biosynthesis. The transient existence of TH-immunoreactive (IR) cells in the caudoputamen (CP) of postnatal mice was proved for the first time by means of the peroxidase-antiperoxidase method, using a specific antibody to TH. TH-IR cells appeared from postnatal day 5 (P5) and the number of their cells reached maximum from P6 to P7, and slowly decreased until P20. At P30, only a few TH-IR cells were detected in the CP. Most number of the TH-IR cells were seen at the central part of the CP. They had round or oval cell bodies and short processes. TH-IR cells lacked other CA-synthesizing enzymes, such as aromatic L-amino acid decarboxylase, dopamine-β-hydroxylase, phenylethanolamine-N-methyltransferase, and dopamine.

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Lectin stainings were performed to characterize the properties of glycoconjugates expressed in normal and malignant endometrial tissues. The specimens examined included 21 cases of normal endometrium and 20 cases of endometrial adenocarcinoma. The effect of prior neuraminidase digestion (Neu) upon the lectin staining was examined as well. Nine lectins were used such as Griffonia simplicifolia agglutinin-I-B4 (GS-I-B4), peanut agglutinin (PNA), Ulex europaeus agglutinin-I (UEA-I), wheat germ agglutinin (WGA), soybean agglutinin (SBA), Dolichos biflorus agglutinin (DBA), Maclura pomifera agglutinin (MPA), Griffonia simplicifolia agglutinin-II (GS-II), and Limax flavus agglutinin (LFA). The results obtained indicated that the lectin reactivities were classified into three groups; those detected in normal as well as neoplastic endometrium (Neu-GS-I-B4, WGA, SBA, LFA), those altered depending upon menstrual phases (PNA, SBA; proliferative phase dominant, WGA, DBA, MPA; secretory phase dominant), and those found only in carcinoma tissues (UEA-I, Neu-PNA). GS-I-B4 showed a unique staining pattern, since the reactivity of this lectin appeared only after neuraminidase digestion. This appears to indicate that sugar chains reactive to Neu-GS-I-B4 might be a tissue specific antigen, as such a reactivity was not demonstrated in other organs.

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Using para-nitrophenyl phosphate as substrate, extralysosomal localization of acid phosphatase activity was investigated in the developing and adult rat cerebellar cortex, particularly in the Purkinje (P) cells. Light microscopic observations revealed that acid para-nitrophenylphosphatase (acid NPPase) activity appeared increasingly in the P cell and molecular layers throughout their postnatal development. Under an electron microscope, the acid NPPase activity was found in the cisterns of nuclear envelope and endoplasmic reticulum of the P cells, in addition to the lysosomal activity. In the synapses on the P cells, the acid NPPase activity was predominantly localized in the presynaptic membrane of such parallel fibers. The activity of nonlysosomal acid NPPase was inhibited by fluoride (0.1mM) and CuSO₄ (1.0mM), but not by L(+)-tartrate (10mM). The present results are discussed in a possible relationship to an activity of phosphotyrosine protein phosphatase in the developing rat cerebellar cortex.

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The activity of poly(adenosine diphosphate-ribose) (poly(ADP-ribose)) synthesis was examined in permeabilized transformed mouse epidermal cells (PAM 212 cells) in culture. PAM cells grown in low (0.02mM) Ca⁺⁺ medium showed a higher activity of poly(ADP-ribose) synthesis compared with the cells grown in normal (0.7mM) Ca⁺⁺ medium. Poly(ADP-ribose) synthesis was completely inhibited by 1mM 3-aminobenzamide, which is a specific inhibitor of poly(ADP-ribose) synthetase. PAM 212 cells grown in medium containing 2% fetal calf serum (FCS) switched from medium containing 15% FCS showed an increase of poly(ADP-ribose) synthesizing activity time-dependently during incubation. After PAM cells grown in normal Ca⁺⁺ medium were cultivated with 1α, 25-dihydroxyvitamin D₃ (1α, 25-(OH)₂-D₃) or TPA, poly(ADP-ribose) synthesizing activity decreased time-dependently, compared with the condition without addition, but an incubation with all-trans-retinoic acid (retinoic acid) did not show such an effect. Immunohistochemical analysis using anti-poly(ADP-ribose) antibody confirmed the increased poly(ADP-ribose) synthesis in nuclei of PAM cells grown in low Ca⁺⁺ medium. These results suggest that poly(ADP-ribose) synthesizing activity is correlated to proliferation and differentiation of epidermal cells.

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Precursor cells in the rapid recovery of Leydig cells after depletion by ethanedimethanesulfonate (EDS) injury to bilateral abdominal testes were examined immunohistochemically 8, 11, and 14 days after an intraperitoneal injection of EDS, 75mg/kg, 2 weeks after cryptorchidization. Compared with shamoperated rats, the repopulation of Leydig cells in cryptorchid rats was accelerated. Row-like arranged fusiform-shaped mesenchymal cells were found in peritubular regions of abdominal testes 14 days after EDS injection. At 11 days after injection, a significant increase of bromodeoxyuridine-incorporated nuclei was noted in peritubular mesenchymal cells of abdominal testes when compared with scrotal testes. In intertubular tissues, both attenuated mesenchymal cells in earlier stages and fusiform-shaped mesenchymal cells in later stages were actin-, desmin-, and vimentin-negative, and the cells later revealed marked pyronophilia. Therefore, division of peritubular mesenchymal cells seems to contribute somewhat to the rapid recovery of abdominal testes, although differentiation through cytoplasmic pyronophilia from a pool of mesenchymal stem cells give rise to the main part of new Leydig cell populations.

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