No abstract

H2O2 stress is shown to produce cataract in cultured rat lenses. The loss of transparency begins in the equatorial region within 24 hours and the entire superficial cortex is opaque by 96 hours. No involvement of the nuclear region is observed. However after an additional 48 hours, the nuclear region becomes opaque. The loss of transparency is accompanied by a large uptake of H2O which occurs gradually over the 96 hour period, complete loss of glyceraldehyde phosphate dehydrogenase (GPD) activity, almost complete loss of non-protein thiol and a slight decrease in protein thiol. Control lenses show no change other than the establishment of a new non-protein thiol base line approximately 60% lower than 0 time levels. The Alcon glutathione peroxidase type mimic, AL-3823A, completely eliminates almost all of the H2O2 induced effects and the lens remains transparent. Utilizing a more severe photochemical model than may be anticipated physiologically with 10 microM riboflavin and exposure to daylight fluorescent lamps, significant concentrations of superoxide and low levels of OH. are produced as well as extraordinarily high concentrations of H2O2 ranging from about 400 to 1000 microM. As with the H2O2 model, opacification begins at the equator but the cataract develops more rapidly, the lens being completely opaque by 68 hours. Hydration, GPD activity, non-protein and protein thiol all decrease more rapidly than in the H2O2 model. AL-3823A prevents loss of transparency until approximately 92 hours and markedly decreases changes in other parameters. At 92 hours, slight loss of transparency is observed. Catalase is somewhat less effective. AL-3823A is shown to also significantly decrease superoxide levels. The marked delay in the onset of changes in lens biochemistry and physiology in the severe photochemical stress model and the maintenance of normal parameters in the H2O2 model in the presence of AL-3823A suggests that such compounds may prevent cataract caused by oxidative stress under physiological conditions.

Because protein phosphorylation is an important reaction involved in the control of a number of diverse biological processes such as cell differentiation and gene expression (1), investigation of phosphorylation reactions in the lens of the eye was initiated. The lens is a particularly inviting tissue for such study. It contains a single layer of epithelial cells, which in the equatorial region are constantly going into terminal differentiation (2). In this process, the cells increase dramatically in volume, extending to both the anterior and posterior side of the tissue. The cells lose their nuclei and gradually their ability to synthesize protein. During terminal differentiation, an entirely new population of cytosol and membrane proteins is produced. The new fibers are constantly displacing older fibers in towards the center of the tissue. This process results in the production of an age-dependent gradient with the oldest, metabolically inactive cells in the center of the tissue and the youngest fibers in the outer perimeter of the tissue in contact with the epithelium.Previous studies on protein phosphorylation in the lens (3) indicate that there is little phosphorylation in the inner regions of the tissue. The protein phosphorylation observed in the outer fiber cells of the tissue gives a distinctly different pattern from that observed in the epithelial cell layer. Most of the observed protein phosphorylation is cAMP dependent.A few of the major cAMP-dependent phosphorylated proteins found in the lens have been identified. They include the major fiber membrane protein MP26 (4, 5), vimentin (6), actin (6), and fodrin (7).Of particular interest is the preliminary finding of a major phosphorylated polypeptide with Mr in the 20,000 range, which appears in the outer fiber layers (the outer cortex) but not in the epithelium (3). This report presents evidence which suggests that this phosphorylated fraction consists of the Al and B1 chains of a-crystallin, one of the major proteins of the fiber cell. The phosphorylation may be involved in the process of terminal differentiation and the organization of the fiber cell. Thus, the major lens proteins, previously thought to only contribute to the uniform refractive index of the tissue, may have other functions and be under stringent metabolic control. MATERIALS AND METHODSA soluble fraction from calf lens outer cortex (LOCSF) was prepared with bovine eyes from approximately 15-week-old calves obtained from a local slaughterhouse. The lenses (1.2-1.6 g) were removed from the eyes and decapsulated by dissection. Outer cortex fiber cells (10-20% by weight) were obtained by placing the decapsulated lenses in a buffer containing 10 mM Hepes, and 50 mM 2-mercaptoethanol, pH 7.4, and stirring vigorously for 5-10 min on ice. The suspension of fiber cells was homogenized by using a Potter-Elvehjem homogenizer and centrifuged at 105,000 x g for 45 min. The supernatant, containing 30-50 mg ofprotein per ml, was collected, divided into aliquots, quick frozen under liquid n...

PurposeThe purpose of the experiments described here was to determine the effects of lipoic acid (LA)-dependent disulfide reduction on mouse lens elasticity, to synthesize the choline ester of LA (LACE), and to characterize the effects of topical ocular doses of LACE on mouse lens elasticity.MethodsEight-month-old mouse lenses (C57BL/6J) were incubated for 12 hours in medium supplemented with selected levels (0–500 μM) of LA. Lens elasticity was measured using the coverslip method. After the elasticity measurements, P-SH and PSSP levels were determined in homogenates by differential alkylation before and after alkylation. Choline ester of LA was synthesized and characterized by mass spectrometry and HPLC. Eight-month-old C57BL/6J mice were treated with 2.5 μL of a formulation of 5% LACE three times per day at 8-hour intervals in the right eye (OD) for 5 weeks. After the final treatment, lenses were removed and placed in a cuvette containing buffer. Elasticity was determined with a computer-controlled instrument that provided Z-stage upward movements in 1-μm increments with concomitant force measurements with a Harvard Apparatus F10 isometric force transducer. The elasticity of lenses from 8-week-old C57BL/6J mice was determined for comparison.ResultsLipoic acid treatment led to a concentration-dependent decrease in lens protein disulfides concurrent with an increase in lens elasticity. The structure and purity of newly synthesized LACE was confirmed. Aqueous humor concentrations of LA were higher in eyes of mice following topical ocular treatment with LACE than in mice following topical ocular treatment with LA. The lenses of the treated eyes of the old mice were more elastic than the lenses of untreated eyes (i.e., the relative force required for similar Z displacements was higher in the lenses of untreated eyes). In most instances, the lenses of the treated eyes were even more elastic than the lenses of the 8-week-old mice.ConclusionsAs the elasticity of the human lens decreases with age, humans lose the ability to accommodate. The results, briefly described in this abstract, suggest a topical ocular treatment to increase lens elasticity through reduction of disulfides to restore accommodative amplitude.

The effects of pH, acetimidate concentration, temperature, and reaction time of methyl acetimidate with sperm whale myoglobulin have been assessed. Reaction at pH 9.8 and 15 degrees C for 30 min with a sixfold excess of methyl acetimidate relative to each amino group yielded six acetimidomyoglobin derivatives which were separated and purified. Reaction with tetrahydrophthalic anhydride revealed the number of amino groups that remained unreacted in each separated component and made possible further subractionation. Modification at the NH2 terminus was quantitated by automated stepwise Edman degradation. The acetimidyl and tetrahydrophthalyl groups, were readily removable. The potentiometric titration of three of the completely deprotected components showed identity with the parent untreated sperm whale myoglobin. The first of two major products was acetimidated at all 19 epsilon-amino groups but not at the NH2 terminus. The second major product bore a blocked NH2 terminus but retained one unmodified epsilon-amino group, identified after modification by trinitrobenzenesulfonate as lysine residue 77. Of the minor components, one was identified as completely acetimidated at all 20 amino groups. The other three minor components appeared to contain irreversible by-products.