Myosin and actin from Escherichia coli K12 C600.

Myosin-like protein and actin-like protein from E. coli formed filaments very similar in structure to those of myosin and actin from skeletal muscle. At 0.2 M KCl, a large number of "thick filaments" of uniform size (about 0.6-0.7 micron long and about 20 nm wide) was present. These thick filaments aggregated as the KCl concentration decreased to less than 0.2 M. Filaments of actin-like protein were decorated with muscle heavy meromyosin, showing "arrowheads". The arrowhead structure disappeared in the presence of ATP. A mixture of E. coli myosin-like protein and rabbit skeletal actin exhibited a gelation phenomenon on the additon of ATP. The phenomenon was reversible and showed ATP specificity. However, the gelation phenomenon was not observed with the mixture of E. coli actin-like protein and E. coli myosin-like protein. These results provide compelling evidence that the E. coli myosin-like protein and actin-like protein we isolated are essentially identical to myosin and actin, respectively.     

Isolation and characterization of adult rat hearts cells.

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca²⁺ at concentrations greater than 10^?5m.     

Microbial Resolution of alpha-Hydroxy Acids by Enantiospecifically Dehydrogenating Bacteria from Soil

Thirty-three bacterial strains were isolated from soil, utilizing optically asymmetric degradation of dl-2-hydroxy-4-methylpentanoic acid (dl-HMPA) as the screening probe. Those strains were distributed in the following group and genera: Coryneform and Bacillus, Pseudomonas, and Streptomyces. Among them, the most potent strains, Bacillus freudenreichii NRS-137KH20B and Brevibacterium albidum NRS-130KH20B, could perform the resolution of more than 30 g of dl-HMPA per liter within 4 to 5 days of fermentation. Optically pure l- and d-HMPA enantiomers were obtained in more than 80% theoretical yield, whereas the transformed enantiomer was almost quantitatively recovered as 2-oxo-4-methyl-pentanoic acid in the culture broth. The enantiospecific dehydrogenation responsible for this resolution reaction had a rather wide substrate specificity on straight or branched aliphatic C(4) to C(16) 2-hydroxy acids, exhibiting the optima at chain lengths of either C(7) or C(5), although the enantiospecificity was not changed by chain length. The process was thus successfully extended to the preparation of optically pure C(5) to C(9) 2-hydroxy acids.     

Nitric oxide production by cultured aortic endothelial cells in response to thiol depletion and replenishment

The requirements and influence of thiols on the production of nitric oxide (NO) were examined in cultured porcine aortic endothelial cells. NO production was diminished when cells were pretreated with thiol-depleting agents (IC50: N-ethylmaleimide, 30 microM; 1-chloro-2,4-dinitrobenzene, 200 microM; diamide, 1.5 mM; diethyl maleate, 20 mM). The depletion of glutathione (45-99% loss at the various IC50 values) and protein thiols (3-25% loss at IC50) showed no consistent relationship to decreased NO production. The effects of the agents on NO production were not linked to altered sensitivity to the stimulant (calcium ionophore A23187; maximal effect at 10 microM), but roughly paralleled the appearance of cell damage (17-44% lactate dehydrogenase release at IC50). The decrease in NO production due to 1-chloro-2,4-dinitrobenzene was partially reversed by cysteine, dithioerythritol, and dihydrolipoate, whereas cystine partially reversed the decrease due to diamide or diethyl maleate. On the other hand, several thiols diminished NO production in control cells. Overall, alterations of NO production did not parallel the depletion or replenishment of either glutathione, protein thiol, or soluble thiol pools, and so the results argue against hypotheses that cellular thiols are either substrates or necessary cofactors in the pathway of NO synthesis in endothelial cells.     

Demonstration of platelet activating factor receptor in guinea pig kidney.

Distribution of platelet activating factor (PAF) receptor was examined in the guinea pig kidney. Northern blot analysis showed a single band electrophoresed just below the 28S rRNA, and the mRNA was richest in the cortex with lesser amounts in the outer and then inner medulla. Scatchard analysis of membrane fraction using [3H]WEB 2086, a specific PAF receptor antagonist, revealed a single binding site with Bmax of 522, 228, 58 fmol/mg protein for the cortex, outer medulla and inner medulla, respectively. Kd values were in the same order of magnitude (10(-8) M). These results indicate the presence of a single class of PAF receptor in the guinea pig kidney which is most abundant in the cortex.     

Occurrence of Gibberella zeae strains that produce both nivalenol and deoxynivalenol.

By single ascospore isolation, several sets of asci containing eight ascospores were isolated from perithecia of Gibberella zeae. Of these sets, seven were investigated for their ability to produce 8-ketotrichothecene mycotoxins on rice grains. Analyses were made with gas chromatography-mass spectrometry and gas chromatography with 63Ni electron capture detection. Of 56 total isolates, 11 produced nivalenol, 4-acetylnivalenol, and deoxynivalenol, 1 produced nivalenol and deoxynivalenol, 7 produced deoxynivalenol and 3-acetyldeoxynivalenol, 19 produced deoxynivalenol and 15-acetyldeoxynivalenol, and 6 produced deoxynivalenol and both 15- and 3-acetyldeoxynivalenol. The remaining 12 isolates produced nivalenol and 4-acetylnivalenol. All isolates of G. zeae that we examined could produce 8-ketotrichothecenes in this investigation. This report is the first to demonstrate the presence of G. zeae isolates producing both nivalenol and deoxynivalenol. In addition, differences in the production between 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol are discussed in relation to culture conditions.     

Effect of EGF administration on EGF receptor mRNA in hCG producing tumor

In this study we examined the expression of EGF receptor mRNA after EGF administration in hCG producing tumor (choriocarcinoma). We transplanted the tissue of choriocarcinoma into female nude mice and investigated the effects of EGF on the growth of tumors, the binding activity of EGF receptor and the expression of EGF receptor mRNA in the tumor tissues. Two doses of EGF 5.0 micrograms, 50 micrograms and phosphate buffered saline as a control were injected subcutaneously every day for four weeks. Removed tumors were used for immunocytochemical studies and EGF receptor mRNA investigations. HCG and EGF receptors were detected immunocytochemically in the tumor. The low dose EGF employed stimulated the tumor growth while the high dose EGF inhibited the tumor growth compared with that of the control group. The binding activity of EGF receptor and the expression of EGF receptor mRNA also changed in accordance with the stimulation or inhibition of tumor growth. The growth of hCG producing tumor by EGF administration appeared to be dependent upon the binding activity of EGF receptor and the expression of EGF receptor mRNA.     

Phalloidin-induced accumulation of myosin in rat hepatocytes is caused by suppression of autolysosome formation

Administration of phalloidin in vivo to rats causes marked changes in the distribution of actin and myosin in hepatocytes, which accompanies reduced bile flow. We have found that in hepatocytes treated with phalloidin for 3 and 7 days, cellular myosin content increased about 1.5-fold and 4.7-fold, respectively. In addition, total cell protein content and several marker enzyme activities were also elevated by 30-120% depending on the duration of phalloidin treatment. These observations allow us to speculate that phalloidin somehow elicits inhibition of cellular protein degradation, which results in the increase of these protein levels. To examine this possibility further, we analyzed leupeptin-induced density shift of phagolysosomes. In normal liver, the injection of leupeptin/E64c caused an increase in the density of both heterolysosomes and autolysosomes, due to retarded digestion of sequestered proteins as a result of the inhibition of lysosomal cathepsins. Accumulation, in these denser autolysosomes, of lactic dehydrogenase, pyruvate kinase, aldolase, and myosin was demonstrated by enzyme assays and immunoblot analysis. In the phalloidin-treated liver, the increase in the density of autolysosomes and the accumulation of above cytoplasmic enzymes were markedly inhibited. However, phalloidin did not affect the shift in the density of heterolysosomes. From these data, we concluded that autolysosome formation was specifically hindered in phalloidin-treated rat hepatocytes, which results in the reduction of autophagic protein degradation and eventual increase in intracellular protein levels.