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M.J. Costello, C.D. Freel, K.O. Gilliland; Identification of Multilamellar Bodies in the Urea Insoluble Fraction of Human Age-Related Nuclear Cataracts . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3502.
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Purpose: To isolate and enrich multilamellar bodies (MLBs) from human age-related nuclear cataracts. MLBs are rare, spherical, micron-sized objects found more frequently in human age-related nuclear cataracts than in aged transparent lenses and are postulated to be significant sources of light scattering. Methods: A rapid micro enrichment procedure was developed to homogenize and dissolve the entire fetal and embryonic nuclei of cataractous lenses in a low calcium, high pH reducing buffer (PBS with DTT). Within 5-10 min even relatively hardened nuclei could be suspended, ready for incubation in 4-7M urea. Following Airfuge centrifugation, the small urea insoluble pellet was recovered and divided. One part was washed with 0.1 M NaOH to produce a clean membrane fraction and one part was not washed yielding membranes with adherent proteins and separate aggregates of protein material. Although small, the pellets are suitable for the preparation of specimens for TEM analysis. Results: All nuclei from cataracts produced similar results. For example, an 83 y.o. hardened nucleus obtained by extracapsular extraction (ophthalmologist grade 3, dark yellow) was dissolved, urea washed and pelleted for TEM fixation (glutaraldehyde or cryo plunging) within 3 hours of receipt. Thin section TEM of the NaOH washed pellet displayed typical membrane profiles including gap junctions and undulating junctional membranes. Large circular profiles with multiple thin concentric bilayers were observed and were probably the desired MLBs. Freeze-fracture images displayed gap junctional plaques and square arrays of aquaporin0. Several examples of large globular objects with very smooth fracture faces typical of pure lipid bilayers and multiple small steps between layers were indicative of the MLB structure. The unwashed pellet produced identical membrane profiles by TEM and extensive protein aggregates of irregular assemblages of 5-40 small spherical objects similar to alpha-crystallin. Conclusions: Single cataractous lens nuclei can be processed to yield membrane pellets containing globular structures with the characteristics of MLBs described previously by light and electron microscopy of intact tissue. The MLB structures were easy to locate and thus appeared to be enriched. The pellets are suitable for biochemical analysis using immunomicroscopic methods and the enrichment procedure is useful for following key changes in nuclear membranes and cytoplasmic protein components during cataract formation.
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