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OXIDATION INDUCED MEMBRANE DAMAGE IN CATARACTOGENESIS

白内障发生过程中氧化引起的膜损伤

基本信息

批准号：
2838255
负责人：
KAILASH C BHUYAN
金额：
$ 26.4万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
1978
资助国家：
美国
起止时间：
1978-09-01 至 1999-11-30
项目状态：
已结题

项目摘要

DESCRIPTION: Oxidative modifications of lens proteins and lipids occur in maturity onset human cataract. Yet, its relation to cataractogenesis remains questionable. Oxidation of membrane proteins precedes that of cytosolic protein in normal human lenses of the elderly; lipids are also more prone to oxidation than proteins, in spite of being critical to maintenance of structural and functional integrity of proteins. Phospholipid hydroperoxides (LOOH) and their aldehydic breakdown products can cause both protein oxidation and increased membrane permeability. The overall objective of the proposed research is to establish peroxidation of membrane lipids as a precataractous event, with the goal of identifying early reactions which occur during cataract progression sequentially, by detecting membrane lipid-derived oxidant species such as conjugated dienes, LOOH, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), by UV spectrophotometry and HPLC in normal lenses of human donor eyes as a function of age. Aminophospholipid MDA (or 4-HNE), protein-NH2-MDA aminophospholipid adducts produced by Schiff-base conjugation of carbonyl groups with NH2 groups, and protein carbonyls produced by Michael type addition reactions of 4-HNE with NH2 groups of lysine residues, histidyl residues and cysteinyl -SH groups of proteins will be measured by TLC, HPLC and spectrofluorometry in normal human lenses. The data may yield definitive evidence of membrane damage due to lipid peroxidation in the precataractous condition. As additional indication of membrane damage due to peroxidation of lipids, analyses of other functionally important membrane proteins such as major intrinsic proteins and less prevalent glycoproteins will be done by SDS-PAGE and western blotting. Correlative studies will be performed using precataractous lenses of Emory mice exposed to in vivo photochemical insult, a condition analogous to man exposed to sunlight. The proposed work could clarify initial mechanisms of cataract progression in man, and be useful in modeling and testing of anticataract drugs.

描述：晶状体蛋白质和脂质的氧化修饰发生在成熟期发病的人类白内障。然而，它与白内障的发生有关仍然值得怀疑。膜蛋白氧化先于膜蛋白氧化。老年人正常晶状体中的胞浆蛋白；脂类也比蛋白质更容易氧化，尽管对维持蛋白质的结构和功能的完整性。磷脂过氧化氢(LOOH)及其醛分解产物可导致蛋白质氧化和膜通透性增加。这个拟议研究的总体目标是建立作为白内障前期事件的膜脂，目的是确定在白内障进展过程中发生的早期反应，由检测膜脂衍生的氧化剂物种，如共轭双烯， LOOH，丙二醛(MDA)和4-羟基壬烯醛(4-HNE)，UV 人供眼正常晶状体的分光光度和高效液相色谱分析年龄的作用。氨基磷脂丙二醛(或4-HNE)，蛋白质-氨基-丙二醛羰基席夫碱偶联制备氨基磷脂加合物含有NH2基团的基团，以及Michael TYPE产生的蛋白质羰基 4-HNE与氨基赖氨酸残基组氨酸的加成反应用TLC、HPLC法测定蛋白质的残基和半胱氨基-SH基团以及正常人类晶状体中的荧光光谱。数据可能会产生脂质过氧化导致的膜损伤的确凿证据白内障前期的情况。作为膜损伤的额外指示对脂质过氧化，其他重要功能膜的分析蛋白质，如主要的内源蛋白和不太常见的糖蛋白将通过SDS-PAGE和Western blotting进行鉴定。相关研究将被用暴露于体内的Emory小鼠白内障前期晶状体进行手术光化学侮辱，一种类似于人类暴露在阳光下的情况。这个拟议的工作可以阐明白内障进展的初步机制人，并在抗白内障药物的建模和测试中有用。