This research program continues to explore, develop and exploit the information content of Raman/resonance Raman/fluorescence spectra of human and animal lenses. The major thrust of this proposal is the application of our new instrumentation and techniques (automated two-dimensional scanning microprobe system) to study normal and cataractous lenses.
The specific aims are: 1) To obtain the precise distribution profiles of all the identifiable fluorophors in human lenses (normal, diabetic, Tibet/Beijing cataracts etc.) with continuously tunable laser excitation wavelenghts (300-800 nm); 3) To obtain the precise distribution profiles of important lens constituents such as -SH, S- S, Trp, H20 and Tyr which have been implicated in cataractogenesis; 3) To establish if certain fluorophors in the human lens are generated by photocatalyzed reactions; 4) To investigate the similarities and difference in fluorophor production between human and animal species; 5) To investigate the time-dependent spectral changes of glutathione-depleted intact lens (treated with 1-chloro-2,4-dinitrobenzene) to oxidative stress (H2O2 and superoxide anions) by multichannel """"""""snap-shot"""""""" technique; 6) To apply the precision difference Raman technqiue to detect subtle structural changes of lens crystallins upon oxidation and in the presence of calcium ion; 7) To investigate the in vitro behavior of the protein -SH groups in rat lens crystallins from very young rat lenses (17-day old); 8) To compare the reactivities of protein -SH groups with glutathione, cysteines, 2- mercaptoethanol and p-hydroxymercuribenzoate between bovine and rat crystallins; 9) To investigate if the following systems capable of producing red fluorophors: (1) Schiffbase adducts from cross-linking of carbonyl groups of malondialdehyde with the reactive amino groups of phospholipids and (ii) browning products from nonenzymatic glycosylation of lens crystallins; 10) To improve the sensitivity and the speed of data collection/transfer (approximately 10 times) in our automated scanning microprobe system.
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