The broad and long range goal of this proposal is to understand and clarify the underlining mechanisms for human senile cataract formation and to develop an appropriate drug intervention scheme to delay cataract progression. Emphasis will be to provide evidences for the hypothesis that under oxidative stress, the excessive formation of protein-thiol mixed disulfide with glutathione (PSSG) and cysteine (PSSC) is one of the early oxidative damage to the protein which may lead to protein-protein disulfide formation, aggregation and insolubilization during cataract progression.
The specific aims are l) .To study the proposed mechanism that PSSG preceded protein-protein aggregation and how this process can be reversed in in vitro and in vivo. 2) To study the importance of PSSG and PSSC in human cataracts, pigmentation and aging of the lens. 3) To investigate how PSSC is formed and its role in cataract formation. 4) To characterize which of the major lens proteins are involved in PSSG and PSSC adducts. 5) To examine the physiological role of protein thiolation (i.e. PSSG, PSSC formation), the control of this process and the effect of aging in normal lenses. The methods to be used for this proposal are l). The sequence of events in oxidative damages to sulfhydryl biomolecules will be studied by three cataract models: H2O2 (cortical) in vitro, hyperbaric 02 (nuclear) in vivo and in vitro, naphthalene (perinuclear) in vivo. 2) .High resolution Amino Acid Analyzer will be used to quantitate PSSG, PSSC and cysteine. 3). hyperbaric O2 or H2O2 induced cataract with BSO (GSH synthesis inhibitor) pretreated lenses will be used to examine how PSSC is made. 4) Immunochemistry and radiolabeling will be employed to characterize the in situ thiolated proteins in the lens. 5) PSSGs made from beta and gammacrystallins will be used to study protein unfolding with cD and glycation by ascorbate. 5) The effect of H202 on PSSG and hexose monophosphate shunt will be correlated to clarify the role of protein thiolation in normal lens. 6) Presence of thiolation regulating enzymes:thioltransferase and the dethiolase/GSH and dethiolase/NADPH systems will be examined in the lens.
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