PROTEINS OF NORMAL AND CATARACTOUS LENSES

本项目的总体目标是评价 氧化应激在人老年性白内障发生发展中的作用 的 假设透镜中抗氧化防御系统的崩溃 可导致促氧化剂水平增加， 将检测透镜蛋白和透镜透明度的最终损失。 长期目标是开发一种动物模型来研究 核性白内障的形成，并有助于发展 抗氧化疗法，以延缓老年性白内障的发病。 透镜或 透镜上皮细胞将受到高压O2的氧化作用的挑战 在体外和体内，H_2O_2和X射线照射。 将使用高压氧 为了研究存在于透镜中的 谷胱甘肽氧化还原循环和抗坏血酸（AA），并确定是否 透镜中GSH的功能是维持AA处于还原状态。 涉及HPLC、电化学和电子自旋的灵敏方法 将采用共振（ESR）测量透镜和房水水平 还原型和氧化型谷胱甘肽、AA、脱氢抗坏血酸和抗坏血酸 自由基（AFR）。 水平和氧化态发生的变化 的两种抗氧化剂将与可能的修改， 透镜蛋白，包括晶状体蛋白组成、形成的改变 酸性蛋白质种类的增加和蛋白质水平的增加 羰基化合物和混合二硫化物。 蛋白质分析方法包括 HPLC、SDS-PAGE、等电聚焦和差异吸收 谱 观察到的O2诱导的蛋白质修饰将与 已经报道在老化的人透镜中发生的那些，和 其被认为与金属催化的氧化侵蚀有关。 将采用缺乏AA的实验性饮食进行研究， 研究这种抗氧化剂在房水中的功能， 透镜，以及评估氧化应激在 形成糖性白内障。 H2 O2诱导的细胞凋亡机制的研究 兔和人透镜上皮细胞的损伤将用 抗氧化剂丁基化羟基甲苯和TEMPOL的使用。 的 过渡金属在透镜氧化过程中的可能催化作用 BC也在调查。 铁和铜对环境的贡献 AFR在眼房水中的形成将通过使用 ESR，此外，铜和铁的放射性同位素将用于 鉴定可能存在于水溶液中的过渡金属络合物 幽默和透镜。 最后，X线白内障的氧化机制将 可以使用脂溶性自旋捕获剂和 羟基自由基清除剂二甲基硫脲。

The overall objective of this project is to evaluate the role of oxidative stress in the development of human senile cataract. The hypothesis that a breakdown in antioxidant defense systems in the lens can lead to increased levels of prooxidants, irreversible modification of lens proteins and eventual loss of lens transparency will be tested. Long-term goals are to develop an animal model to investigate the formation of nuclear cataract and to contribute to the development of antioxidant therapies to delay the onset of senile cataract. Lenses or lens epithelial cells will be challenged oxidatively with hyperbaric O2 in vitro and in vivo, H2O2 and X-irradiation. Hyperbaric O2 will be used to study the relationship which exists in the lens between the glutathione redox cycle and ascorbic acid (AA) and to determine whether a function of GSH in the lens is to maintain AA in the reduced state. Sensitive methods involving HPLC, electrochemistry and electron spin resonance (ESR) will be employed to measure lens and aqueous humor levels of reduced and oxidized glutathione, AA, dehydroascorbate and ascorbyl free radical (AFR). Changes occurring in the levels and oxidation states of the two antioxidants will bc correlated with possible modifications to lens proteins including alterations in crystallin composition, formation of acidic protein species and increases in the levels of protein carbonyls and mixed disulfides. Methods of protein analysis will include HPLC, SDS-PAGE, isoelectric focussing and difference-absorption spectroscopy. Observed 02-induced protein modifications will bc compared to those which have been reported to occur in the aging human lens, and which are believed to be related to metal-catalyzed oxidative attack. Studies with an experimental diet, deficient in AA, will be employed to investigate the function of this antioxidant in the aqueous humor and lens, as well as to evaluate a possible role for oxidative stress in the formation of sugar cataract. Studies of the mechanism of H202-induced damage in rabbit and human lens epithelial cells will bc performed with the use of the antioxidants butylated hydroxytoluene and TEMPOL. The possible catalytic role of transition metals in lens oxidative processes will also bc investigated. The contribution of iron and copper to the formation of AFR in the aqueous humor will be determined with the use of ESR, and, in addition, radioisotopes of copper and iron will bc used to identify transition metal complexes which may be present in the aqueous humor and lens. Finally, the oxidative mechanism of X-ray cataract will be explored with the use of a lipid soluble spin-trapping agent and the hydroxyl radical scavenger dimethylthiourea.