Jiro Ohta

Studies on the amoebo-flagellate transformation inPhysarum polycephalum

Flagellar formation in the true slime mold,Physarum polycephalum, involves a sequence of events during which amoebae are changed into flagellate cells. In the present study a series of inhibitors thought to inhibit RNA and protein synthesis and microtubule assembly were added in an attempt to characterize the metabolic processes associated with this amoebo-flagellate transformation.Proflavin (inhibitor of cellular RNA synthesis), puromycin, cycloheximide and streptomycin (inhibitors of protein synthesis), blocked the transformation; however, actinomycin D (inhibitor of DNA-dependent RNA synthesis) did not block this transformation.On the other hand, 2-mercaptoethanol and dithiothreitol did block flagella formation, but even high concentrations of colchicine failed to have such an effect.Flagellate formation was more strongly inhibited by inhibitors of oxidative phosphorylation than by other respiratory inhibitors; this suggests that oxidative phosphorylation takes part in the energy metabolism of this transformation.

Expression of UDP-glucose: poriferasterol glucosyltransferase in the process of differentiation of a true slime mold, Physarum polycephalum

An expression of UDP-glucose:poriferasterol glucosyltransferase activity associated with differentiation of a true slime mold, Physarum polycephalum, from haploid myxoamoebae to diploid plasmodia was demonstrated. In the haploid cells, this enzyme activity was not detected, but after conjugation of the myxoamoebae, the enzyme activity was expressed and increased definitely. In the plasmodial stage, high enzyme activity was maintained constantly. The enzyme was partially purified (35-fold purification, and 28% yield), and molecular weight of 72,000, pH optimum of 7.0, and some characteristics were demonstrated.

Changes in phospholipid composition and phospholipase D activity during the differentiation of Physarum polycephalum.

Changes in phospholipid composition and phospholipase D activity were observed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum. In the amoeboid stage, the main components of phospholipid fraction were phosphatidylethanolamine (PE, 43.3%), phosphatidylcholine (PC, 28.8%) and phosphatidylinositol (PI, 8.0%), but in the plasmodial stage, PC was dominant (40.7%) and other main components were PE (31.5%) and phosphatidic acid (PA, 11.0%). The specific activity of phospholipase D in the plasmodia was 5.7-times higher than that in the myxoamoebae when measured in the presence of Ca2+ at the alkaline pH. In the amoeboid stage, phospholipase A activity (A1 or A2) was detected at the alkaline pH with Ca2+. Phospholipase D activity in the plasmodia was characterized: pH optimum was 6.0; Ca2+ was required for the reaction and Ba2+ could substitute partly for Ca2+; PE was the best substrate for the hydrolytic activity and PC and PI were not appreciably hydrolyzed; and all detergents tested inhibited the enzyme activity.

Studies on cyst formation inPhysarum polycephalum myxamoebae

Encystment of myxamoebae ofPhysarum polycephalum was induced by transferring the amoebae to a high salt medium of 1/60 M Sørensen buffer (pH 6.0) containing 0.125 M NaCl, 1.6 mM MgCl2 and 0.18 mM CaCl2.The induction of cysts was blocked by inhibitors of protein synthesis, such as puromycin, cycloheximide and streptomycin. However, inhibitors of RNA synthesis, such as actinomycin D, proflavin and 8-azaguanine did not block the transformation. These results suggest that in the cyst formation,de novo RNA synthesis is not involved, whereas protein synthesis is required.Cyst formation was more strongly inhibited by inhibitors of oxidative phosphorylation than by other respiratory poisons. It seems that oxidative phosphorylation takes part in the energy supply of this differentiation.

Visualization method for sulfolipids and its application to the determination of nanomole quantities of lipid sulfur

A simple and sensitive visualization method for sulfolipids on a thin-layer chromatogram is described. By spraying with an acidic solution of azure A, a complex was formed between an anionic sulfolipid and a blue cationic compound. After the unbound dye was washed out by brief soaking in methanol, sulfolipids were visualized as clear dark-blue bands on a light-blue background. As little as 0.5 nmol could be detected. Sulfolipid-dye complex was estimated by densitometry or colorimetric measurement after extraction with chloroform/methanol. For the quantitative determination of sulfolipids having long sugar chains, it is necessary to treat thin-layer chromatography plates with acetic anhydride before color development. Of the other tissue lipids not containing sulfuric acid ester that were tested none were stained significantly. A linearity of quantitative determination was observed over the range of 1-8 nmol.