Separation of the yellow chromophores in individual brunescent cataracts

Quantitative changes in the 330 nm absorbing chromophores and 350/450 nm fluorophores of water-soluble (WS) and water-insoluble (WI) proteins of individual human cataract lenses were characterized and compared with aged normal human lens. Twenty-five brunescent cataract lenses from India were selected from five different stages (types I-V) based upon the color of the lens. The WS and WI proteins from each lens were collected and subjected to an extensive enzymatic digestion procedure under argon. The lens protein digests were separated by Bio-Gel P-2 size-exclusion chromatography and individual peaks were analyzed further by reversed-phase HPLC. The total WI proteins increased and the total WS protein decreased with the development of cataract, especially in the late stages of cataract (III-V). The total 330 nm absorbance and 350/450 nm fluorescence of the WI fraction also increased, however, the A(330) and fluorescence per mg lens protein were constant except for type V (black) lenses. Bio-Gel P-2 chromatography separated the chromophores and fluorophores into four fractions. The main fraction (designated as peak 2+3) from the cataract WI proteins was several times higher than that present in aged normal human lens WI proteins. A significant increase of this fraction was observed in WI proteins, but not in WS proteins with cataract development. Similarly, fractions 1 and 4 in the WI proteins also increased gradually but fraction 5 did not. Reversed-phase HPLC resolved fraction (2+3) of the water-insoluble sonicate supernatant proteins into four 330 nm absorbing peaks and eight fluorescent peaks. Among these peaks, a late-eluting peak (peak 8) increased 10 to 15-fold with the progress of cataract, and accounted for 80% of the total chromophores in type V lenses. This peak may represent limit digests of advanced glycation end-products (AGEs) derived protein cross-links. HPLC profiles of fraction 5 from both WS and WI proteins showed numerous new peaks which were not observed in either WS protein from cataract or WI proteins from aged normal human. The severe coloration and the higher levels of numerous novel chromophores and fluorophores in brunescent cataractous lenses reveal the possibility that a different chemistry occurs during cataract development.     

Chromatographic comparison of the UVA sensitizers present in brunescent cataracts and in calf lens proteins ascorbylated in vitro.

The water-insoluble (WI) fraction from aged human lenses contains yellow chromophoric sensitizers, which generate reactive oxygen species (ROS) when irradiated with UVA light. The WI proteins from type I to V brunescent cataract lenses were assayed for UVA-dependent superoxide anion synthesis. Rates varied from 8.4-15 nMol h(-1)mg protein(-1), but there was no significant difference in specific activity between cataract types. When calf lens soluble proteins were incubated with ascorbic acid for 4 weeks and dialyzed, they were capable of generating 30-40 nMol h(-1)mg protein(-1)superoxide anion when irradiated with UVA light. Two preparations each of brunescent cataract WI proteins and bovine lens proteins ascorbylated in vitro were extensively digested with proteolytic enzymes and the released amino acids separated by normal phase HPLC. The elution profiles of the digests were very similar based upon the absorbance at 330 nm and fluorescence at 350 nm excitation/450 nm emission. Each peak was pooled and analyzed for the UVA-dependent generation of both superoxide anion and singlet oxygen. Every peak exhibited sensitizer activity, and the UVA-dependent ROS generation was roughly proportional to the absorbance at 330 nm. In addition, the ratio of superoxide anion to singlet oxygen generated was similar with both preparations. These data argue that it is the brown, fluorescent compounds which accumulate during aging and cataract formation that are responsible for the UVA-dependent ROS formation, and that these browning products may be similar to the advanced glycation endproducts produced by ascorbylation of lens proteins under oxidative conditions. This work also presents an initial report of a chromatographic method to separate the UVA-sensitizers present in each of these protein preparations without the use of acid or base hydrolysis.     

Glycation of lens membrane intrinsic proteins.

Changes occurring at the membrane are believed to be the decisive factors in the initiation of diabetic cataract. During diabetic hyperglycemia lens crystallins were shown to undergo glycation. Several studies indicated that glycation brings about protein conformational changes thus implicated in cataractogenesis. Since the membrane proteins are the first targets for glycation, in this study we measured the glycation of alkali washed urea-insoluble membrane proteins from control and diabetic rats by two different methods, phenyl-boronate affinity chromatography and [3H]NaBH4 reduction, and confirmed by amino acid analysis. There was a significant increase in the glycation of membrane proteins in diabetic cataract lenses when compared to controls. It appears that lysine is the major site of glycation. Concomitant to early glycation, there was an increase in non-tryptophan fluorescence (Ex: 350 nm/Em: 440 nm) in the diabetic lens membrane proteins suggesting the presence of advanced glycation mediated protein cross-links. In order to identify whether the major membrane intrinsic protein, MIP26, undergoes glycation, we isolated MIP26 along with its degradatory product MIP22 as one peak on molecular sieve HPLC. HPLC isolated MIP26/MIP22 was further separated on SDS-PAGE followed by slicing and counting. This analysis revealed that MIP26 and MIP22 were more or less equally glycated in controls, however, in diabetic rats glycation of MIP22 was glycated slightly higher than MIP26. Moreover, the proportion of MIP22 increased by about 2-fold in diabetic lenses compared to controls. Thus it appears that major glycation sites are still retained in MIP22 in diabetic rat lenses. In vitro glycation studies with bovine lens membranes were also done using 14C glucose, followed by SDS-PAGE and autoradiography. The major protein glycated in vitro also seems to be MIP26. Interestingly, MIP22 was less glycated than MIP26 in vitro.     

氧化性白内障晶状体中蛋白和细胞的改变

目的探讨将氧化性白内障动物作为模型研究人老年性白内障机制的可行性。方法用过氧化氢(H2O2)诱发大鼠晶状体产生白内障,分析晶状体相对灰度值、晶状体水化程度,Folin酚法测量水溶性蛋白(WSP),电泳及染色分析WSP条带,流式细胞术测定晶状体上皮细胞(LECs)凋亡。结果6、12和24h时晶状体相对灰度值减小(P<0.05);WSP比例减少(P<0.05);WSP的相对分子质量为25000、29000和30000的信号减弱;晶状体水化程度增加(P<0.01);凋亡LECs增加(P<0.01)。结论H2O2诱发大鼠晶状体产生的白内障,其蛋白和细胞的改变与人老年性白内障相似,将氧化性白内障动物作为模型研究人老年性白内障具可行性。     

The reaction of proteins with 3-hydroxyanthranilic acid as a possible model for senile nuclear cataract in man

Proteins, including lens proteins, were incubated in the presence of 3-hydroxyanthranilic acid (30 HA) under oxidizing conditions. Samples were monitored for alterations in color, fluorescence, sulfhydryl content, lysine availability, methionine content, tryptophan content and protein size. Incubation of proteins with 30 HA produced rapid brown coloration and a correspondingly rapid decrease in sulfhydryl content. Alpha-, beta- and gamma-crystallins were all found to react with 30 HA. An increase in protein fluorescence (excitation 340/emission 425 nm) accompanied the color development. No significant decrease in the content of tryptophan or any other amino acid was detected by amino acid analysis. The levels of available lysine were not affected significantly by treatment with 30 HA. Oxidation of methionine to methionine sulfoxide and the covalent cross-linking of polypeptides was obtained by subsequent treatment of the tanned proteins with H2O2. The modifications observed are very similar to those found in the senile nuclear cataract lens.     

Exposure of rabbit lens to hyperbaric oxygen in vitro: regional effects on GSH level

Previous studies have indicated that in vivo exposure to hyperbaric O2 may be associated with the development of nuclear cataract. In the present work, in vitro effects of hyperbaric O2 on rabbit lenses were investigated following culture of the lenses in an atmosphere of 99% O2 at pressures ranging between 1 and 100 atm. Treatment with O2 resulted in a significant decrease in the level of reduced glutathione (GSH) in the lenses even at the lower pressures studied (less than 8 atm). At 100 atm O2 the loss of GSH was 85% after a 3 hr exposure. At 8 atm O2 a significant drop in GSH concentration was shown to occur in the lens nucleus prior to loss of the tripeptide in the superficial cortex. O2-treated lenses became hazy in appearance, especially at the higher pressures, but did not become densely opaque. Pressures of N2 up to 100 atm had no effect on either lens transparency or on the concentration of GSH. Although oxidized glutathione (GSSG) was detected in the whole lens at pressures of O2 as low as 4 atm, no change in GSH level or evidence for GSSG accumulation was observed in the capsule-epithelium of the lens at pressures as high as 50 atm O2. Ninety percent of the GSSG present in lenses after exposure to 100 atm O2 could be reconverted to GSH by subsequent culture of the lenses under normal conditions. Exposure of lenses to 50 atm O2 produced a three-fold stimulation of hexose monophosphate shunt activity, equal to that which has been reported for treatment of lenses with 0.06 mM H2O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-activated proteolysis in the lens nucleus during selenite cataractogenesis

A single injection of 20 mumol sodium selenite/kg body weight in 10-day-old rats caused severe nuclear cataract within 4 days. By 4 days postselenite injection, nuclear calcium levels increased from 0.4 to 6.8 mmol/kg lens dry weight. The purpose of these experiments was to determine if this calcium increase was associated with proteolysis specifically in the lens nuclear region. Sodium dodecyl sulfate polyacrylamide electrophoresis of lens nuclear proteins following selenite injection showed: loss of 30, 27, and 26 K molecular weight polypeptides in the soluble fraction, loss of 83, 52, 30, 27, and 26 K polypeptides in the insoluble fraction, and loss of the major 26 K membrane protein. Gel chromatography of nuclear soluble proteins indicated a decrease in beta H and beta L crystallins following selenite injection. Two-hour in vitro incubation of nuclear lens homogenates with calcium duplicated many of the proteolytic changes occurring in lenses in vivo following selenite injection. Calcium induced proteolysis in vitro was inhibited by EGTA, leupeptin, and iodoacetate but was not inhibited by phenylmethylsulfonyl fluoride. These properties are similar to calcium activated protease (CAP) from other tissues. Activation of CAP, and subsequent degradation of nuclear proteins, may be causes of selenite cataract.     

Glutathione: a vital lens antioxidant.

The reducing compound glutathione (GSH) exists in an unusually high concentration in the lens where it functions as an essential antioxidant vital for maintenance of the tissue's transparency. In conjunction with an active glutathione redox cycle located in the lens epithelium and superficial cortex, GSH detoxifies potentially damaging oxidants such as H2O2 and dehydroascorbic acid. Recent studies have indicated an important hydroxyl radical-scavenging function for GSH in lens epithelial cells, independent of the cells' ability to detoxify H2O2. Depletion of GSH or inhibition of the redox cycle allows low levels of oxidant to damage lens epithelial targets such as Na/K-ATPase, certain cytoskeletal proteins and proteins associated with normal membrane permeability. The level of GSH in the nucleus of the lens is relatively low, particularly in the aging lens, and exactly how the compound travels from the epithelium to the central region of the organ is not known. Recently, a cortical/nuclear barrier to GSH migration in older human lenses was demonstrated by Sweeney et al. The relatively low ratio of GSH to protein -SH in the nucleus of the lens, combined with low activity of the glutathione redox cycle in this region, makes the nucleus especially vulnerable to oxidative stress, as has been demonstrated with use of in vivo experimental animal models such as hyperbaric oxygen, UVA light and the glutathione peroxidase knockout mouse. Effects observed in these models, which are currently being utilized to investigate the mechanism of formation of human senile nuclear cataract, include an increase in lens nuclear disulfide, damage to nuclear membranes and an increase in nuclear light scattering. A need exists for development of therapeutic agents to slow age-related loss of antioxidant activity in the nucleus of the human lens to delay the onset of cataract.     

Raman spectroscopic evidence for nuclear disulfide in isolated lenses of hyperbaric oxygen-treated guinea pigs

Our laboratory treats guinea pigs with hyperbaric oxygen (HBO) as a model for investigating the formation of nuclear cataract. Previous analyses of lens supernatants using this model have shown an increase in disulfide (-SS-) and loss of sulfhydryl (-SH) in the lens nucleus of O(2)-treated animals. In this paper, we have used the non-invasive technique of Raman spectroscopy to confirm these findings in intact, freshly-excised lenses. Guinea pigs were treated 3 times per week with HBO for a total of 50 (4 months of treatment) or 85 (7 months of treatment) times to induce an increased level of lens nuclear light scattering. Intact lenses were analyzed by Raman spectroscopy using a 514.5 nm laser and collecting the scattered light in a 90 degrees geometry. The laser beam was focused either in the lens nucleus or equatorial cortex. Changes in the levels of -SS- (503 cm(-1)) and -SH (2577 cm(-1)) vibrations were measured. Raman spectra were analyzed by fitting Lorentzian profiles to the observed data in the -SS- and -SH regions. -SS- levels in the O(2)-treated nucleus were found to have increased by a factor of 2.1 (p=0.0001) and 2.5 (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. Based on previous biochemical analyses, the -SS- increase was due mainly to the formation of protein disulfide (PSSP) with contribution also from protein/thiol mixed disulfides, but not from oxidized glutathione. -SH levels in the O(2)-treated nucleus decreased by 13% (p=0.007) and 35% (p=0.001) after 50 and 85 HBO treatments, respectively, compared to age-matched controls. No significant increase in -SS- or loss of -SH was observed in the lens cortex of the O(2)-treated guinea pigs. The Raman spectroscopy results rule out the possibility that artifactual production of -SS- and loss of -SH occurred during homogenization of lenses in previous studies. The data provide additional evidence to support a link between O(2), disulfide-crosslinking of lens crystallins in the nucleus, and nuclear cataract.     

Reduced expression of 1-cys peroxiredoxin in oxidative stress-induced cataracts

1-cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin family with a single conserved cysteine residue, reduces a broad spectrum of hydroperoxides. This study was undertaken to examine changes in 1-cysPrx expression in human cataract samples, human lens epithelial (HLE B3) cell line, and rat organ-cultured lenses in response to oxidative insult induced by H2O2 or transforming growth factor-beta1 (TGF-beta1). Expression of 1-cysPrx mRNA and protein in HLE B3 cells increased in response to 2-8 ng ml(-1) TGF-beta1 and 50-75 microm H2O2 and then decreased below the control level at high doses (10 ng ml(-1) TGF-beta1 and 100-150 microm H2O2), as determined by Northern blot and immunoblot analysis. This reduction coincided with the decrease of cell viability. Immunoreactive 1-cysPrx protein was measured in capsulorrhexis specimens obtained from patients with anterior subcapsular cataract (ASC), nuclear sclerosis (NS), cortical spokes (CS), posterior subcapsular cataract (PSC), or white mature cataract (WC) at the time of cataract surgery. Significant reduction of 1-cysPrx protein was observed in ASC, PSC, and WC samples, but there was no statistical difference in CS and NS samples relative to normal control. Also, rat lens explants were cultured with 10 ng ml(-1) TGF-beta1 for approximately 5 days or 500 microm H2O2 for approximately 2 days. Subsequently, expression of 1-cysPrx mRNA and protein in the lens capsules was evaluated. Rat lens explants treated with TGF-beta1 or H2O2 developed a cataract similar to human ASC or WC, respectively, which resulted in a markedly decreased expression of 1-cysPrx mRNA and protein. Collectively, these findings show that expression patterns of 1-cysPrx gene in the lens are changed in response to oxidative stress, a major factor in the etiology of cataract.     

The role of protein-thiol mixed disulfides in cataractogenesis

Protein-thiol mixed disulfides in lenses have been implicated in the mechanism of protein-protein disulfide and other cross-linking leading to protein aggregation. The methodology for the detection and quantitation of protein-thiol mixed disulfides has been successfully established in our laboratory. Examination of mixed disulfides at different stages during development of a cataract may give relevant information on the mechanism of cataractogenesis, and whether oxidation is a part of that mechanism. In this study we investigated the involvement of mixed disulfides in cataract formation by using the H2O2-exposed lens as a model. Rat lenses, after being exposed to 0.5 mM H2O2 in culture showed an inverse relationship between the GSH loss and the protein-GSH formation with no effect on the protein-cysteine level. The H2O2-induced protein modification was also demonstrated indirectly by isoelectric focusing. The rate of protein-GSH production is dependent on the time of exposure and the concentration of H2O2. Age also plays some role as the lens GSH level decreases and the protein-thiol mixed disulfides increase as the animal becomes older. Lenses of older rats did not display more susceptibility to H2O2-induced mixed disulfide formation. The two protein-thiol mixed disulfides have a well-defined pattern of distribution in the rat lens. Most of the protein-GSH was found in the cortex and the water-soluble protein fraction whereas more protein-cysteine was found in the nucleus and water-insoluble protein fraction. Lens of older rat has more protein-cysteine as well as more water-insoluble proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quercetin attenuates UV- and H(2)O(2)-induced decrease of collagen type I in cultured human lens epithelial cells.

Ultraviolet (UV) radiation is related to cataract formation. The dynamics of matrix proteins play crucial roles in cell proliferation, cell migration, and the remodeling of lens capsule and, possibly, cataract formation. However, the change of dynamics of matrix proteins, such as collagens, in lens cells in response to UV radiation has not been investigated. Using cultured human lens epithelial cells, we, for the first time, demonstrate that UV radiation induces a decrease of collagen type I in a time- and dose-dependent manner. Hydrogen peroxide (H(2)O(2)) also induces a collagen type I decrease in a similar pattern. We observed that UV and H(2)O(2) induce JNK and its downstream component, c-Jun, activation in both a time- and dose-dependent manner. The pharmacologic inhibitor of JNK or JNKi inhibits UV-induced JNK and c-Jun activation and attenuates a UV-induced decrease of collagen type I. Quercetin, a well known antioxidant, also protects against a UV- and H(2)O(2)-induced decrease of collagen type I in a dose-dependent manner. Quercetin inhibits UV- and H(2)O(2)-induced JNK and c-Jun activation. Collectively, we conclude that quercetin attenuates both a UV- and H(2)O(2)-induced decrease of collagen type I via the inhibiting of JNK/c-Jun activity. Understanding the cellular-signaling pathways involved in the UV- and H(2)O(2)-induced decrease of collagen type I may reveal potential therapeutic targets for the UV-induced cataract.     

Age-related nuclear cataract-oxidation is the key

Age is by far the biggest risk factor for cataract, and it is sometimes assumed that cataract is simply an amplification of this aging process. This appears not to be the case, since the lens changes associated with aging and cataract are distinct. Oxidation is the hallmark of age-related nuclear (ARN) cataract. Loss of protein sulfhydryl groups, and the oxidation of methionine residues, are progressive and increase as the cataract worsens until >90% of cysteine and half the methionine residues are oxidised in the most advanced form. By contrast, there may be no significant oxidation of proteins in the centre of the lens with advancing age, even past age 80. The key factor in preventing oxidation seems to be the concentration of nuclear glutathione (GSH). Provided that nuclear GSH levels can be maintained above 2 mm, it appears that significant protein oxidation and posttranslational modification by reactive small molecules, such as ascorbate or UV filter degradation products, is not observed. Adequate coupling of the metabolically-active cortex, the source of antioxidants such as GSH, to the quiescent nucleus, is crucial especially since it would appear that the cortex remains viable in old lenses, and even possibly in ARN cataract lenses. Therefore it is vital to understand the reason for the onset of the lens barrier. This barrier, which becomes apparent in middle age, acts to impede the flow of small molecules between the cortex and the nucleus. The barrier, rather than nuclear compaction (which is not observed in human lenses), may contribute to the lowered concentration of GSH in the lens nucleus after middle age. By extending the residence time within the lens centre, the barrier also facilitates the decomposition of intrinsically unstable metabolites and may exacerbate the formation of H(2)O(2) in the nucleus. This hypothesis, which is based on the generation of reactive oxygen species and reactive molecules within the nucleus itself, shifts the focus away from theories for cataract that postulated a primary role for oxidants generated outside of the lens. Unfortunately, due to marked variability in the lenses of different species, there appears at present to be no ideal animal model system for studying human ARN cataract.     

Biochemical changes in selenite cataract model measured by high-resolution MAS H NMR spectroscopy

PURPOSE: To correlate certain levels of lens opacification with high-resolution magic-angle spinning proton nuclear magnetic resonance (HR-MAS (1)H NMR) spectroscopy analysis of the biochemical changes in rat lenses in a selenite cataract model. METHODS: Selenite cataract was induced by injecting 13-day-old Sprague-Dawley rat pups with a single subcutaneous dose of sodium selenite (3.28 mg/kg in 0.9% sodium chloride solution). Lens opacification was observed using a photographic slit-lamp microscope at selected time-points 3, 6 and 9 days after selenite injection and was then graded (levels 0, 1 and 2). The animals were killed after the slit-lamp microscopy, lenses were removed and HR-MAS (1)H NMR spectra from intact lenses were obtained. Relative changes in metabolite concentrations were determined after comparison with matched lenses from untreated animals. RESULTS: Photographic slit-lamp microscopy revealed different stages of cataract in all animals treated with selenite. In the high quality HR-MAS (1)H NMR spectra of the lenses, more than 30 different metabolites were identified in each lens. With the exception of taurine, the concentrations of all amino acids showed a significant increase (p < 0.05) in the second level of cataract. By contrast, glutathione (GSH), succinate and phosphocholine concentrations were significantly reduced. CONCLUSIONS: For the first time, this study demonstrates the potential to correlate the level of lens opacification with the biochemical changes obtained with HR-MAS (1)H NMR spectroscopy analysis in a selenite cataract model.     

Thiol regulation in the lens.

The high content of glutathione (GSH) in the lens is believed to protect the thiols in structural proteins and enzymes for proper biological functions. The lens has both biosynthetic and regenerating systems for GSH to maintain its large pool size (4-6 mM). However, we have observed that, in aging lenses or lenses under oxidative stress, the size of GSH pool is diminished; and some protein thiols are being S-thiolated by oxidized nonprotein thiols to form protein-thiol mixed disulfides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC). We have shown in an H2O2-induced cataract model that PSSG formation precedes a cascade of events starting with protein disulfide crosslinks, protein solubility loss, and eventual lens opacification. Recently, we discovered that this early oxidative damage in protein thiols could be spontaneously reversed in H2O2 pretreated lenses if the oxidant was removed in time. This dethiolation process is likely mediated through a redox regulating enzyme, thioltransferase (TTase), which has been discovered recently in the lens. To understand if the role of oxidative defense and repair is the physiological function of TTase in the lens, we cloned the TTase gene and purified the recombinant human lens TTase. Although TTase required GSH for its activity, TTase was far more efficient in dethiolating lens proteins than GSH alone. It favored PSSG over PSSC and dethiolated gamma-crystallin-S-S-G better than the alpha-crystallin counterparts. Furthermore, TTase showed a remarkable resistance to oxidation (H2O2) in cultured rabbit lens epithelial cells when GSH peroxidase, GSH reductase, and glyceraldehyde-3-phosphate dehydrogenase were severely inactivated. We further showed that activity loss in those SH sensitive enzymes could be attributed to S-thiolation, but reactivation via dethiolation could be attributed to TTase. We conclude that TTase can regulate and repair the thiols in lens proteins and enzymes through its dethiolase activity, thus contributing to the maintenance of the function of the lens.     

Redox regulation in the lens

The high content of glutathione (GSH) in the lens is believed to protect thiols in structural proteins and enzymes for proper biological functions. The lens has both biosynthetic and regenerating systems for GSH to maintain its large pool size. However, ageing lenses or lenses under oxidative stress show an extensively diminished size of GSH pool with some protein thiols being S-thiolated by oxidized non-protein thiols to form protein-thiol mixed disulfides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC) or protein-S-S-gamma-glutamylcysteine. It was shown in an H(2)O(2)-induced cataract model that PSSG formation precedes a cascade of events before cataract formation, starting with protein disulfide crosslinks, protein solubility loss and high molecular weight aggregation. Furthermore, this early oxidative damage in protein thiols can be spontaneously reversed in H(2)O(2) pretreated lenses if the oxidant is removed in time. This dethiolation process appears to have mediated through a redox-regulating enzyme, thioltransferase (TTase), which is ubiquitously present in microbial, plant and animal tissues, including the lens. The GSH-dependent, low molecular weight (11.8 kDa) cytosolic enzyme plays an important role in oxidative defense and can modulate key metabolic enzymes in the glycolytic pathway. The enzyme repairs oxidatively damaged proteins/enzymes through its unique catalytic site with a vicinal cysteine moiety, which can specifically dethiolate protein-S-S-glutathione and restore protein free SH groups for proper enzymatic or protein functions. Most importantly, it has been demonstrated that thioltransferase has a remarkable resistance to oxidation (H(2)O(2)) in cultured human and rabbit lens epithelial cells under oxidative stress conditions when other oxidation defense systems of GSH peroxidase and GSH reductase are severely inactivated. A second repair enzyme, thioredoxin (TRx), which is NADPH-dependent, is widely found in many lower and higher life forms of life. It can dethiolate protein disulfides and thus is an extremely important regulator for redox homeostasis in the cells. Thioredoxin has been recently found in the lens and has been shown to participate in the repair process of oxidatively damaged lens proteins/enzymes. These two enzymes may work synergistically to regulate and repair thiols in lens proteins and enzymes, keeping a balanced redox potential to maintain the function of the lens.     

Influence of exposure time and pupil size on a Shack-Hartmann metric of forward scatter

PURPOSE: To determine the influence of exposure time and pupil size on a Shack-Hartmann (S/H) derived metric of forward scatter (MAX_SD) using a physical model of nuclear cataract. METHODS: A physical model eye was developed and mounted to a S/H wavefront sensor. The eye model consisted of a lens, variable pupil, simulated cataract, and retina. Located behind the pupil, a cuvette contained one of five polystyrene microsphere solutions simulating five levels of nuclear cataract severity. Cataract severity was described using a S/H derived metric of forward scatter (MAX SD), which measures aspects of forward scatter contained in the S/H lenslet point spread functions (PSF). To determine the impact of exposure time and pupil size, measurements of MAX_SD were regressed against cataract severity for three different exposure times and three different pupil sizes. RESULTS: MAX_SD was well correlated to cataract severity. Exposure time had the largest influence, and pupil size had the smallest influence on the forward scatter metric. When pupil size and exposure time were allowed to vary and image saturation was allowed to occur, MAX SD explained 83% of the variance in cataract severity. Excluding images where saturation occurred, holding optimal exposure time constant, and varying pupil size, MAX_SD explained 97% of the variance in cataract severity. CONCLUSIONS: The ability of the forward scatter metric derived from S/H measurements to predict cataract severity for a longitudinal study is optimized by selecting a patient-specific exposure at the initial cataract assessment to avoid saturation and maximize the dynamic range of the system. This patient-specific exposure should be used in all future visits.     

The effect of H2O2 on lens epithelial cell glutathione

Investigation of lens epithelial cells indicates that under normal conditions, essentially all of the detectable cellular glutathione is in a reduced state. However, exposure to levels of H2O2 in the range found in the aqueous fluid of cataract patients causes rapid, very large changes in the glutathione redox ratios. Immediately following short-term exposure to 0.15-0.2 mM H2O2, reduced glutathione drops to 19% of its normal level and the remainder of the total glutathione is found in the oxidized form. Within the next few minutes, the redox ratio returns to normal. However, total glutathione levels remain approximately 20% below normal even one hour after exposure to H2O2. With exposure to a higher concentration of H2O2, a greater loss of glutathione is observed. The results suggest that the glutathione redox ratios change dramatically as a result of oxidative insult but quickly return to normal when the oxidative insult is removed. The formation of mixed glutathione-protein disulfide was also observed but only after long-term (1 hour) exposure to a high level (0.6 mM) of H2O2.     

Regional distribution of free calcium in selenite cataract: relation to calpain II

The purpose of this experiment was to assess the roles of free, intracellular calcium and calcium-dependent neutral protease (calpain II, EC.34.22.17) in selenite nuclear cataract. Free calcium ion concentrations within lens nuclear fibers during selenite cataractogenesis increased to 3 microM on day 2 post-injection (clear lens) and to 108 microM at day 4 (nuclear cataract). Calpain II is known to be activated in vitro by calcium levels above 50 microM. Calpain II activity was present in the lens nucleus at time periods preceding formation of selenite cataract. These data suggested that after selenite injection, calpain II was activated by elevated free calcium in the nucleus, and that calpain II-induced proteolysis of nuclear proteins was an important mechanism in selenite cataract. Calpain II levels were also observed to decrease in the nucleus during selenite cataractogenesis, probably due to autolysis. This was supported by the finding that incubation of purified lens calpain II with 100 microM calcium caused partial inactivation of the protease.     

Analysis of proteins during recovery from lens opacity--analysis of selenite cataract model using Sprague-Dawley and Wistar rat

BACKGROUND AND PURPOSE: The cataract in Sprague-Dawley rats injected with selenite is a dense nuclear opacity that appears by 4 or 5 days after selenite injection and becomes irreversible by 7 days. Injection of Wistar rats with selenite resulted in a similar nuclear opacity by 4 or 5 days that began to recover transparency by 7 days. In this report, the cytoplasmic proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in lenses from Sprague-Dawley and Wistar rats at 4 and 7 days after injection. RESULTS: In the opaque lens cells, degradation of the 31 kDa protein and cytoskeletal proteins (vimentin, spectrin, and actin) was observed during cataract development using SDS-PAGE and western blot analysis. During recovery from opacity, the decreased 31 kDa protein and the vimentin increased. CONCLUSION: The results suggest that the 31 kDa protein and the vimentin may be important for recovery of transparency in a reversible model of cataract formation.