OXIDATION INDUCED MEMBRANE DAMAGE IN CATARACTOGENESIS

描述：透镜蛋白和脂质的氧化修饰发生在 成熟期白内障 然而，它与白内障发生的关系 仍然值得怀疑。 膜蛋白的氧化先于 老年人正常晶状体中的胞质蛋白; 比蛋白质更容易氧化，尽管对 维持蛋白质的结构和功能完整性。 磷脂过氧化氢（LOOH）及其降解产物 可导致蛋白质氧化和膜渗透性增加。 的 拟议研究的总体目标是建立 膜脂质作为一个precataractous事件，与识别的目标， 在白内障进展过程中相继发生的早期反应， 检测膜脂质衍生的氧化剂物质如共轭二烯， LOOH、丙二醛（MDA）和4-羟基壬烯醛（4-HNE）（UV法） 用分光光度法和高效液相色谱法测定正常人晶状体中的晶状体蛋白 年龄的函数。 氨基磷脂MDA（或4-HNE）、蛋白质-NH 2-MDA 羰基席夫碱共轭生成的氨基磷脂加合物 具有NH 2基团的基团，以及由Michael型产生的蛋白质羰基 4-HNE与赖氨酸残基、组氨酰 蛋白质的半胱氨酸残基和半胱氨酰-SH基团将通过TLC、HPLC 和荧光分光光度法。 数据可能会产生 由于脂质过氧化导致的膜损伤的明确证据， 前白内障状态 作为膜损伤的额外指示， 脂质过氧化，其他功能重要的膜分析 蛋白质如主要内在蛋白质和不太普遍的糖蛋白 将通过SDS-PAGE和蛋白质印迹法进行。 相关研究将在 使用暴露于体内的Emory小鼠的前白内障晶状体进行 光化学损伤，一种类似于人暴露在阳光下的状态。 的 拟议的工作可以澄清白内障进展的初步机制， 人，并可用于建模和测试抗白内障药物。

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.