ANALYSIS OF MICRODISSECTED CATARACTOUS HUMAN LENSES

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The long term objectives of this research project are to understand the causes and mechanisms which lead to the formation of cataract in humans. The specific aims are: (1) To start a long term clinicobiochemical investigation of aging-related and drug-related human cataracts. In these studies, a comprehensive presurgical evaluation of the patient will be performed. The progression of cataract in the patient will be followed clinically, and will be documented using standardized slit lamp procedures, polarized retroillumination photography, and Scheimpflug photography. After cataract surgery, the lens will be microdissected and evaluated biochemically as described under aims 2 and 3. (2) To microdissect single human cataractous lenses and to separate with the aid of a microscope opague areas and adjacent normal areas. High performance liquid chromatography (HPLC) will be used to study the properties of the water soluble proteins, urea soluble proteins, and the membrane proteins in both the clear and opaque sections. Other techniques will include SDS-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid analysis, tryptic peptide mapping of radioiodinated protein fractions employing the two-dimensional system of electrophoresis and chromatography followed by autoradiography. (3) To analyze the major nonprotein low molecular weight constituents in opaque and clear sections of human lenses. (4) To develop new methods for retrieving lens material during extracapsular surgery. To approach taken will be tap the aspiration lines of the equipment used during surgery. (5) To identify and characterize a 23,000 dalton crystallin which is selectively degraded and modified in many human cataractous lenses, and to identify and study the proteolytic enzymes which are involved, in part, in this process. (6) To complete the nucleotide sequencing of cDNA clones of the lens plasma membrane main intrinsic protein (MIP-26). (7) To complete the nucleotide analysis of the cow beta 23/26 crystallin. (8) To study the effects of diet and aging on the differential loss of gamma-crystallin in mice which are given a reduced calorie diet.