Polysaccharide formed histochemically from glucose 1-phosphate by the enzyme activity and native glycogen were observed under the electron microscope.
Polyglucose newly formed only by phosphorylase in rat muscle fibers was stained blue with iodine and appeared to be a finer granular evidence showing ambiguous particles with a diameter from 100Å to 150Å, while native glycogen stained red brown with iodine was demonstrable in mono- particulate form with diameter from 200Å to 400Å.
Polysaccharides synthesized by phosphorylase and branching enzyme in rat liver and hepatoma cells were stained red purple with iodine and were demonstrable in the same ambiguous granular form with a diameter from 100Å to 200Å, while native glycogen appeared to form so-called glycogen rosettes.
In the methacrylate-embedded sections, the newly synthesized polyglucose was more sensitive to the electron beam and thereby was more easily degraded and missed in the cells than native glycogen. When using the Epon embedding the resistance of both substances to the electron beam increased markedly. Nevertheless, the newly formed polyglucose was less resistant to the beam than native glycogen. Furthermore, the synthe-sized polyglucose had difficult staining with lead hydroxide, while glycogen stained densely with it. It was, therefore, newly found that polyglucose synthesized from glucose 1-phosphate by the enzyme activity is obviously different from native glycogen, contrary to Leloir's in-vitro observation.

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During the course of cytochemical studies on ornithine carbamoyl-transferase (OCT) activity in the briefly fixed rat liver with glutaraldehyde perfusion, carbamoyl phosphate, one of the substrate of OCT, was found to be hydrolyzed enzymatically in the endoplasmic reticulum and the nuclear envelope of hepatocytes independently of OCT activity. The nature of the activity was analyzed: (1) The activity was abolished by pretreatment of the sections at pH 5.0 and 37°C. (2) Heat treatment at 50°C inactivated the activity. (3) Cold acetone and sodium fluoride also destroyed the activity. (4) The pH optimum was between pH 6.0 and 7.0. These results were compared to the characteristics of glucose 6-phosphatase and acyl-phosphatase.

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Homogenates of various organs of rats were electrophoresed by the method of Ornstein and Davis and stained with the buffered nitro blue tetrazolium solution containing α-glycerophosphate, glutamate, hydro-cortisone, dehydroepiandrosterone, androsterone, isocitrate, malate, lactate, or 6-phosphogluconate in addition to either nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate.
Several bands of dehydrogenase activity were demonstrated with each substrate, but some of them were active even in the absence of substrate. A considerably high activity of nothing dehydrogenase was found in homogenates of rat organs, particularly, kidney, heart, and brain when nicotinamide adenine dinucleotide was used as coenzyme. This complicated the separation of substrate-specific dehydrogenases on gels. Consequently, only the positive reactions of some bands observed with α-glycerophosphate, malate, isocitrate, and 6-phosphogluconate as substrates were regarded as specific for the substrate-specific dehydrogenases.

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Remarkable increase of quantity of HCG in earlier stage of normal pregnancy has been requested in further investigation on its biological and immunological properties. Normal human embryo presented the production of immunoglobulins in the medullar thymus which was the initial responsive organ among the lymphoid apparatus of human embryo in the 12th week of gestation. The so-called blood thymic barrier was apt to be broken by continuous strong stimulation of HCG to promote the histological and functional development of the medullar thymus in production of human embryonic immunoglobulins.
The detection was done by fluorescent antibody technique on normal human embryos which were obtained at legitimate induced abortion or at spontaneous abortion due to mainly incompetent cervix.

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A method for the histochemical demonstration of fatty acid synthesizing enzymes was deviced in mouse tissue, utilizing the characteristic nature of calcium salts of saturated fatty acid. Fresh frozen sections are incubated in a solution containing capryl-CoA, malonyl-CoA, NADPH, CaCl₂, glucose 6-phosphate, and other factors. Capryl-CoA, malonyl-CoA and NADPH undergo the enzymatic reaction and become higher fatty acids which are precipitated as insoluble calcium salts. Although calcium phosphate, which is formed by the decomposition of substrate and coenzyme, is also precipitated in sections, it is completely excluded by treatment with 0.1 M acetate buffer (pH 5.4) for 15 min, and calcium salts of higher fatty acids which remain stable are visualized by a method similar to the visualization procedures of Gomori's tween method for lipase. The reactions have been found in hepatic cells of mice throughout the entire lobules, sometimes also in periportal or centrilobular areas. Tubular epithelial cells of the kidney have also shown the reactions. A very week reaction is seen in heart muscle. The effect of inhibitor and fixation on enzyme activity, and changes in reaction in ethionine and carbon tetrachloride-induced fatty livers are discussed. These results are consistent with the biochemical data on fatty acid synthesizing enzymes.

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Fatty acid synthesizing enzyme activity was studied histochemically in the hepatic lobules of young male mice with fatty livers induced by ethionine, carbon tetrachloride or ethanol. In fatty livers induced by ethionine or carbon tetrachloride, even when the fatty changes were very slight, enzyme activity was decreased throughout the entire lobules and in the centrilobular areas there was sometimes very little or no activity. In livers with mild to moderate fatty changes very little enzyme activity was found only in the peripheral areas and in some cases no enzyme activity was observed throughout the lobules. Severe fatty changes caused complete loss of enzyme activity. In ethanol-induced fatty liver, no change in enzyme activity was observed even in livers with moderate to somewhat severe fatty changes. But severe fatty liver developed when the dose of ethanol was increased, and enzyme activity became negative. The change in intensity and distribution of enzyme activity in fatty livers induced by ethionine and by carbon tetrachloride was similar, whereas the change was different in fatty liver induced by ethionine or carbon tetrachloride from that in fatty liver induced by ethanol. In fatty livers induced by ethionine and carbon tetrachloride, an inverse relationship was observed between the distribution of fat droplets and fatty acid synthesizing enzyme activity.

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