Tamm-Horsfall glycoprotein was isolated and characterized, and then was studied using the fluorescent antibody technique. The cytoplasma of the tubule cell was well stained and the glomeruli were remained completely unstained. The fluorescent materials were observed also in tubular lumina.
Electron microscopic observations were made of the mossy fiber endings distributed in the hippocampal formation of rats receiving dithizone and of normal controls. The mossy fiber endings were noted to be enlarged to form terminal synaptic knobs complex in shape, each surrouding a dendritic spine. The synaptic knob contained synaptic vesicles. The portion of the presynaptic membrane of the knob facing a dendritic spine appeared to be markedly thick whereas that of the presynaptic membrane in contact with the smooth surface of a dendrite was not notably thick. Sections taken at 15 and 30minutes after administration of dithizone frequently showed remarkably dense accumulations of synaptic vesicles in parts of the terminal knobs of mossy fibers. At 1 and 1.5hours after dithizone, there was evidence of drastic diminution of the synaptic vesicles in the mossy fiber endings and, concomitantly with it, the dendritic spines were seen to have decreased. The morphologic features of the mossy fiber ending, however, returned almost completely to the normal state by 3 to 5hours after dithizone. An attempt was made to investigate the interrelation of these ultra-structural alterations with zinc and the mechanism of action of dithizone on the mossy fiber ending has been discussed.
Glucose-6-phosphatase activity can be demonstrated ultracytochemically in surgical specimen of human kidney fixed by immersion in dilute aldehyde solution. Tissues fixed for 30min. in 2% glutaraldehyde or 5min. in 1% paraformaldehyde plus 3% glutaraldehyde solution and incubated for 15min. in Wachstein-Meisel's medium at 37°C revealed satisfactory results. The enzyme activity was confined to proximal convoluted tubule cells. Subcellular localizations were smooth and rough endoplasmic reticulum as well as nuclear envelopes. All the plasma membranes including brush borders showed no reaction products. Golgi apparatuses remained unreactive. The enzyme localizations in proximal tubule cells of human kidney were essentially same as those in hepatic parenchymal cells of various animals and in proximal tubule cells of rat kidney in the literature. This investigation showed that G6Pase can be used as a cytochemical marker for the human kidney study.
The activity of steroid 3β-ol dehydrogenase (steroid 3β-ol DH) was studied enzyme-histochemically using the following transplantable tumors of mice which had been kept in our laboratory. Gardner's 107 testicular interstitial cell tumor and the transplantble granulosa cell tumor, originated from the steroid hormone-producing cells, always showed a positive activity of the enzyme. It revealed that the former still retains the enzyme activity even to the present time, despite of serial transplantations in over ten years. The enzyme activities of the transplantable pulmonary tumors showed a wide range from highly positive to negative. The fact that some of them showed a highly positive reaction, in relation to hyperestrogenism found in the hosts at the stage when the tumors became histologically anaplastic, was an especially interesting finding. The enzyme activity was also positive in some transplantable liver cell tumors. The enzyme activity was completely negative in the osteosarcomas (519OS and AKR/MSOS).
Two rate limiting enzymes of glycolysis and gluconeogenesis, i.e, low-Km hexokinase (HK) and glucose 6-phosphatase (G6Pase), respectively, in liver tissues of patients with liver diseases, including acute hepatitis (convalescent stage), chronic hepatitis (active and inactive forms) and chronic hepatitis with sublobular hepatic necrosis and cirrhosis of the liver, were demonstrated histochemically. Generally, an intense staining of HK and a weak staining of G6Pase were observed in the degenerated hepatic cells in the lobule. In chronic hepatitis, active form, the HK activity was increased in the whole lobule with an emphasis in the central area, whereas the G6Pase activity was decreased in the central area. Chronic hepatitis with sublobular hepatic necrosis, which has been assumed to progress rapidly into the cirrhosis of the liver, was characterized by an uneven distribution of HK and G6Pase within the hepatic lobule; the activity of the former being increased and the latter decreased at the site of hepatic cell injuries. In cirrhosis of the liver, the HK activity was increased and G6Pase activity was retained well in the well defined nodule, whereas the latter was significantly decreased in the nodule of swollen and irregular hepatic cells with marked infiltration of round cells in the connective tissue.
Ribose-5-phosphate isomerase was histochemically demonstrated in cryostat sections of the human placenta and the bovine heart muscle containing a conduction system. Suitable conditions for histochemical reaction of enzyme activity in those tissues were determined. The incubation mixture containing 50mg ribose-5-phosphate, 10mg NADP, 10ml Nitro-BT (1mg/ml), 10ml propandiol-HCl buffer (0.2M pH 8.0), 0.04ml 6-phosphogluconate dehydrogenase solution (1mg/ml), 2ml 3% gelatine solution and 2mg MnCl2 was proposed, while two control procedures were prepared, using the incubation mixture without ribose-5-phosphate and without 6-phosphogluconate dehydrogenase.
For electron microscopic demonstration of glycogen particles in tissue sections, a new histochemical staining method is introduced as a substitute for stains such as periodic acid thiosemicarbazide silver proteinate. It is based on the fact that glycogen particles can be demonstrated with alkaline bismuth (A·Bi) solution alone and without periodic acid pretreatment. A. The staining mechanism involves a direct reaction where vic-glycol groups in glycogen compounds are oxidized into carboxyl groups and bismuth is reduced. The latter combines with the carboxyl groups to effect increased electron opacity of glycogen particles under electron microscopic observations. B. The glycogen particles can be observed even if periodic acid treatment is added on condition that the treatment be stopped within 10-15min. But glycogen particles disappear when the treatment lasts more than 30min. The treatment is not necessary even in theory because the A·Bi stainable vic-glycol groups are gradually decreased in proportion to the duration of the treatment. C. The technique is adapted to the observation under high magnification of the mode of α-D-glucopyranose arrangement in glycogen compounds because the bismuth deposit has a high electron opacity due to the high atomic number of bismuth and is positioned only in the vic-glycol group of the glycogen compound. D. The staining technique is remarkably simple as staining needs to be carried out only once, in contrast with previous methods. This A·Bi staining technique permits excellent high contrast observation of glycogen particles in the cytoplasm of liver cells and myocardial muscle cells which could be seen to consist of fine granular substructures.