We have done histochemical analyses of the enzymes of carbohydrate metabolism in the femoral vein of the rat. These enzymes were phosphorylase, UDPG-glycogen transferase, UDPG-pyrophosphorylase (belonging to glycogenesis and glycogenolysis), and glucose-6-phosphatase, aldolase, lactate dehydrogenase (belonging to the anaerobic process), and isocitrate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase (belonging to the aerobic process). The localization of the various enzyme activity was studied histochemically in the three layers of the normal venous wall. The results of the experimental studies were as follows. 1. The venous wall has a low or moderate enzyme activity in both the aerobic and anaerobic processes in the intima. 2. The venous wall also has a high enzyme activity in both the aerobic and anaerobic processes in the media. 3. Enzyme activity of the venous wall is lower in the adventitia than in the media or in the intima. We speculate that this may be considered to be related to active energy metabolism in the venous wall.
The ability of excision repair of a single Ehrlich ascitic cancer cell was quantitated by cytofluorometry of unrepaired pyrimidine dimer-FITC complex per single nuclear Feulgen DNA. UV-irradiated Ehrlich ascitic cancer cells were made react with antibodies to denatured UV-DNA using the antisera which were absorbed with MBSA and heat denatured DNA. Then the immunofluorescent staining specific for pyrimidine dimers was combined with subsequent Feulgen nuclear reaction using the method reported in the previous paper (Fukuda et al., 1976). Then simultaneous cytofluorometric measurement of the amounts of pyrimidine dimer-FITC complex and nuclear Feulgen DNA was performed on a single tumor cell at various times of incubation after UV-irradiation. Before the occurrence of excision repair of UV-DNA lesions, the amount of pyrimidine dimers per unit amount of nuclear DNA was found equal in all tumor cells at various stages of cell cycle. After incubation for 3 hours, unrepaired pyrimidine dimers were largest in amount in polyploid cells and least in diploid cells; some diploid cells had no nuclear fluorescence of FITC with a faint fluorescence in the cytoplasm. It might indicate their completion of excision repair of UV-induced pyrimidine dimers followed by transport of them to the cytoplasm within the period. Thus the potency of excision repair of a cell in S phase which has not been demonstrated by the detection of unscheduled DNA synthesis could be quantitated using the immunofluorescent-Feulgen double staining. The order of the increase in the magnitude of excision repair in Ehrlich ascitic cancer cells at different stages of cell cycle was polyploid cells<G2 and S phase cells<diploid G1 cells. When non-absorbed antisera were used, a significant fluorescence was found in the nuclei of some unirradiated large Ehrlich ascitic cancer cells. The specific fluorescence was suspected to have some special relation to naturally occuring single-stranded DNA in the tumor cells.
Thiamine-deficient diet was given to male Donryu rats (ca. 100g.) and changes in acid phosphatase (ACPase) and thiamine pyrophosphatase (TPPase) activity were observed ultracytochemically in the nerve cells of the cerebral cortex up to 3 weeks after the onset of the experiment. The control rats (on normal diet containing thiamine) were also kept simultaneously. It was demonstrated by morphological preparations that the lysosomes were increased in number and enlarged in size in nerve cells as well as neuroglial cells of thiamine-deficient rats, and it was confirmed in the cytochemical preparations that these lysosomes possessed intense ACPase activity. Besides, many vacuolated lysosomes were also seen with ACPase activity indicating an increase in the lysosomal ACPase activity. In addition, the enzymatic distribution in the Golgi apparatus was also slightly increased. Overall increase in ACPase activity is thus noted in contrast to that of controls. Increase is related to autolysis. While TPPase activity revealed in the Golgi apparatus was inclined to decline in thiamine-deficient rat suggesting the decreased rate of thiamine metabolism. These enzymatic changes may be attributed to the early deficiency impairment of thiamine.
A method which facilitates the separation of the conjugated γ-globulin from both non-labeled antibody and unreacted peroxidase by use of DEAE-cellulose chromatography was shown in this paper. Using the new method of conjugation reported by Nakane and Kawaoi (1974), the isoelectric point of the peroxidase-labeled antibody was found appreciably to shift toward lower pH than that of the original antibody. Paying attention to the difference of the isoelectric point between labeled and unlabeled antibodies, DEAE-cellulose chromatography was performed to isolate labeled antibody. The enzymelabeled antibody was eluted with 0.025M-0.05M phosphate buffer (pH 8.0), while the non-labeled antibody was eluted at the concentration of 0.005M-0.01M phosphate buffer (pH 8.0) and the modified peroxidase was eluted at higher concentration of 0.25M phosphate buffer (pH 8.0).
A new method was developed for cytospectral characterization of leucocytes after staining with Wright solution. The spectral types of various vertebrate leucocytes were compared by this method. To characterize vertebrate leucocytes their absorption maximum (A max) and relative absorption (RA) spectra (A/A max, %) were recorded. From the values of A max and the absorptions at 600nm (RA 600) and 460nm (RA 460), the RA spectra were divided into 7 types (I, II-a, II-b, III-A, III-a, III-b, IV). The leucocyte types of individual species could be classified into certain types, but these types differed in different species. However, even in different species, most thrombocytes were classified as type III-A, mammalian eosinophils as type III-b and those of lower vertebrates as type IV. In general the spectral types changed progressively from higher to lower animal species. The blood of lower vertebrates seemed to contain immature cells judging from the RA spectra of these cells. From our data this spectral classification seems promising for comparison of vertebrate leucocytes and should be useful in their cytoanalysis.
An autoradiographic method was applied in the study of esterase localization in the ultrastructure of rat adrenal glands. Reaction with 3H-diisopropyl fluorophosphate was used to demonstrate esterase activity in various intracellular compartments. The silver grains were observed over lipid granules, mitochondria and smooth-surfaced endoplasmic reticulum, each of which is envolved in steroidogenesis. No silver grains could be detected over the nuclei or over lysosomes. The results are in agreement with those obtained by an ultrahistochemical heavy metal method.