The broad objective of this research is determining the molecular basis of cataracts produced by inhibition of lens cholesterol biosynthesis. This goal is significant because it may help protect the public from cataracts potentially induced by hypocholesterolemic drugs and can provide information on links between damage to lens membrane and protein insolubilization. The rodent cataract induced by U18666A is a model for examining relationships between insult to lens membranes and opacification. Determining the mechanism of cholesterol-cataracts requires an understanding of the role of cholesterol in lens membrane structure and the regulation of lens cholesterol biosynthesis.
A specific aim i s describing the impact of cholesterol on the fluidity of human lens membrane phospholipids. The effect of cholesterol on the trans to gauche isomerization of phospholipid hydrocarbon chains will be measured using ch2-infrared stretching band frequencies. Oxidation of cholesterol can alter lens membrane structure. Thus, the PI plans to describe the cholesterol oxide composition of human lenses with aging and cataracts using capillary-gas chromatography. Whether lovastatin alters lens cholesterol in man will be addressed by measuring the cholesterol content of superficial cortex from lens of control and treated humans. These lenses are available postmortem from organ donor banks. Translation of HMG CoA reductase mRNA may be the ultimate factor controlling lens cholesterol biosynthesis. A major aim of this proposal is describing how translation of this critical lens mRNA is regulated. An anti-sense mRNA probe will be produced to assess degradation of the message as fiber cells complete elongation. RACE assays will be used to determine the length of the 5'-untranslated region of the reductase mRNA, since this structure may promote translation control And, a RT-PCR system will be used to examine the role of non-sterol isoprene precursors of cholesterol in regulating translation of the mRNA. The basis of the increased binding of protein to lens membrane in cataracts will also be studied. The goal is to describe the role of altered lens membrane structure in accounting for the increased binding. The capacity of membrane from U18666A and human cataracts to bind a model substrate, rat 3H- crystallin, will be measured using an established binding assay. Alpha crystallin, the major extrinsic protein of lens membrane, may facilitate the binding of other proteins. Alpha crystallin binds to membrane at both low and high affinity sites. By using bifunctional crosslinking agents we hope to identify the membrane components responsible for crystallin binding at these sites.
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