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OXYGEN FREE RADICAL PRODUCTION AND SCAVENGING IN E. COLI

大肠杆菌中氧自由基的产生和清除

基本信息

批准号：
3445472
负责人：
JOSEPH P MARTIN
金额：
$ 4.74万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-09-30 至 1986-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3445472
关键词：
Escherichia coli bacterial genetics catalase cell death cell membrane electron microscopy fatty acid metabolism free radicals hydrogen peroxide membrane lipids microorganism metabolism molecular cloning peroxidation plasmids superoxide dismutase superoxides tissue /cell culture

项目摘要

The proposed research will elucidate the roles of superoxide dismutase and catalase in preventing oxygen toxicity in Escherichia coli and determine the nature and the toxic effects of oxygen free radical species in vivo. In addition, a bacteriological plate assay will be developed to rapidly screen drugs, toxic compounds and naturally occurring plant and bacterial compounds for their abilities to generate oxygen free radicals in cells. The Carbon-Clarke clone bank will be screened for chimeric plasmids containing superoxide dismutase or catalase. Appropriate plasmids will be amplified, purified and the superoxide dismutase and catalase genes will be excised and subcloned into additional plasmid vehicles. These plasmids (PSC101, COL E1, PBR322) appear in different copy numbers and transformation of recipient cells will give strains that overproduce superoxide dismutase or catalase in variable amounts. The strains will be examined for their relative abilities to resist oxygen toxicity due to hyperoxic oxygen and to compounds that exacerbate oxygen toxicity. Damage will be assayed as a loss of viability and as the stimulation of membrane lipid perioxidation. Lipid peroxidation will serve as an indicator for the production of toxic oxygen free radicals within the bacteria. The degree of protection afforded the cells by either superoxide dismutase or catalase will indicate the nature of the oxygen radical species involved in damage. Overproducing strains will also be compared to control strains in a plate assay under anaerobic and aerobic conditions to examine their relative resistances to the toxic effects of redox active compounds. The chief goals of the work are to elucidate the molecular basis of oxygen toxicity in cells, to understand the enzymatic scavenging system that has evolved to detoxify oxygen, and to develop a method of identifiying compounds in the human environment that exacerbate oxygen toxicity. The proposal involves biochemistry, molecular biology, and microbiology and is directed to solving problems of drug and environmental toxicology. These studies should clarify the mechanisms by which oxygen radicals mediate: 1) red blood cell lysis in G6PDH deficient individuals by antimalarial drugs and by dietary substances, 2) the cardiotoxicity of antitumor antibiotics, 3) the destruction of the lung capillary beds following exposure to some herbicides.

拟议的研究将阐明超氧化物歧化酶和过氧化氢酶预防大肠杆菌氧中毒的作用及其测定体内氧自由基种类的性质和毒性作用。此外，将开发细菌学平板测定法以快速检测筛选药物、有毒化合物以及天然存在的植物和细菌化合物具有在细胞中产生氧自由基的能力。 Carbon-Clarke克隆库将筛选嵌合质粒含有超氧化物歧化酶或过氧化氢酶。合适的质粒将是扩增、纯化超氧化物歧化酶和过氧化氢酶基因切除并亚克隆到额外的质粒载体中。这些质粒（PSC101、COL E1、PBR322）以不同的拷贝数出现，并且受体细胞的转化将产生过量生产的菌株不同量的超氧化物歧化酶或过氧化氢酶。菌株将是检查它们抵抗氧毒性的相对能力高氧和加剧氧毒性的化合物。损害将被测定为活力丧失和膜刺激脂质过氧化。脂质过氧化可作为脂质过氧化的指标细菌内产生有毒的氧自由基。学位超氧化物歧化酶或过氧化氢酶为细胞提供保护将表明参与损伤的氧自由基种类的性质。过量生产的菌株还将与平板中的对照菌株进行比较在厌氧和有氧条件下进行测定以检查其相关性对氧化还原活性化合物的毒性作用的抵抗力。酋长这项工作的目标是阐明氧毒性的分子基础在细胞中，了解已经进化到的酶清除系统解氧，并开发一种鉴定化合物的方法人类环境加剧氧毒性。该提案涉及生物化学、分子生物学和微生物学，旨在解决药物和环境毒理学问题。这些研究应阐明氧自由基介导的机制：1）红色抗疟药物对 G6PDH 缺陷个体的血细胞溶解和通过膳食物质，2) 抗肿瘤抗生素的心脏毒性，3) 暴露于某些物质后，肺毛细血管床受到破坏除草剂。