An NAD:cysteine ADP-ribosyltransferase is present in human erythrocytes

A novel enzymatic activity, i.e., the catalysis of the formation of ADP-ribosylcysteine, was found in the cytosol of human erythrocytes. The NAD:cysteine ADP-ribosyltransferase was partially purified by sequential chromatographic steps on phenyl-Sepharose, phosphocellulose, and Sepharose CL-6B. The enzyme has an apparent molecular weight of 27,000 +/- 3,000, as determined by gel permeation. The formation of ADP-ribosylcysteine was associated with the stoichiometric release of nicotinamide from NAD. The enzyme was found to be highly specific toward cysteine and cysteine methyl ester as ADP-ribose acceptors. S-Benzoyl-L-cysteine, cystine, histidine, glutamic acid, arginine, arginine methyl ester, and agmatine were ineffective as acceptors for this enzyme.     

Escherichia coli produces linoleic acid during late stationary phase.

Escherichia coli produces linoleic acid in the late stationary phase. This was the case whether the cultures were grown aerobically or anaerobically on a supplemented glucose-salts medium. The linoleic acid was detected by thin-layer chromatography and was measured as the methyl ester by gas chromatography. The linoleic acid methyl ester was identified by its mass spectrum. Lipids extracted from late-stationary-phase cells generated thiobarbituric acid-reactive carbonyl products when incubated with a free radical initiator. In contrast, extracts from log-phase or early-stationary-phase cells failed to do so, in accordance with the presence of polyunsaturated fatty acid only in the stationary-phase cells.     

藏红花花瓣和雄蕊挥发油化学成分GC-MS分析及比较

采用水蒸气蒸馏法从藏红花花瓣和雄蕊中提取挥发油,用GC-MS技术结合计算机检索对其二者化学成分进行分离和鉴定,用色谱峰面积归一化法计算各组分的相对含量.花瓣中共鉴定出16种化合物,主要成分为正二十六烷(11.60％)、正十五烷(11.31％)、棕榈酸甲酯(10.82％)、油酸甲酯(10.35％)、2,4-二叔丁基苯酚(9.63％)、亚油酸甲酯(7.18％)、藏红花醛(5.66％);雄蕊中共鉴定出20种化合物,主要成分为油酸甲酯(30.83％)、亚油酸甲酯(24.12％)、环已醇(16.80％)、硬脂酸甲酯(12.88％)、棕榈酸甲酯(8.97％)、花生酸甲酯(1.18％)、苯并噻唑(1.01％).     

花生△12脂肪酸脱氢酶基因高效表达载体构建与原核表达

花生是世界范围内广泛种植的重要油料作物之一,其种子中富含油酸和亚油酸。△^12脂肪酸脱氢酶（FAD2）是亚油酸合成的关键酶,催化油酸（18：1）在△^12位上脱氢生成亚油酸（18：2）,但由于△^12脂肪酸脱氢酶本身的特性,目前还没有有效的方法将其纯化并在蛋白水平作进一步的研究,尚需对其结构和功能之间以及表达调控进行更深入全面的研究。本文利用从花生中克隆的△^12脂肪酸脱氢酶基因（GenBank接受号为AY1006）构建高效表达载体,把花生△^12脂肪酸脱氢酶基因全长序列插入到大肠杆菌高效表达载体pRSETB中,构建了pRSET／HO-A融合表达载体,并转化到大肠杆菌表达菌BL21（DE3）pLysS中,在IPTG诱导下,pRSET／HO-A融合表达载体在BL21（DE3）pLysS菌株中高效表达了△^12脂肪酸脱氢酶。利用Clon-Tech蛋白纯化Kit进一步分离了目的蛋白,同时加入外源性底物油酸在20℃温育6h后,进行脂肪酸甲酯化处理,通过气相色谱（GC）和气相色谱,质谱（GC-MS）分析表明,所编码的酶具有△^12脂肪酸脱氢酶的活性,能将外源性的底物油酸转化为亚油酸,转化率为11．8％。花生△^12脂肪酸脱氢酶基因的原核表达目前国内外还未见报导,本实验为其进一步的大量纯化和结构功能分析奠定了基础。     

Cyclization of natural allene oxide in aprotic solvent: formation of the novel oxylipin methyl cis-12-oxo-10-phytoenoate

Allene oxide, (9Z,11E)-12,13-epoxy-9,11-octadecadienoic acid (12,13-EOD), was prepared by incubation of linoleic acid (13S)-hydroperoxide with flaxseed allene oxide synthase (AOS) and purified (as methyl ester) by low temperature HPLC. Identification of pure 12,13-EOD was substantiated by its UV and (1)H NMR spectra and by GC-MS data for its methanol trapping product. The methyl ester of 12,13-EOD (but not the free carboxylic acid) is slowly cyclized in hexane solution, affording a novel cyclopentenone cis-12-oxo-10-phytoenoic acid. Free carboxylic form of 12,13-EOD does not cyclize due to the exceeding formation of macrolactone (9Z)-12-oxo-9-octadecen-11-olide. The spontaneous cyclization of pure natural allene oxide (12,13-EOD) into cis-cyclopentenone have been observed first time.     

Evidence in the formation of conjugated linoleic acids from thermally induced 9t12t linoleic acid: a study by gas chromatography and infrared spectroscopy

Thermally induced isomerisation leading to the formation of conjugated linoleic acids (CLAs) has been observed for the first time during the thermal treatment of 9t12t fatty acid triacylglycerol, and methyl ester. Fifteen microlitre portions of the triacylglycerol sample containing 9t12t fatty acid (trilinoelaidin) were placed in micro glass ampoules and sealed under nitrogen, then subjected to thermal treatment at 250 °C. The glass ampoules were removed at regular time intervals, cut open, and the contents were analysed by infrared spectroscopy using a single reflectance attenuated total internal reflectance crystal accessory. The samples were then subjected to derivatisation into their methyl esters. The methyl esters of the isomerised fatty acids were analysed by gas chromatography. The same procedure was repeated with methyl ester samples containing 9t12t fatty acid (methyl linoelaidate). Each sample was subjected to infrared measurements and gas chromatographic analysis after appropriate dilution in heptane.The results show that the thermally induced isomerisation of 9t12t fatty acids from both triacylglycerol molecules and methyl esters give identical CLA profiles as those found for the thermally induced isomerisation of 9c12c fatty acids. The infrared spectrometry provides additional evidence confirming the formation of CLA acids during thermal treatment. A mechanism for the formation of the CLAs from 9t12t fatty acid molecules is also formulated for the first time. This mechanism complements the pathways of formation of CLAs from 9c12c fatty acids during thermal treatment.     

Evidence for participation of cytochrome b5 in microsomal delta-6 desaturation of fatty acids.

The delta-6 desaturation of linoleic acid to gamma-linolenic acid and oleic acid to 6,9-octadecadienoic acid by rat liver microsomes was investigated. Using a specific antibody prepared against purified rat liver cytochrome b5, we demonstrated that cytochrome b5 participated in delta-6 desaturation of both fatty acids. The reaction products were identified as their methyl ester derivatives by argentation thin-layer chromatography, gas-liquid chromatography, and reductive ozonolysis followed by gas-liquid chromatography.     

Chelation-controlled regioselectivity in the lanthanum-promoted monobenzoylation of monosaccharides in water

Monosaccharides are selectively converted to monobenzoates in a base-catalyzed reaction with benzoyl methyl phosphate (BzMP) and a lanthanum salt in water. Yields are reported in terms of formation of the ester, which competes with hydrolysis of BzMP, to give an estimate of the efficiency of the conversion of the sugar. Higher conversions can be achieved using excess reagent. Regioselectivity is influenced by the structure of the glycoside. For example, the reaction leads to different product distributions from alpha- and beta-anomers of the glycosides. The reaction combination provides a basis for efficient ester formation in specific geometric situations, providing a means of identification as well as modification.     

楮实子油的化学成分及含量分析

楮实子为桑科植物构树[Broussonetia papyrifera(L.)Vent.]的干燥成熟果实,在中国分布广泛[1],具有补肾清肝、明目、利尿的功效,临床上用于治疗腰膝酸软、虚劳骨蒸、头晕目昏、目生膜、水肿胀满,还可以治疗顽癣、神经性皮炎、湿疹等皮肤病,<名医别录>载为上品[2].楮实子不仅具有开发治疗老年性疾病新药的潜力,还可以制成美容保健类的产品[3-5].     

Occurrence of a New Lipoxygenase Isoenzyme in Germinating Barley Embryos

Partially purified preparations of lipoxygenase from the germinating barley embryos converted linoleic acid to 9- and 13-hydroperoxy linoleic acids in the ratio of approximately 3:1, while the similar preparations from the ungerminated embryos converted linoleic acid mainly to 9-hydroperoxy linoleic acid.

Isoelectric focusing of the partially purified preparations of the germinating embryos revealed the presence of the two lipoxygenase active peaks, having isoelectric point at pH 4.9 and 6.6, respectively. The former peak (barley lipoxygenase-1) was identical to lipoxygenase of the ungerminated embryos, but the latter peak (barley lipoxygenase-2) was found only in the germinating embryos. The newly found isoenzyme, barley lipoxygenase-2, converted linoleic acid mainly to 13-hydroperoxy linoleic acid, and could oxidize esterified derivatives of linoleic acid (methyl linoleate and trilinolein) much strongly than barley lipoxygenase-1.     

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.     

Hydroperoxide isomers and ketohydroxy product from oxidation of linoleic acid by eggplant lipoxygenase

Hydroperoxides produced by oxidation of linoleic acid with purified eggplant lipoxygenase were separated by TLC and analysed by IR spectroscopy. The methyl hydroxystearates from the enzymatically produced hydroperoxides were analysed by MS and GLC. Both analyses indicated that the eggplant enzyme converted linoleic acid almost exclusively (96%) into the 13-hydroperoxy isomer whereas the 9-hydroperoxy isomer was only a minor product (4%). HPLC of the methyl ester of the isolated hydroperoxides showed three components. Each component was collected, reduced to methyl hydroxystearate and characterized by GLC, MS and IR analysis. The components were identified as 13-hydroperoxy cis-trans isomer (92.8%), 13-hydroperoxy trans-trans isomer (2.6%) and 9-hydroperoxy cis-trans isomer (4.6%). A polar by-product present in the reaction mixture was identified by IR, ¹H NMR, and MS (of the toluene-p-sulphonyl derivative) as 13-hydroxy-12-oxo-octadec-cis-9-enoic acid.     

Sequential enzymes of linoleic acid oxidation in corn germ: lipoxygenase and linoleate hydroperoxide isomerase

Linoleic acid oxidation catalyzed by lipoxygenase (lipoxidase) activity in extracts of defatted corn germ does not terminate in the product, linoleic acid hydroperoxide, unless the lipoxygenase is first partially purified. If purification is not attempted, the hydroperoxide product exists only as a barely detectable intermediate in the synthesis of three products. One of these was identified as 9-hydroxy-10-oxo-cis-12-octadecenoic acid formed from the hydroperoxide by the enzyme, linoleate hydroperoxide isomerase. Another product, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, is believed to be formed by an isomerase also. The third product was the linoleate ester of one of the hydroxy-oxo-fatty acids, 9-(cis-9,cis-12-octadecadienoyl)-10-oxo-cis-12-octadecenoic acid. It is not known if the synthesis of the ester is enzyme-catalyzed. When a mixture of 13-hydroperoxy-cis-9,trans-11-octa-decadienoic acid and 9-hydroperoxy-trans-10,cis-12-octa-decadienoic acid from soybean lipoxygenase oxidation of linoleic acid was used as a substrate, 13-hydroxy-12-oxo-cis-9-octadecenoic acid and 9-hydroxy-12-oxo-trans-10-octadecenoic acid were formed as the major products of catalysis by linoleate hydroperoxide isomerase(s) from corn. Smaller quantities of 9-hydroxy-10-oxo-cis-12-octadecenoic acid and 13-hydroxy-10-oxo-trans-11-octadecenoic acid were also formed.     

Vitamin B12-dependent Methionine Biosynthesis and Its Metabolic Role in Corynebacterium simplex ATCC 6946, a Vitamin B12-producing and Hydrocarbon-utilizable Bacterium

A vitamin B₁₂-dependent N⁵-methyltetrahydrofoIate-homocysteine methyltransferase was found in cell-free extracts of Corynebacterium simplex ATCC 6946 grown aerobically in a medium containing hydrocarbon as a sole carbon source and the enzyme was partially purified. Absolute requirements for S-adenosylmethionine and an appropriate reducing system were observed for the transmethylation from N⁵-methyltetrahydrofolate. The same preparation catalyzed also the formation of methionine from homocysteine and methyl-B₁₂ under both aerobic and anaerobic conditions. The concentration of cobalt ion in the growth medium had a pronounced effect on the intracellular vitamin B₁₂ level and the activity of the vitamin-dependent methionine-synthesizing system in the bacterium. The relationship between the methionine synthesis and the methyl branched-chain fatty acid formation was discussed.     

Selective inhibition of the insulin-stimulated phosphorylation of the 95,000 dalton subunit of the insulin receptor by TAME or BAEE

Added N alpha-p-tosyl-l-arginine methyl ester or N alpha-benzoyl-l-arginine ethyl ester inhibited the stimulation by insulin of phosphorylation of the 95,000 dalton subunit of the insulin receptor both in a partially purified insulin receptor fraction from rat adipocytes and in a highly purified insulin receptor preparation from human placenta. N-alpha-p-tosyl-l-lysine chloromethyl ketone, N alpha-p-tosyl-l-lysine methyl ester, or N-acetyl-l-phenylalanine ethyl ester were much less potent, while N-benzoyl-1-alanine methyl ester was without effect. Inhibition of the phosphorylation by the arginine analogues did not require preincubation of the insulin receptor with inhibitors in the presence of insulin prior to phosphorylation. Inhibition by N alpha-p-tosyl-l-arginine methyl ester was decreased by preincubation of the receptor fraction with cold ATP and MnCl2. These results suggest that N alpha-p-tosyl-l-arginine methyl ester inhibits an initial ATP and Mn2+ dependent reaction in insulin-stimulated phosphorylation process.     

Stereospecificity of the hydroxyeicosatetraenoic and hydroxyoctadecadienoic acids produced by cultured bovine endothelial cells

Characterization of the stereospecificity of the derivatives of arachidonic acid and linoleic acid produced by endothelial cells is needed to define the enzymatic origin of these compounds and their role in vascular physiology. In studies utilizing two bovine endothelial cell lines (CPAE and AG04762), both free 15-hydroxyeicosatetraenoic acid (15-HETE) and 11-hydroxyeicosatetraenoic acid (11-HETE) were generated during incubations with exogenous arachidonic acid and both free 9-hydroxyoctadecadienoic acid (9-HODE) and 13-hydroxyoctadecadienoic acid (13-HODE) were generated during incubations with exogenous linoleic acid. Esterification of 15-HETE, 9-HODE and 13-HODE during these incubations was demonstrated. The analyses included reversed-phase high performance liquid chromatography of the free acid and its methyl ester and chiral separation of the methyl ester on straight phase chiral columns. The ratio of 9-HODE/13-HODE averaged 2.7 in the chromatographic analyses of the extracts of the incubations with linoleic acid. The combined production of 13-HODE and 9-HODE from linoleic acid was four times greater than that of 15-HETE and 11-HETE from arachidonic acid. With regard to the products of the CPAE endothelial cell line, the S/R ratio of the stereoisomers averaged 1.5 for free 15-HETE, 5.7 for free 13-HODE and 0.2 for free 9-HODE. The 11-HETE had strict (R) stereospecificity. The products from the AG04762 endothelial cell line had similar stereochemistry. All these stereochemical findings point to the activity of a cyclooxygenase rather than that of a lipoxygenase.     

Signal transduction leading to appressorium formation in germinating conidia of Magnaporthe grisea: effects of second messengers diacylglycerols, ceramides and sphingomyelin

The inhibition of appressorium formation in germinating conidiospores of Magnaporthe grisea on an inductive surface by glisoprenin A could be reversed in a competitive manner by 1,2-dioctanoylglycerol, a known activator of protein kinase C. Dioctanoylglycerol and linoleic acid methyl ester induced appressorium formation on a noninductive surface. The effect of ceramides as inducers was heavily dependent on the fatty acid in the molecule. Ceramide IIIa containing linoleic acid was a good inducer whereas others had only weak effects. Sphingomyelin, however, inhibited appressorium formation induced by 1,16-hexadecanediol and to a lesser extent after induction with 8-(4-chlorophenylthio)-adenosine-3′,5′-monophosphate. The results are in agreement with our previous findings that in appressorium formation two signal transducing pathways are involved.     

Comparison of three GC/MS methodologies for the analysis of fatty acids in Sinorhizobium meliloti: development of a micro-scale, one-vial method

Three protocols for fatty acid analysis in Sinorhizobium meliloti were improved by the addition of a number of standards/controls and a silylation step which allowed the determination of recoveries, extents of conversion of lipids to fatty acid methyl esters (FAMEs) and extents of side reactions. Basic hydrolysis followed by acid-catalyzed methylation and transmethylation with sodium methoxide, were the best for the analysis of 3-hydroxy- and cyclopropane fatty acids, respectively. A micro-scale, one-vial method that employed sodium methoxide/methanol was equally efficient and on a 1000-fold smaller scale than standard methods. Because this method avoids aqueous extractions, 3-hydroxybutanoic acid was detected as its trimethylsilyloxy methyl ester along with FAMEs.     

Biochemical characterization of MelJ and MelK

Melithiazol and myxothiazol are two myxobacterial metabolites that are highly efficient electron transport inhibitors of the respiratory chain. MelJ and MelK encoded in the melithiazol biosynthetic gene cluster were recently shown to be involved in the formation of the methyl ester from a hypothetical amide intermediate. In vivo studies suggest that the structurally highly similar amide myxothiazol A can be used as a substrate mimic of the hypothetical melithiazol amide to characterize the hydrolase MelJ. Both enzymes were produced in Escherichia coli as intein chitin fusion proteins and were purified using affinity chromatography. MelJ was found to catalyse the conversion of the amide myxothiazol to free myxothiazol acid. The formerly unknown myxothiazol acid was purified and used as a substrate for the methyl transferase MelK which methylates the compound using S-adenosyl-methionine as cosubstrate. Sequence analyses suggest that MelJ and MelK are members of the amidase signature family and of a new subclass of methyltransferases, respectively. Kinetic analyses point at a very high substrate specificity for both enzymes. Furthermore, the in vitro reconstitution of a unique mechanism of methyl ester formation found in myxobacteria is reported.     

Stimulation of hematopoiesis and bone marrow suppressor cells by the subcutaneous injection of linoleic acid

The effects of injection of linoleic acid into C57Bl/6 mice on hematopoietic and immunological parameters were examined. Administration of linoleic acid stimulated hematopoiesis as it increased spleen weight and cellularity, increased the number of bone marrow and splenic granulocytic-monocytic progenitor cells, and increased the colony stimulating factor activity in the serum of the treated mice. Associated with the hematopoietic stimulation in linoleic acid-treated mice was a decline in the spleen cell blastogenic responses and the appearance of bone marrow suppressor cells which were inhibitory to normal spleen cell blastogenesis. The linoleic acid-induced bone marrow suppressor cells resembled cells of the monocyte lineage in that they were sensitive to treatment with L-leucine methyl ester, partially sensitive to treatment with anti-Ia antibodies and complement, and their suppressor activity was minimized by indomethacin, a prostaglandin synthesis inhibitor. These results suggest that administration of linoleic acid results in hematopoietic stimulation and, concurrently, in the appearance of suppressor cells in the bone marrow. The bone marrow suppressor cells resemble immature cells of the monocyte lineage and appear to mediate their suppressive effects through the production of prostaglandins.