ANALYSIS OF MICRODISSECTED CATARACTOUS HUMAN LENSES

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The long term objectives are to understand the causes and mechanisms which lead to the formation of cataract in humans. The specific aims are: 1. To continue our long term clinicobiochemical investigation of aging-related and drug-related human cataracts. A comprehensive presurgical evaluation of the patient will be performed, and the progression of cataract in the patient will be followed clinically, and will be documented using slit-lamp procedures, polarized retroillumination, and Scheimpflug photography. Following cataract surgery, the lens will be microdissected and evaluated biochemically. 2. To study the biochemical properties of freshly excised human lens epithelium following cataract surgery. Special attention will be given to glutathione reductase and other flavoenzymes. Since flavoenzymes depend on dietary riboflavin, the status of dietary riboflavin will be examined in patients with cataract by monitoring glutathione reductase activity in the red blood cells of these patients. Other key enzymes will also be studied. 3. To study the effects of thyroid hormones (T3 and thyroxine), beta-blockers (such as propranolol) and steroids (such as prednisone) on enzyme activity in human lens epithelium. Lens organ culture of bovine, rabbits, and rats will also be used to study the effects of the hormones and drugs. 4. To synthesize short peptides based on the known sequence of the various crystallins and other lens-specific proteins. These peptides will be used to produce antibodies specific to these peptides, and then will be used to probe cataractous human lens. 5. To study the stability of lens crystallins. The thermal stability of human lens crystallin as a function of age will be studied using circular dichroism (CD), absorption, and fluorescence spectroscopy. 6. To continue and study the structure and function of the lens plasma membrane major intrinsic polypeptide (HIP-26), and to try to crystallize it for X-ray diffraction studies. The possibility that MIP-26 has a binding site for cGMP will be explored. 7. To elucidate the physico-chemic properties and to immunolocalize the newly discovered lens fiber-specific intrinsic membrane protein MP-17. This will include purification of the protein characterization of its secondary and tertiary structure using CD and in situ localization. 8. To examine the cataractous and clear lenses of rats with inherited retinal degeneration (RCS). The cataract formation is a secondary phenomenon, these rats are an excellent animal model for oxidative insults to the lens which may cause cataract.