OXYGEN FREE RADICAL PRODUCTION AND SCAVENGING IN E. COLI

这项拟议的研究将阐明超氧化物歧化酶和 过氧化氢酶在防止大肠杆菌氧中毒中的作用及测定 氧自由基的性质及其在体内的毒性作用。 此外，细菌学平板分析将迅速发展到 筛选药物、有毒化合物和自然产生的植物和细菌 化合物在细胞中产生氧自由基的能力。 Carbon-Clarke克隆库将对嵌合质粒进行筛选 含有超氧化物歧化酶或过氧化氢酶。合适的质粒会是 扩增、纯化和超氧化物歧化酶和过氧化氢酶基因将 切下并亚克隆到额外的载体中。这些质粒 (PSC101、COL E1、PBR322)以不同的拷贝数和 受体细胞的转化将产生过度生产的菌株 不同量的超氧化物歧化酶或过氧化氢酶。这些菌株将是 检查它们抵抗氧毒性的相对能力，这是由于 高氧氧和加剧氧毒性的化合物。毁伤 会被检测为活力的丧失和膜的刺激 脂质过氧化。脂质过氧化将作为一种指标 细菌内产生有毒的氧自由基。学位 由超氧化物歧化酶或过氧化氢酶为细胞提供的保护 将表明与损害有关的氧自由基物种的性质。 高产菌株也将与培养皿中的对照菌株进行比较。 厌氧和好氧条件下检测其相对分子质量 对氧化还原活性化合物毒性作用的抵抗力。酋长 这项工作的目标是阐明氧毒性的分子基础。 在细胞中，为了了解酶的清除系统已经进化到 解毒氧气，并开发一种鉴定化合物的方法 加剧氧毒性的人类环境。该提案涉及 生物化学、分子生物学和微生物学，并针对 解决药物和环境毒理学问题。这些研究 应阐明氧自由基的调节机制：1)红色 抗疟药物对G6PDH缺陷者血细胞的裂解作用 通过饮食物质，2)抗肿瘤抗生素的心脏毒性，3) 暴露于某些污染物后对肺毛细血管床的破坏 除草剂。

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.