Inhibition of microsomal lipid peroxidation by cytosolic protein in presence of ADP and high concentration of Fe2+

Microsomal lipid peroxidation induced by NADPH, but not by ascorbate, was found to be inhibited by liver cytosol. This inhibition was not dependent on glutathione and was enhanced by ADP in presence of Fe2+ at a concentration of 50 microM or higher. ATP was also effective, but not AMP or cyclic AMP. The cytosolic factor appeared to be a protein as it was heat-labile (greater than 70 degrees C), was non-dialyzable and was precipitated by ammonium sulfate and acetone. It was stable for several months in frozen state and also when heated at 50 degrees C for 10 min. The inhibition by the cytosolic protein was obtained by producing a lag in the activity of lipid peroxidation and was reversed by ceruloplasmin but not by catalase, cytochrome c, hemoglobin or superoxide dismutase. This inhibitory effect by cytosol was limited to formation of lipid peroxides whereas oxygen uptake and NADPH oxidation remained unaffected. Regulation of lipid peroxidation by nucleotide-Fe complexes and cytosolic proteins is indicated by these studies.     

Effect of Copper on Growth of Yeast

The effect of copper was tested on the growth of many strains of yeast. Plate culture on density gradient agar of copper was used for estimating the growth response to copper. Growth in many strains was more strongly inhibited by the copper-aquo complex than by the copper-amino acid complex. Debaryomyces hansenii IFO 023 was found a suitable strain for the present study, because it was not resistant, not producing H₂S, and copper absorption by this strain was similar to that of the resistant strain. Growth of yeast cells in medium containing copper was affected by pH and concentration of amino acid in medium. Absorption of copper into intact cells was almost saturated for the initial few minutes. It was also affected by the addition of amino acid to copper solution. Our results indicated that the growth response of yeast to copper was closely related to copper absorption into cells. About 60 percent of copper absorbed into cells was distributed in the soluble fraction of the cell homogenate which was obtained by centrifugation at 105,000 g for 60 min.     

Activation of Intracellular Proteinases of Yeast

The existence of the inactive precursors of yeast proteinases B and C was confirmed in the autolysate of baker’s yeast and they were named as pro-proteinases B and C, respectively. The active and inactive forms of proteinase C were two distinct proteins, separable by chromatographical procedures. The two precursors were markedly activated by incubation at pH 5 or by treatment with denaturing agents, e.g. urea, dioxane, acetone and certain alcohols.

These activations were also observed with extracts from acetone-dried cells and from mechanically destructed cells, but the activation of proteinase A was not demonstrated under any conditions tested. Therefore, it was assumed that most of proteinases B and C exist in vivo as inactive precursors, whereas proteinase A originally exists in an active form.

Pro-proteinase C, the latent form of yeast proteinase C, was partially purified from the autolysate of baker’s yeast. It was strongly activated by incubation at pH 5 or by treatment with urea or dioxane. The former activation was prevented by treatment to inactivate yeast proteinase A, which co-existed with the pro-enzyme in the present preparation, but was promoted by addition of purified proteinase A. Thus, it was confirmed that A could activate pro-proteinase C. Furthermore, it was found that activation could be caused by extremes in pH or by heating to 55~60°C, accompanied by the simultaneous destruction of the enzyme produced. Pro-proteinase C was stable over a range of pH 5 to 8 after 60 min incubation at 50°C.     

Biotechnological potential of sulfate-reducing bacteria for transformation of nitrocellulose

The biotransformation of NC by Desulfovibrio sp. was studied. The mass of NC was decreased by 4.9-9.3%. The rate of NC transformation was between 46 and 73 mg NC per mg of bacterial protein in 10 days. Moreover, N content (%N) in the remaining NC was reduced by 2-12%. The inhibitory effect of NC was clearly expressed when the growth of D. desulfuricans 1388 in lactate/sulfate medium was initiated. The growth rate of bacteria was 1.5-fold greater when NC was not added (0.074 and 0.05 h(-1) respectively). The transformation of NC by D. desulfuricans was accompanied by the appearance of nitrate in the culture liquid, the amount of which reached the peak by the 8th day.     

Influence of the oxygenation state of the culture media on the level of non esterified fatty acids and of cholesterol in heart cells of the newborn rat]

The uptake of non esterified fatty acids (NEFA), from a medium supplemented with 10% of calf serum, by rat myocardial cells cultured during 8 days, without renewal of the medium, was identical whatever the values of partial pressures of oxygen in the medium. Cellular cholesterol level was reduced when air or oxygen (+5% CO2) was blown into culture bottles; it was enhanced by gassing with nitrogen 24 h later. Cellular NEFA level was reduced by air, but not by oxygen blowing; it was enhanced by gassing with nitrogen 24 h later. The variations observed may originate from pertubations of NEFA beta-oxidation.     

Purification of lysophospholipase of Vibrio parahaemolyticus and its properties.

Lysophospholipase [EC 3.1.1.5] was solubilized from the cells of Vibrio parahaemolyticus with Triton X-100 and purified by the following procedure; precipitation with ammonium sulfate, acid treatment and ion exchange column chromatography using DEAE-cellulose, DEAE-Sephadex A-50, and CM-cellulose, successively. The purified preparation was shown to be homogeneous by polyacrylamide gel disk electrophoresis. The isoelectric point of the enzyme was found to be around pH 3.64 by isoelectric focusing electrophoresis, and its molecular weight was estimated to be 89,000 at pH 7.6 by gel filtration on Sephadex G-200. The minimal molecular weight (15,000) was found at pH 3 by gel filtration on Sephadex G-100 and also by SDS-polyacrylamide disk electrophoresis. The enzyme hydrolyzed 1-acyl-GPC, 1-acyl-GPE, 2-acyl-GPE, and lysocardiolipin but did not attack monoacylglycerol, triacylglycerol, or phosphatidylcholine at all. The enzyme activity required no bivalent cations, and was unaffected by reagents specific to SH-groups, although it was inhibited by Hg2+. The enzyme activity was completely inhibited by preincubation with diisopropylfluorophosphate. The enzyme lost its activity on preincubation with either 1% SDS or 8 M urea at 37 degrees C for 30 min, but the activity lost with urea was recovered by dialysis against distilled water.     

Stabilization of invertase by modification of sugar chains with chitosan

Chitosan was linked to invertase by covalent conjugation to periodate-activated carbohydrate moieties of the enzyme. The thermostability of modified enzyme was enhanced by about 10?°C. The half-life at 65?°C was increased from 5 min to 5 h. The enzyme stability was enhanced by 20% at pH below 3.0. The half-life of denaturation by 6 M urea was increased by 2 h.     

癌基因ras对β-1,4-半乳糖基转移酶活性的调节

 研究癌基因ras对细胞表面的 β 1,4 半乳糖基转移酶活性的调节 构建Ha ras表达载体并转染NIH 3T3细胞株 ,测定细胞表面和细胞内 β 1,4 半乳糖基转移酶活性和其mRNA的水平 结果发现ras使NIH 3T3细胞表面的 β 1,4 半乳糖基转移酶活性降低 ,而高尔基体内的活性不变 此外用Northern印迹检测后发现 ,ras不能改变细胞内 β 1,4 半乳糖基转移酶的mRNA水平 这说明癌基因ras能够调节细胞表面β 1,4 半乳糖基转移酶活性 ,但不能改变其转录水平     

Conditions of formation of the heparin-fibronectin-collagen complex and the effect of plasmin

The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4 microM heparin and 0.9 microM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3 microM. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaCl as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.     

Analysis of the regulation of gene expression during Bacillus subtilis sporulation by manipulation of the copy number of spo-lacZ fusions.

The control of expression of the Bacillus subtilis spoIIA locus was analyzed by titrating gene expression against gene copy number. A plasmid integrated into the B. subtilis chromosome and carrying the spoIIA control region fused to Escherichia coli lacZ was forced to form tandem repeats by the selection of clones that grow on high levels of chloramphenicol, the antibiotic against which the plasmid determines resistance. DNA from the clones was digested with BglII, which did not cut in the reiterated region, and the size of the fragment was determined by orthogonal-field-alternation gel electrophoresis to determine the copy number. Most clones had fairly homogeneous copy numbers. Gene expression was monitored by beta-galactosidase activity. The results indicate that spoIIA was under positive control by a moiety present at about five copies per chromosome. Spore formation was not affected by amplification, so spoIIA-lacZ reiteration did not sequester a molecule required elsewhere for sporulation.     

小蜡果皮水溶性色素的提取及光敏感性

以去离子水和不同体积分数乙醇从小蜡果皮中浸提红色素,用光谱扫描法检测该红色素的光吸收特性,比较不同浸提时间及不同体积分数乙醇对浸提效果的影响,并用暴露方式进行光敏感性测定。结果显示,小蜡果皮色素在纯水中的溶解性最好,属水溶性色素,延长浸提时间可提高色素的浸出率,但杂质质量分数也随之提高,紫外线对色素的色度影响最大,直接照射可使色素的色度大幅度下降。     

膜分离技术在硫酸软骨素分离方面的应用

介绍了硫酸软骨素的生产工艺 ,应用膜分离技术对现有工艺进行了改进 ,使硫酸软骨素纯度达 95 .2 % ,收率提高3% ,并且简化了操作     

Regulation of the proliferation of primary rat hepatocytes by eicosanoids

DNA synthesis in primary adult rat hepatocyte cultures was promoted by epidermal growth factor (EGF), arachidonic acid, and prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha). Growth promotion by EGF was blocked by 0.1 mM indomethacin and 1 mM aspirin, without affecting cell viability. If verapamil was present in the medium when EGF was added, the growth response was inhibited. Hepatocytes stimulated by EGF or arachidonic acid released PGE2 and PGF2 alpha into the culture medium. This was diminished if 0.1 mM indomethacin was also in the medium. The importance of autocrine regulation of hepatocyte growth by prostaglandins is discussed.     

Alteration of rheological properties of human erythrocytes by crosslinking of membrane proteins

The crosslinking of membrane proteins of human erythrocytes by diamide (diazene dicarboxylic acid bis(N,N-dimethylamide) ) was quantified by 4% polyacrylamide gel electrophoresis in 1% sodium dodecyl sulfate. The relation between the crosslinking of membrane proteins and erythrocyte functions (rheological and oxygen transporting) was quantitatively examined. (i) The crosslinking of membrane protein was induced by diamide, without changing the shape and the contents of intracellular organic phosphates (adenylates and 2,3-diphosphoglycerate). The intensity of spectrin 2 in SDS-polyacrylamide gel electrophoresis decreased proportionally to diamide concentration. The percentage decrease in spectrin 2 (using band 3 as an internal standard) was the most appropriate indicator for crosslinking ("% crosslinking'). (ii) The suspension viscosity of erythrocytes increased in proportion to the percentage of crosslinking, in the range of applied shear rates of 3.76-752 s-1. (iii) Erythrocyte deformability (measured by a high-shear rheoscope) was reduced by the crosslinking. The change was detectable even at 5% crosslinking. (iv) Rouleaux formation (measured by a television image analyzer combined with a low-shear rheoscope) was inhibited by the crosslinking. The inhibition was also sensitively detected at more than 5% crosslinking. (v) Hemoglobin in erythrocytes was chemically modified by higher dose of diamide (probably by the binding of diamide with sulfhydryl groups). Also the oxygen affinity of hemoglobin increased and the heme-heme interaction decreased. (vi) The reduction of the crosslinking of membrane proteins by dithiothreitol apparently reversed the intensity of spectrin bands in SDS-polyacrylamide gel electrophoresis and the erythrocyte functions (the suspension viscosity and the deformability), though not completely.     

The effect of Ca++ on cyclic nucleotide phosphodiesterases of superior cervical ganglion.

Cyclic nucleotide phosphodiesterase was examined in canine and bovine superior cervical ganglia. Activity in crude supernatant fractions was only slightly stimulated by Ca++ despite the presence of protein activating factor. Three forms of phosphodiesterase were resolved from bovine ganglia supernatant extracts by chromatography on DEAE-cellulose. The first enzyme eluted, (DI), was almost completely specific for cyclic GMP, while the other two (DII and DIII), hydrolyzed both cyclic AMP and cyclic GMP; all were free of heat-stable protein activator. Each enzyme was inhibited by low concentrations of Ca++ in the assay medium. Inhibition by Ca++ was reversed by addition of protein activator, but activity did not increase above the control level. Cyclic AMP hydrolysis by enzyme DII was stimulated by micromolar concentrations of cyclic GMP. This stimulation was reduced by Ca++ unless protein activator was present.     

Subunit Structure of Glucoamylase of Saccharomyces diastaticus

Extracellular glucoamylase produced by a starch-fermenting yeast, Saccharomyces diastaticus 5106-9A, was purified. The enzyme was found to be heterogeneous in molecular weight, ranging from approximately 80K to 66K as estimated by gel filtration, and consisted of two subunits, H and Y. The molecular weight of subunit H was heterogeneous and was determined to be approximately 68K, 59K, and 53K by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of subunit Y was 14K, estimated by the same gel. the molecular weight of the deglycosylated form of subunit H was 41K, suggesting that the heterogeneity of the enzyme was due to glycosyl moieties of subunit H. Subunits H and Y were separated by gel filtration in the presence of sodium dodecyl sulfate. Subunit Y seemed to be hydrophobic, since it was insoluble in an aqueous buffer without detergent.     

Involvement of calmodulin in phagocytotic respiratory burst of leukocytes

The superoxide release of guinea pig exudate leukocytes induced by phagocytosis or by stimulation with cytochalasin D, digitonin or calcium ionophore A23187 was completely inhibited by the inhibitors of calmodulin-stimulated processes such as trifluoperazine at 10 μM. A particulate NADPH-dependent superoxide-forming enzyme from the cytochalasin D-stimulated cells was also inhibited by the inhibitors and by EGTA. The activation of heart phosphodiesterase by a boiled extract of the cells which was dependent on calcium ions and abolished by trifluoperazine was observed. These results suggest the presence of calmodulin in leukocytes and its possible role in the stimulation of the superoxide formation.     

An electron-microscopical analysis of capture and initial stages of penetration of nematodes byArthrobotrys oligospora

A detailed analysis was made of the capture and subsequent penetration of nematodes by the nematophagous fungusArthrobotrys oligospora using different electron-microscopical techniques. Capture of nematodes by this fungus occurred on complex hyphal structures (traps) and was effectuated by an adhesive coating, present on these trap cells. The adhesive layer was largely fibrillar in nature and was absent on cells of normal hyphae. Following capture, penetration hyphae were formed at those sites where the trap cell wall was anchored to the nematode cuticle by the adhesive. New walls of these hyphae were formed underneath the original trap cell walls, which were partly hydrolysed to allow growth and development of the penetration tubes through the adhesive coating towards the cuticle. Our observations indicated that the cuticle of the nematode was subsequently penetrated by the penetration tubes by mechanical means. After penetration a large infection bulb was formed from which trophic hyphae arose. Cytochemical experiments indicated that the sites of penetration of the cuticle were intensely stained for acid phosphatase activity. At later stages of infection activity of this enzyme was present throughout the nematode contents; the enzyme was most probably secreted by complex membranous structures associated with the cytoplasmic membrane of the infection bulb and the trophic hyphae.     

Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin.

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.     

Uptake and output of various forms of choline by organs of the conscious chronically catheterized sheep.

The net uptake and output of plasma unesterified choline, glycerophosphocholine, phosphocholine and lipid choline by organs of the conscious chronically catheterized sheep were measured. There was significant production of plasma unesterified choline by the upper- and lower-body regions and the alimentary tract and uptake by the liver, lungs and kidneys. The upper- and lower-body regions drained by the venae cavae provided the bulk (about 82%) of the total body venous return of plasma unesterified choline. Production of plasma unesterified choline by the alimentary tract was approximately balanced by the plasma unesterified choline taken up by the liver, and was almost equal to the amount of choline secreted in the bile. There was a considerable amount of glycerophosphocholine in the liver and there was production of plasma glycerophosphocholine by the liver and uptake by the lungs and kidneys. Glycerophosphocholine was higher in the plasma of sheep than in that of rats. Plasma phosphocholine was produced by the alimentary tract and kidneys. There was production of plasma lipid choline by the upper- and lower-body regions drained by the venae cavae. The results suggest that the sheep synthesizes substantial amounts of choline in ectrahepatic tissues and has the capacity for extensive retention and recycling of bile choline. These observations, coupled with a slow turnover of the endogenous choline body pool, explain the low requirement of sheep for dietary choline in contrast with non-ruminant species.