Histochemical detection of l-gulonolactone: phenazine methosulfate oxidoreductase activity in several mammals with special reference to synthesis of vitamin C in primates

Summary An improved detection of activity of l-gulonolactone oxidase, which is responsible for the final oxidative step in the synthetic process of l-ascorbate from glucose in animals, was achieved using phenazine methosulfate and cyanide. Cold acetone fixation eliminated non-specific deposition of formazan on lipid droplets. The specificity of the method was tested and proven by a biological control, histochemical controls, inhibitors and activators. By application of the method, strong reactivity was found in the cytoplasm of centrilobular parenchymal cells of livers of the opossum, rat, ground squirrel and flying squirrel. Staining of dog liver was moderate and centrilobular. Prosimians were strongly positive: The centrilobular localization was found in the tree shrew and galago; slow lorises and some pottos showed strong reactivity in centrilobular cells and some peripheral cells as well. These prosimians seem to be able to synthesize l-ascorbate as many lower mammals are. On the contrary, true simians (i.e. the squirrel monkey, spider monkey, rhesus monkey and chimpanzee) were negative as guinea pigs were, suggesting their probable inability for l-ascorbate synthesis.Visiting scientist from the Department of Anatomy, Tokyo Medical and Dental University, Tokyo, Japan. T. R. Shanthaveerappa in previous publications, also fellow, Department of Anesthesiology, Emory University.     

Histochemical studies on urate oxidase in several mammals with special reference to uricolytic ability of primates

Summary Strong reactivity for urate oxidase was found in the liver parenchymal cells of the prosimians (i.e. the tree shrew, slow loris, potto and galago) as well as those of lower mammals. The liver parenchymal cells of the platyrrhine monkeys (i.e. the marmoset, owl monkey, squirrel monkey, capuchin monkey and spider monkey) were moderately positive. There was no preferential distribution of granular reaction products in zones of liver lobules of these species. The prosimians and platyrrhine monkeys seem to be uricolytic as lower mammals are. On the other hand, the old world monkeys (i.e. Java monkey and rhesus monkey) and the apes (i.e. the orang-utan and chimpanzee) were histochemically negative.     

Release of erythrocytes from the spleen during exercise and splenic constriction by adrenaline infusion in the rainbow trout

Erythrocyte-supplying function of the spleen was examined in the rainbow troutSalmo gairdneri under exercise. The spleen showed remarkable reduction, about 70% in weight and about 85% in hemoglobin content, after forced exercise of 15 min. The amount of erythrocytes released from the spleen was 2.33 ml/kg body, and this amount corresponds to about 20% of the total volume of circulating erythrocytes in resting condition. No damage was observed at the spleen, splenic artery and splenic vein after the exercise. Examination of the vascular system by a corrosion casting method showed that no place other than the venous circulation exists for the erythrocytes released from the contracted spleen. The spleen was strongly constricted by infusion of adrenaline into the organ. These facts imply that the fish spleen supplies stored hemoglobin into the circulating blood in response to an increased demand of oxygen during exercise, under the control of the sympathetic nervous system.     

Survival of cultured cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification

Cultured cells and somatic embryos derived from the mesophyll tissue of asparagus (Asparagus officinalis L.) were cryopreserved by vitrification. The vitrification solution (PVS) contains (w/v) 22% glycerol, 15% ethylene glycol, 15% propylene glycol and 7% DMSO in Murashige-Skoog medium enriched with 0.5M sorbitol. After initial cryoprotection with sorbitol supplemented MS medium containing 12% ethylene glycol, cells or embryos were exposed stepwise to 85% PVS at 0°C. They were loaded into 0.5 ml transparent straws, and were then plunged directly into liquid nitrogen. After rapid warming, PVS was removed and diluted stepwise. The highest survivals of vitrified cells and embryos were about 65 and 50%, respectively. Surviving embryos developed into plantlets.Abbreviations DMSO dimetyl sulfoxide - PVS vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - MS Murashige-Skoog salt medium - NAA naphthalene acetic acid - BA 6-benzyladenine     

Binding of kinesin to stress fibers in fibroblasts under condition of microtubule depolymerization

The localization of kinesin in EBTr (bovine embryonic trachea fibroblast) cells was studied by indirect immunofluorescence microscopy using an affinity-purified antibody against bovine adrenal kinesin. It has already been shown that in interphase cells a part of kinesin is located on microtubules and the rest diffusely distributed throughout the cytoplasm [Murofushi et al., 1988]. When microtubules were depolymerized with cold or colchicine treatment, antikinesin antibody-stained fibrous components distinct from microtubules. These fibrous structures were considered to be stress fibers because they were stained with rhodamine-phalloidin and because the fibrous staining with antikinesin antibody was completely lost by treating the cells with cytochalasin D along with colchicine. When cold-treated cells in which a major part of kinesin had been localized on stress fibers were incubated at 37 degrees C, kinesin reappeared on reconstituted microtubules. These observations strongly suggest that kinesin has affinity not only to microtubules but also to stress fibers in culture cells.