Glucose-sensitive adenylate cyclase in toluene-treated cells of Escherichia coli B.

Toluene treatment of Escherichia coli B makes it possible to measure adenylate cyclase activity directly using [alpha-32-P]-ATP as substrate. In contrast to French press extracts, the activity of adenylate cyclase in toluene-treated cells shows many of the characteristics of the enzyme seen in the intact cell. In both toluene-treated and intact cells the activity of adenylate cyclase is inhibited at least 85% by glucose, while in French press extracts the enzyme activity is much lower and is not sensitive to inhibition by glucose. In toluene-treated cells, glucose inhibits at 10 muM, and the effect is rapid in onset and readily reversible. The activity is not inhibited by glucose 6-phosphate suggesting that glucose is responsible for the inhibition. The measurement of the activity and sensitivity to glucose of adenylate cyclase in toluene-treated cells requires the presence of potassium phosphate in the assay medium. Since it does not increase the activity or sensitivity of the enzyme in the French press extract, it is suggested that potassium phosphate is required for the maintenance of cellular integrity necessary for the activity and sensitivity of adenylate cyclase.     

Ammonium inhibition of nitrogenase activity in Herbaspirillum seropedicae.

The effect of oxygen, ammonium ion, and amino acids on nitrogenase activity in the root-associated N2-fixing bacterium Herbaspirillum seropedicae was investigated in comparison with Azospirillum spp. and Rhodospirillum rubrum. H. seropedicae is microaerophilic, and its optimal dissolved oxygen level is from 0.04 to 0.2 kPa for dinitrogen fixation but higher when it is supplied with fixed nitrogen. No nitrogenase activity was detected when the dissolved O2 level corresponded to 4.0 kPa. Ammonium, a product of the nitrogenase reaction, reversibly inhibited nitrogenase activity when added to derepressed cell cultures. However, the inhibition of nitrogenase activity was only partial even with concentrations of ammonium chloride as high as 20 mM. Amides such as glutamine and asparagine partially inhibited nitrogenase activity, but glutamate did not. Nitrogenase in crude extracts prepared from ammonium-inhibited cells showed activity as high as in extracts from N2-fixing cells. The pattern of the dinitrogenase and the dinitrogenase reductase revealed by the immunoblotting technique did not change upon ammonium chloride treatment of cells in vivo. No homologous sequences were detected with the draT-draG probe from Azospirillum lipoferum. There is no clear evidence that ADP-ribosylation of the dinitrogenase reductase is involved in the ammonium inhibition of H. seropedicae. The uncoupler carbonyl cyanide m-chlorophenylhydrazone decreased the intracellular ATP concentration and inhibited the nitrogenase activity of whole cells. The ATP pool was not significantly disturbed when cultures were treated with ammonium in vivo. Possible mechanisms for inhibition by ammonium of whole-cell nitrogenase activity in H. seropedicae are discussed.     

Phenylalanine hydroxylase in melanoma cells.

A pigmented subclone of Cloudman S91 melanoma cells, PS1-wild type, can grow in medium lacking tyrosine. This ability is conferred by phenylalanine hydroxylase activity, and not by tryptophan hydroxylase, tyrosine hydroxylase or tyrosinase activities, although the latter activity is also present in these cells. Conversion of phenylalanine to tyrosine was measured in living cells by chromatographic identification of the metabolites of [14C]phenylalanine and in cell extracts using a sensitive assay for phenylalanine hydroxylase. Phenylalanine hydroxylase activity in melanoma cell extracts was identified by its inhibition with p-chlorophenylalanine and not with 6-fluorotryptophan, 3-iodotyrosine, phenylthiourea, tyrosine or tryptophan; and by adsorption with antiserum prepared against purified rat liver phenylalanine hydroxylase, and migration of immunoprecipitable activity with authentic phenylalanine hydroxylase subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Papain solubilization of the Epstein-Barr virus-induced membrane antigen.

The Epstein-Barr virus (EBV)-induced membrane antigen (MA) was successfully solubilized from the membranes of viable EBV-infected Raji cells by treatment with papain (5 to 6 U per 1 X 10(7) to 2 X 10(7) cells). The loss of MA from viable cells was monitored by membrane immunofluorescence and antibody-dependent cellular cytotoxicity. Soluble MA was demonstrated in papain digests through inhibition of antibody-dependent cellular cytotoxicity and by inhibition of the binding of anti-MA antibodies to cells as detected by use of 125I-labeled staphylococcal protein A. Approximately 75% of the MA activity in the extracts was not sedimentable at 100,000 X g,, indicating that the majority of EBV MA activity that was released by this procedure was associated with small-molecular-weight material. Antiserum prepared from an owl monkey immunized with these papain extracts contained antibody to MA and neutralizing antibodies, but lacked detectable antibodies against viral capsid antigens and EBV-induced early antigens.     

小桐子提取物除草活性的生物测定

为全面了解小桐子(Jatropha curcas L. )提取物的除草活性,以萝卜(Raphanus sativus L. )、苋(Amaranthus tricolor L. )、苏丹草[Sorghum sudanense (Piper) Stapf]和黑麦草(Lolium perenne L. )为实验材料,对小桐子果壳和枝叶的水、乙醇(体积分数95%)、正丁醇、乙酸乙酯、氯仿和石油醚粗提物的除草活性进行了生物测定,并从中筛选出抑制作用最强的水粗提物进行进一步的活性组分分离及其除草活性的生物测定.测定结果显示,小桐子果壳和枝叶的6种溶剂提取物(10 g·L~(-1))对供试的4种植物幼苗的根长和茎高均有不同程度的抑制作用,其中水粗提物和乙醇(体积分数95%)粗提物的抑制作用较强,且水粗提物对供试的4种植物幼苗的根长和茎高的抑制作用均在75%以上,显著高于其他溶剂粗提物(P<0.05);石油醚粗提物的抑制作用最小,均在10%以下.小桐子果壳和枝叶水粗提物的石油醚、氯仿、乙酸乙酯、正丁醇和水萃取物(10 g·L~(-1))对萝卜和苏丹草幼苗的根长和茎高均有不同程度的抑制作用,其中水、正丁醇和乙酸乙酯萃取物的抑制作用显著高于氯仿和石油醚萃取物,抑制率均在70%以上;水萃取物的抑制作用最强,抑制率均在80%以上;石油醚萃取物的抑制作用最小,抑制率均在10%以下.研究结果表明,小桐子果壳和枝叶的水粗提物具有一定的除草活性,其有效成分为极性较大的组分.     

黄芩提取物抗氧化性及其对肝癌HepG-2细胞的抑制作用

本研究探讨黄芩提取物的抗氧化能力以及对肝癌细胞HepG-2生长的影响。通过分光光度法、邻苯三酚自氧化法测定不同黄芩提取物对DPPH自由基、超氧阴离子的清除效果,以此考察其抗氧化性能;同时采用MTT法测定不同黄芩提取物对HepG-2细胞的生长影响来研究其抗肿瘤作用。结果显示,3种不同的黄芩提取物均具有较好的抗氧化性能,并对肝癌细胞的生长具有明显的抑制作用,黄芩正丁醇部分、黄芩乙酸乙酯部分及黄芩石油醚部分对肝癌细胞作用72h后,对HepG-2细胞的最大抑制率分别达到57.2%、51.8%和35.2%。3种黄芩提取物具有较强的抗氧化活性,且对肝癌细胞的生长有一定的抑制作用。     

Reversal of 2-bromoethanesulfonate inhibition of methanogenesis in Methanosarcina sp.

2-Bromoethanesulfonate (BES) inhibition of methanogenesis from methanol by resting-cell suspensions or cell extracts of Methanosarcina was reversed by coenzyme M. BES inhibition of methylcoenzyme M methylreductase activity in cell-free extracts was reversed by methylcoenzyme M but not by coenzyme M. Methanol/coenzyme M methyltransferase activity was not inhibited by 10 microM BES. Inhibition of methylreductase by BES and 3-bromopropionate was competitive with methylcoenzyme M, but inhibition by 2-bromoethanol exhibited mixed kinetics. The Ki values for the inhibitors in cell-free extracts were similar to the concentrations which inhibited intact cells. BES-resistant mutants of strain 227 were apparently permeability mutants because in vitro assays showed that mutant and parent strain methylreductases were equally sensitive to BES.     

Metabolism of hypoxanthine in isolated rat hepatocytes.

The hepatic metabolism of hypoxanthine was investigated by studying both the fate of labelled hypoxanthine, added at micromolar concentrations to isolated rat hepatocyte suspensions, and the kinetic properties of purified hypoxanthine/guanine phosphoribosyltransferase from rat liver. More than 80% of hypoxanthine was oxidized towards allantoin; less than 5% of the label was incorporated into the purine mononucleotides, and a similar proportion appeared transiently in inosine. The maximal velocity of oxidation (approx. 750nmol/min per g of cells) was in close agreement with the known activity of xanthine oxidase in liver extracts. In contrast, the maximal velocity of the incorporation of labelled hypoxanthine into mononucleotides reached only 30nmol/min per g of cells, compared with an activity of hypoxanthine/guanine phosphoribosyltransferase, measured at substrate concentrations analogous to those prevailing intracellularly, of 500nmol/min per g of cells. Hypoxanthine incorporation into the mononucleotides was decreased by allopurinol, anoxia and ethanol, despite inhibition of its oxidation under these conditions; it was increased by incubation of the cells in supraphysiological concentrations of Pi. Allopurinol and anoxia decreased the concentration of phosphoribosyl pyrophosphate inside the cells by respectively 40 and 60%, ethanol had no effect on the concentration of this metabolite and Pi increased its concentration up to 10-fold. The kinetic study of purified hypoxanthine/guanine phosphoribosyltransferase showed that a mixture of ATP, IMP, GMP and GTP, at the concentrations prevailing in the liver cell, decreased the V max. of the enzyme 6-fold, increased its Km for hypoxanthine from 1 to 4 microM and its Km for phosphoribosyl pyrophosphate from 2.5 to 25 microM. In the presence of 5 microM-hypoxanthine and 2.5 microM-phosphoribosyl pyrophosphate, the mixture of nucleotides inhibited the activity of purified hypoxanthine/guanine phosphoribosyltransferase by 95%. It is concluded that this inhibition results in a limited participation of hypoxanthine/guanine phosphoribosyltransferase in the control of the production of allantoin by the liver.     

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.     

Alkene monooxygenase from Mycobacterium: a multicomponent enzyme.

A NADH- or NADPH-dependent alkene monooxygenase (AMO) activity has been detected in cell-free extracts of the ethene-utilizing Mycobacterium E3 and Mycobacterium aurum L1. The activity was not linear with protein concentration in the assay suggesting AMO is a multicomponent enzyme. The inhibition pattern of AMO activity was very similar to the inhibition patterns published for the three-component soluble methane monooxygenases. Fractionation of crude extracts revealed that combination of two fractions was required to restore alkene monooxygenase activity. The first fraction was inhibited by acetylene, indicating it contained an oxygenase component. The second fraction contained reductase activity which was absent from non-induced cells. This reductase activity is probably the NADH-acceptor reductase of AMO.     

Purification of an endonuclease from adenovirus-infected KB cells.

This report describes the purification of an endonuclease from extracts of adenovirus-type-2-infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus-infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus-infectedcells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated. The endonuclease in crude extracts from uninfected or adenovirus-infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus-type-2-infected KB cells. A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.     

反枝苋水浸提液与挥发油对黄瓜根尖的影响

采用悬空气法研究了在入侵植物反枝苋(Amaranthus retroflexus L.)水浸提液和挥发油作用下,黄瓜根缘细胞活性、根冠果胶甲基酯酶(PME)、根尖过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)以及丙二醛(MDA)含量的变化规律.结果表明:反枝苋水浸提液对黄瓜根的生长无显著性影响而挥发油显著抑制黄瓜根的生长,且随浓度增大抑制作用显著增强.PME活性随着水浸提液浓度的增大呈先上升后下降趋势,而随着挥发油浓度的升高呈现逐渐上升的趋势;水浸提液和挥发油均降低了对根缘细胞的存活率,这种抑制作用随浓度的增加而增大;随着处理液浓度增大,黄瓜根尖中MDA含量、CAT活性整体表现为增加,SOD活性先升高后降低,POD活性与对照差异不显著.反枝苋挥发油的化感效应大于水浸提液的化感效应.     

红树植物桐花树叶片多酚提取物对酪氨酸酶活性抑制及抗自由基和抗菌活性分析

采用超声波辅助-乙酸乙酯提取方法获得桐花树〔Aegiceras corniculatum(Linn.)Blanco〕叶片多酚提取物,研究了该提取物对酪氨酸酶活性的抑制作用及其动力学特征,并分析了该提取物对DPPH·自由基的清除作用及其抗菌活性。结果显示:多酚提取物得率约为(122.0±31.4)mg·g-1,提取物中多酚含量约为(521.8±17.2)mg·g-1。该提取物对酪氨酸酶活性的抑制作用呈明显正相关的量效关系,IC50为0.650 g·L-1,与阳性对照槲皮素对酪氨酸酶活性的抑制能力相近;该提取物通过降低酶活性实现对酪氨酸酶活性的抑制,且该抑制作用具有可逆性;随提取物质量浓度的提高酶促反应Km值增大、vm值减小,其动力学特征符合混合Ⅰ型抑制类型;对游离酶的抑制常数Ki为0.833 g·L-1,对酶-底物络合物的抑制常数Kis为1.823 g·L-1,表明该提取物与游离酶的亲和力大于其与酶-底物络合物的亲和力。随质量浓度提高,该提取物对DPPH·自由基的清除率逐渐增大并在0.000~0.600 g·L-1范围内呈明显量效关系,且IC50为0.304 g·L-1,与0.036 g·L-1槲皮素等效,显示该多酚提取物对自由基的清除能力较强。随质量浓度提高,该提取物对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)和枯草芽孢杆菌(Bacillus subtilis)的抑菌圈直径均逐渐增大,显示该提取物对3种供试菌均有抑菌作用,最小抑菌浓度均为25 g·L-1。结果表明:通过深入的开发研究,桐花树叶片多酚提取物可作为兼具辅助防腐抑菌功能的新型酪氨酸酶抑制剂。     

Biological activities from extracts of endophytic fungi isolated from Viguiera arenaria and Tithonia diversifolia

A total of 39 endophytic fungi have been isolated from Viguiera arenaria and Tithonia diversifolia, both collected in S?o Paulo State, Brazil. The isolates were identified based on their ribosomal DNA sequences. The ethyl acetate (EtOAc) extracts of all endophytic fungi were evaluated for their antimicrobial, antiparasitic and antitumoral activity. Antimicrobial screening was conducted using an agar diffusion assay against three pathogenic microorganisms: Staphylococcus aureus, Escherichia coli and Candida albicans. Antiparasitic activity was determined by enzymatic inhibition of gGAPDH of Trypanosoma cruzi and adenine phosphorybosiltransferase (APRT) of Leishmania tarentolae. Antitumoral activity was tested against human T leukemia cells by the Mosmann colorimetric method. All extracts showed activity in at least one assay: 79.5% of the extracts were cytotoxic against leukemia cells, 5.1% of the extracts were active against S. aureus, 25.6% against E. coli and 64.1% against Candida albicans. Only one extract showed promising results in the inhibition of parasitic enzymes gGAPDH (95.0%) and three were found to inhibit APRT activity. The cytotoxic extract produced by the strain VA1 (Glomerella cingulata) was fractionated and yielded nectriapyrone and tyrosol. Nectriapyrone showed relevant cytotoxic activity against both human T leukemia and melanoma tumor cell lines.     

Inhibition of plant telomerase by telomere-binding proteins from nuclei of telomerase-negative tissues

The activity of telomerase in plant cells is precisely regulated in response to changes in cell division rate. To explore this regulatory mechanism, the effect on telomerase activity of protein extracts from nuclei of telomerase-negative tissues was examined. An inhibition of telomerase activity was found which was species-non-specific. This inhibition was due to proteins which form salt-stable, sequence-specific complexes with the G-rich telomeric strand and reduce its accessibility, as shown by gel retardation and by terminal transferase (TdT) extension of G-rich telomeric and non-telomeric (substrate) primers. A 40 kDa polypeptide was detected by SDS-PAGE after cross-linking the complex formed by extracts from tobacco leaf nuclei. Such proteins may be involved in regulation of telomerase activity in plants.     

Glucosaminephosphate synthase of human liver.

Although human liver contains glucosaminephosphate synthase (glucosaminephosphate isomerase (glutamine-forming), EC 5.3.1.19), its activity is rapidly lost during the course of extraction. The inactivation, however, is largely prevented if the extraction medium contains isopropanol at 1% concentration; using these "stabilized" extracts, the glucosaminephosphate synthase activity of human liver has been shown to be similar to the activity previously reported in rat liver. The enzyme precipitated from these extracts by (NH4)2SO4 is inhibited by UDP-N-acetylglucosamine, the concentration required to produce a half-maximal inhibition being 6 muM. These results seem to be sufficient to postulate that glucosaminephosphate synthase is important for UDP-N-acetylglucosamine synthesis in human liver. In contrast to the rat liver enzyme, the (NH4)2SO4-precipitated human liver enzyme is resistant to trypsin and undergoes no conversion reaction when incubated with glucose 6-phosphate.     

Feedback inhibition of nitrogenase.

No inhibition of nitrogenase activity by physiological levels of NH4+ or carbamyl phosphate was observed in extracts of Azotobacter vinelandii. All of the 15N2 reduced by cultures which received no NH4+ was found in the cells. By contrast, more than 95% of the 15N2 reduced by cultures which had been given NH4+ was found in the medium. Failure to examine the culture medium would lead to the erroneous conclusion that N2 fixation is inhibited by NH4+. Nitrogenase in a derepressed mutant strain of A. vinelandii was fully active in vivo in the presence of NH4+. The addition of NH4Cl to N2-fixing cultures resulted in no decrease in the N2-reducing activity of intact cells of Klebsiella pneumoniae or Clostridium pasteurianum and only a small (15%) decrease in A. vinelandii. Therefore, no significant inhibition of nitrogenase by NH4+ or metabolites derived from NH4+ exists in A. vinelandii, K. pneumoniae, or C. pasteurianum.     

A kinetic study of glycerophosphate acyltransferase of rat adipocytes in relation to its control by noradrenaline.

Glycerophosphate acyltransferase present in an extract of rat adipocytes is strongly inhibited by excess palmitoyl-CoA. This inhibition is released by serum albumin but an excess of serum albumin is inhibitory, particularly at low palmitoyl-CoA concentrations. An optimal activity is reached when the ratio palmitoyl-CoA/albumin is in the range of 3-6. In the absence of albumin, oleic acid inhibits the activity at all palmitoyl-CoA concentrations. This inhibition is released by albumin and, inversely, oleic acid releases the inhibition by high concentrations of albumin. Another effect of fatty acids is to favour the inactivation of the glycerophosphate acyltransferase in extracts of adipocytes kept at 0 degree C. This inactivation is time-dependent and cannot be reversed by the addition of albumin to the assay mixture. Treatment of adipocytes with noradrenaline had no effect on the activity of the enzyme as long as the cells had been separated from fatty acids and albumin. With extracts of unwashed cells, the effect of noradrenaline on both the activity and stability of glycerophosphate acyltransferase could be explained by the presence of fatty acids in the extract.     

Enzyme activity and bacteriophage infection. I. Breakdown of adenosinetriphosphate

Experiments have been performed on the apyrase activity of E. coli, strain B. Although the dependence on pH and substrate is similar to that of rat tissue, the bacterial extracts are inhibited by Ca⁺⁺ and stimulated by Mg⁺⁺. In bacterial extracts the rate of phosphate release decreases in the course of the reaction, possibly owing to product inhibition. With multiple bacteriophage infection, the apyrase activity of the intact cells increased several fold, and the activity of extracts increased about 30 per cent. It is suggested that the changes could be attributed to an increase in the amount of enzyme although other alternatives cannot be precluded at present.     

Inhibition of purine nucleoside phosphorylase and mitogen-stimulated transformation in immunocompetent murine spleen cells by formycin B.

Formycin B inhibits competitively purine nucleoside phosphorylase activity in murine spleen cell extracts. It also inhibits inosine phosphorolysis by intact spleen cells. Differentiation and proliferation of these cells, stimulated by concanavalin A or lipopolysaccharide, are appreciably reduced by culture with formycin B. Appreciable inhibition occurs at concentrations of formycin B which do not alter cell viability. The transformation of lipopolysaccharide-stimulated cells is more sensitive to inhibition by formycin B than that of concanavalin A-stimulated cells. Characterization of this system should increase our understanding of the relationship between purine nucleoside phosphorylase activity and immune responses.