PROTEINS OF NORMAL AND CATARACTOUS LENSES

该项目的总体目标是评估 氧化应激在人类老年性白内障发生发展中的作用这个 假设晶状体中抗氧化防御系统的崩溃 会导致促氧化剂水平增加，不可逆的修饰 将测试晶状体蛋白质和晶状体透明度的最终损失。 长期目标是开发一种动物模型来研究 核性白内障的形成和发展 延缓老年性白内障发病的抗氧化剂治疗。镜片或 晶状体上皮细胞将受到高压氧的氧化挑战 体外和体内，H_2O_2和X射线照射。将使用高压氧 为了研究存在于晶状体中的 谷胱甘肽氧化还原循环和抗坏血酸(AA)，并确定 晶状体中GSH的功能是维持AA处于还原状态。 涉及高效液相、电化学和电子自旋的灵敏方法 将使用共振(ESR)来测量晶状体和房水的水平 还原和氧化的谷胱甘肽、AA、脱氢抗坏血酸和抗坏血酸 自由基(AFR)。水平和氧化状态发生的变化 这两种抗氧化剂中的Bc与可能的修饰有关 晶状体蛋白质包括晶状体蛋白组成、形成的变化 酸性蛋白质的种类和蛋白质水平的增加 羰基和混合二硫化物。蛋白质分析方法将包括 高效液相色谱、十二烷基硫酸钠-聚丙烯酰胺凝胶、等电聚焦和差示吸收 光谱学。观察到的02诱导的蛋白质修饰将进行BC比较 那些据报道发生在老化的人类晶状体中的基因，以及 它们被认为与金属催化的氧化攻击有关。 对缺乏AA的实验性饮食的研究将被用于 研究这种抗氧化剂在房水中的功能和 晶状体，以及评估氧化应激在 糖性白内障的形成。H_2O_2诱导的机制研究 对兔和人晶状体上皮细胞的损伤作用 抗氧化剂丁基羟基甲苯和坦普尔的使用。这个 过渡金属在晶状体氧化过程中的可能催化作用 还将对BC进行调查。铁和铜对人类健康的贡献 房水中AFR的形成将通过使用 ESR，此外，铜和铁的放射性同位素将用于公元前 确定水溶液中可能存在的过渡金属络合物 幽默和镜头。最后，X射线白内障的氧化机制将 通过使用脂溶性自旋捕捉剂和 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.