Gas Chromatography Mass Spectrometric Analysis of Monohydro-peroxide Isomers and Their Secondary Oxidation Productsof Methyl Linoleate and Methyl Linolenate

Emulsions of methyl linoleate monohydroperoxides (18:2-monoHP) and methyl linolenate monohydroperoxides (18: 3-monoHP) were incubated with ferrous sulfate and ascorbic acid. Gas chromatography mass spectrometric analysis of the trimethylsilyl and te^butyldimethylsilyl derivatives of the reaction products showed that isomerization and secondary oxidation happen competitively during decomposition of 18:2-monoHP, while the secondary oxidation reaction proceeds preferentially and little isomerization is observed in 18: 3-monoHP. It is suggested that 18:3-monoHP is more susceptible to secondary oxidation than 18:2-monoHP because of 18:3 specific secondary oxidation resulting in hydroperoxy-cyclic peroxides and dihydroperoxides. Moreover, an experiment using ¹⁸0₂ has demonstrated that molecular oxygen is scrambled by isomerization and secondary oxidation. It was confirmed that molecular oxygen is attached preferentially to the C-13 position in the 9-monoHP isomer and C-9 position in the 13-monoHP isomer during degradation of 18:2-monoHP.     

昆虫几丁质合成酶及其抑制剂

几丁质合成酶(CS)是几丁质合成的关键酶,它具有3个结构域:结构域A、结构域B和结构域C,其中结构域B是催化域。根据氨基酸序列的差异,几丁质合成酶分为两类:CS-A及CS-B,分别在表皮及围食膜基质中催化合成几丁质。关于几丁质合成有2种假想模型。有多种抑制剂可以抑制几丁质的合成,其中核苷肽抗生素类及核苷磷酸类作用于CS的催化部位,是竞争性抑制剂,其它抑制剂的作用机理仍不明确。     

Heterogeneity of β2-Microglobulin in Human Urine

Purification and characterization of β₂-microglobulin from human urine was performed. The yield was 30.1%, and 150.4 mg of β₂-microglobulin was obtained. The final preparation of β₂-microglobulin obtained showed three bands on disc gel electrophoresis at pH 9.5, and all of them have immunological activity. However, these three bands migrated as a single band on disc gel electrophoresis at pH 4.3. It is concluded that the three bands observed on disc gel electrophoresis at pH 9.5 were charge isomers. The isoelectric points of isomers were determined by isotachophoresis and two of them were 5.4 and 5.9 respectively, while the other one was not determined.     

Studies on zymogenicity and solubilization of chitin synthase from Candida albicans

Abstract The zymogenic form of the chitin synthase present in mixed membrane preparations was extracted by digitonin treatment. The residual extracted membranes exclusively retained the basal activity. Trypsin activation of the zymogenic form of the enzyme did not modify the digitonin solubilization characteristics of the original zymogenic form, suggesting significant differences between 'in vivo' activation of chitin synthase and that carried out by trypsin 'in vitro'.     

几丁质合酶在人类致病真菌中的研究进展

抑制真菌细胞壁的合成常作为防治真菌感染的安全有效手段。几丁质是真菌细胞壁及隔膜的重要结构成分,几丁质合酶是催化几丁质合成的关键酶。真菌细胞中几丁质合酶家族的不同成员在调控几丁质的合成中存在着差异,因此产生不同的生物学效应。本文通过综述几丁质合酶在人体三大条件致病真菌白色念珠菌、烟曲霉、新生隐球菌中的研究进展,分析了几丁质合酶对真菌致病性影响的机制,总结了几丁质合酶调控真菌细胞增殖、形态转换、病原菌与宿主的相互作用和细胞壁损伤诱导的补偿效应,展望了抗真菌感染的新策略及关于真菌几丁质合酶的未来研究方向。     

甜菜夜蛾几丁质合成酶基因A和B启动子的克隆

几丁质不仅是昆虫的表皮和围食膜的主要成分,也是一个非常关键的害虫控制靶标,主要通过几丁质合成酶（chitin synthase,CHS）基因合成。本文在克隆甜菜夜蛾Spodoptera exigua的两个几丁质合成酶基因（SeCHSA和SeCHSB）cDNA和基因组序列的基础上,从基因的5′末端设计特异性引物和构建特定的基因组文库, 采用PCR的方法获得了5′端侧翼序列。通过5′RACE的方法确定SeCHSA和SeCHSB基因的转录起始位点后,获到了启动子序列。这为研究昆虫几丁质合成和转录调控奠定了基础。     

Chitin synthase 2 gene sequence of Malassezia species.

Nucleotide sequences of the chitin synthase 2 (CHS2) gene of seven species, Malassezia furfur, M. globosa, M. obtusa, M. pachydermatis, M. restricta, M. slooffiae and M. sympodialis, were analyzed for their phylogenetic relationship. About 620-bp genomic DNA fragments of the CHS2 gene were amplified from these Malassezia species by polymerase chain reaction (PCR) and sequenced. The CHS2 nucleotide sequences of these Malassezia species showed more than 95% similarity between the species. A phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of seven Malassezia species revealed that the species were genetically distinct from each other.     

甜菜夜蛾cDNA文库的构建

以甜菜夜蛾Spodoptera exigua(Hübner)幼虫为材料建立了cDNA表达文库,经检测文库的初始滴度为1.1×105pfu/mL,重组率为97%,扩增后文库的滴度为5.4×107pfu/mL。用设计的2对引物筛选该文库,得到468 bp的1个片段,分析后证实是几丁质合成酶基因I保守区域的1个片段。该cDNA文库为进一步筛选甜菜夜蛾功能基因奠定了基础。     

Modification at the 5-Position of 4-Deoxypyridoxol and α4-Norpyridoxol

In an attempt to relate structure to anticoccidial activity, a number of 5-modified analogs of 4-deoxypyridoxol (4-DOP) and α⁴-norpyridoxol have been synthesized and their biological activities examined. The compounds prepared include the 5-(3-hydroxypropyl), 5-(2-hydroxyethyl), 5-(1-hydroxyethyl), formyl and acetyl analogs of 4-DOP, and 5-(3-hydroxypropyl), formyl, ethoxycarbonyl, carbamoyl and hydroxyl analogs of α⁴-norpyridoxol. Among these compounds, 4-deoxyisopyridoxal and α⁴-norisopyridoxal were found to exhibit anticoccidal activity.     

Immunolocalization of chitin synthases in the phytopathogenic dimorphic fungus Ustilago maydis

Conserved polypeptides of the chitin synthase genes UmCHS3 and UmCHS6 from the phytopathogenic fungus Ustilago maydis were utilized as immunogens to obtain polyclonal antibodies that were purified by affinity procedures. Because of their similarities at the regions encoded by either polypeptide, it was concluded that anti-Chs3 antibodies recognized both Chs3 and Chs4 chitin synthases, whereas anti-Chs6 antibodies recognized Chs6 and Chs8 polypeptides. These antibodies were used to analyze the localization of the corresponding chitin synthases in U. maydis cells, using both indirect immunofluorescence microscopy and immunoelectron microscopy with colloidal-gold-labeled secondary antibodies. It was observed that chitin synthase proteins were accumulated both in the surface and in the cytoplasm of the fungal cells. Electron microscopy images revealed the accumulation of clusters of gold particles in vesicles, providing evidence for the possible origin and destination of chitin synthases in the fungal cells.     

Biochemical characterization of chitin synthase activity and inhibition in the African malaria mosquito,Anopheles gambiae

Abstract Chitin synthase (CHS) is an important enzyme catalyzing the formation of chitin polymers in all chitin containing organisms and a potential target site for insect pest control. However, our understanding of biochemical properties of insect CHSs has been very limited. We here report enzymatic and inhibitory properties of CHS prepared from the African malaria mosquito, Anopheles gambiae. Our study, which represents the first time to use a nonradioactive method to assay CHS activity in an insect species, determined the optimal conditions for measuring the enzyme activity, including pH, temperature, and concentrations of the substrate uridine diphosphate N‐acetyl‐d ‐glucosamine (UDP‐GlcNAc) and Mg⁺⁺. The optimal pH was about 6.5–7.0, and the highest activity was detected at temperatures between 37°C and 44°C. Dithithreitol is required to prevent melanization of the enzyme extract. CHS activity was enhanced at low concentration of GlcNAc, but inhibited at high concentrations. Proteolytic activation of the activity is significant both in the 500 ×g supernatant and the 40 000 ×g pellet. Our study revealed only slight in vitro inhibition of A. gambiae CHS activity by diflubenzuron and nikkomycin Z at the highest concentration (2.5 μmol/L) examined. There was no in vitro inhibition by polyoxin D at any concentration examined. Furthermore, we did not observe any in vivo inhibition of CHS activity by any of these chemicals at any concentration examined. Our results suggest that the inhibition of chitin synthesis by these chemicals is not due to direct inhibition of CHS in A. gambiae.     

Stereoselectivity in Metabolism of the Optical Isomers of Cyanofenphos (O-p-Cyanophenyl O-Ethyl Phenylphosphonothioate) in Rats and Liver Microsomes

The racemic, (+)- and (—)-forms of cyanofenphos (O-p-cyanophenyl O-ethyl phenylphosphonothioate) were rapidly metabolized in the rat by cleavage of P-O-aryl linkage, cleavage of P-O-alkyl linkage and conjugation of p-cyanophenol with sulfuric acid. There was a marked difference in the proportion of the major urinary metabolites, p-cyanophenol and p-cyanophenyl sulfate, with three forms of cyanofenphos,

The three forms of cyanofenphos were metabolized at almost equal rates in rat liver microsomes-NADPH system. (+)-Cyanofenphos underwent oxidation of P=S to P = O and cleavage of P-O-aryl linkage predominantly. In contrast, the (?)-isomer was converted to the corresponding oxon analog by mixed function oxidase, and then the oxon analog was rapidly hydrolyzed to p-cyanophenol by mícrosomaî arylesterase-type enzyme. This microsomal enzyme had a remarkable selectivity in hydrolyzing (?)-cyanofenphos oxon versus the ( + )-isomer. Stereoselectivity in the metabolism of the cyanofenphos isomers in the rat appears likely to be mainly due to selective hydrolysis of the (?)-oxon analog by the arylesterase-type enzyme.     

朱砂叶螨几丁质合成酶基因1全长cDNA的克隆与表达分析

【目的】克隆朱砂叶螨Tetranychus cinnabarinus几丁质合成过程中的关键酶几丁质合成酶基因,并检测该基因在朱砂叶螨生长发育不同阶段的相对表达量。【方法】本研究采用逆转录聚合酶链反应(RT-PCR)以及c DNA末端快速扩增(RACE)技术首次克隆获得朱砂叶螨几丁质合成酶基因1的全长c DNA序列(命名为Tc CHS1,Gen Bank登录号为KM242062),并使用实时荧光定量PCR技术首次检测了Tc CHS1基因在朱砂叶螨生长发育不同阶段的相对表达量。【结果】朱砂叶螨Tc CHS1基因的c DNA全长为4 881 bp,包括198 bp的5'非翻译区(5'-UTR),4 425 bp的开放阅读框(ORF),258 bp的3'非翻译区(3'-UTR),开放阅读框编码1 474个氨基酸,预测其蛋白质分子质量约为168.35 ku,理论等电点为6.26。其包含EDR和QRRRW这2个几丁质合成酶基因的标签序列。氨基酸序列同源性分析结果表明:Tc CHS1与其他昆虫该基因编码蛋白的氨基酸序列相似度在50%左右,与二斑叶螨Tetranychus urticae的氨基酸相似度最高(98%),与西方盲走螨Metaseiulus occidentalis的相似度为55%。分子系统进化的结果也表明Tc CHS1与其他昆虫的CHS1聚在一起,并且和二斑叶螨具有最近的亲缘关系。荧光定量分析表明Tc CHS1基因在朱砂叶螨生长发育的不同阶段(卵、幼螨、第1若螨、第2若螨、雌成螨和雄成螨)均有表达,在卵和雌成螨中的表达量较高,在第2若螨的表达量最低。【结论】Tc CHS1基因可能在朱砂叶螨生长发育过程中具有重要作用。     

In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

Abstract The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix (PM), composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double‐stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins (insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1) and the chitin biosynthetic or modifying enzymes (chitin synthase‐B and chitin deacetylase 1) in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing (by 24 h) for the gene encoding chitin deacetylase 1 and a slower rate of silencing (by 72 h) for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase‐B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double‐stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.     

A nonradioactive,high throughput assay for chitin synthase activity

Wheat germ agglutinin (WGA) binds with high affinity and specificity to several sites on chitin polymers. Based on these properties we have modified and adapted a previously patented (U.S. patent 5,888,757) nonradioactive, high throughput screening assay for antimicrobial agents, making it suitable as a quantitative enzymatic assay for the activity of individual chitin synthase isozymes in yeast. The procedure involves binding of synthesized chitin to a WGA-coated surface followed by detection of the polymer with a horseradish peroxidase-WGA conjugate. Horseradish peroxidase activity is then determined as an increment in absorbance at 600 nm. Absorbance values are converted to amounts of chitin using acid-solubilized chitin as a standard. The high sensitivity (lower limit of detection about 50 ng chitin), low dispersion (lower than 10%), and high throughput (96-well microtiter plate format) make this assay an excellent substitute for the conventional radioactive chitin synthase assay in cell-free extracts. We have applied this method to the differential assay of chitin synthase activities (Chs1, Chs2, and Chs3) in cell-free extracts of Saccharomyces cerevisiae. Analysis of Chs3 activity in chitosomal and plasma membrane fractions revealed that Chs3 in the plasma membrane fraction is about sixfold more active than in the chitosome.