OXIDATION INDUCED MEMBRANE DAMAGE IN CATARACTOGENESIS

描述：晶状体蛋白和脂质的氧化修饰发生在 成熟期发病的人类白内障。 然而，它与白内障发生的关系 仍然值得怀疑。 膜蛋白的氧化先于膜蛋白的氧化 老年人正常人晶状体中的胞质蛋白；脂质也 尽管对蛋白质至关重要，但它比蛋白质更容易氧化 维持蛋白质结构和功能的完整性。 磷脂氢过氧化物 (LOOH) 及其醛分解产物 可以导致蛋白质氧化和膜通透性增加。 的 拟议研究的总体目标是确定 膜脂作为白内障前兆事件，目的是确定 在白内障进展过程中依次发生的早期反应， 检测膜脂衍生的氧化剂，例如共轭二烯， LOOH、丙二醛 (MDA) 和 4-羟基壬烯醛 (4-HNE)，通过 UV 检测 分光光度法和高效液相色谱法对人类供体眼睛的正常晶状体进行检测 年龄的函数。 氨基磷脂 MDA（或 4-HNE）、蛋白质-NH2-MDA 羰基席夫碱共轭反应产生的氨基磷脂加合物 具有 NH2 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、组氨酰基的 NH2 基团的加成反应 蛋白质的残基和半胱氨酰-SH基团将通过TLC、HPLC测量 和正常人类晶状体的分光荧光测定法。 数据可能会产生 脂质过氧化导致膜损伤的明确证据 白内障前期状况。 作为膜损坏的附加指示 脂质过氧化，其他功能重要膜的分析 蛋白质，例如主要内在蛋白质和不太常见的糖蛋白 将通过 SDS-PAGE 和蛋白质印迹完成。 相关研究将 使用暴露于体内的埃默里小鼠白内障前晶状体进行 光化学损伤，类似于人类暴露在阳光下的情况。 的 拟议的工作可以阐明白内障进展的初步机制 人，并可用于抗白内障药物的建模和测试。

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.