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1.  Nucleophilic compounds decrease advanced glycation end products (AGEs) from ascorbic acid in the hSVCT2 transgenic mouse model of lenticular aging 
Senile cataracts are associated with oxidation, fragmentation, cross-linking, insolubilization, and yellow pigmentation of lens crystallins. This process is partially explained by advanced glycation endproducts (AGEs) from ascorbic acid (ASA), as unequivocally demonstrated in our hSVCT2 transgenic mouse(PNAS 103:16912, 2006). We now present the first pharmacological intervention study against ascorbylation in these mice.
Five groups of mice (10 mice/group) were fed from two to nine months a diet containing 0.1% (wt/wt) aminoguanidine (AG), pyridoxamine (PM), penicillamine (PA), and nucleophilic compounds NC-I and NC-II. AGEs were determined in crystallin digests using HPLC, LC-MS or GC-MS. In vitro incubations of lens protein extract with ASA or dehydroascorbic aicd (DHA) were also performed.
ASA level increased ~10 fold in all groups and was unaffected by treatment. AGEs were several fold increased in transgenic compared to control lenses. Body weight, food intake, lenticular glutathione and glycated lysine level were unaltered. In vitro, all compounds inhibited AGE formation. In vivo, NC-I and NC-II significantly decreased protein fluorescence at λex335/em385 (p=0.045, 0.017, respectively) and λex370/em440 (p=0.029, 0.007, respectively). Other inhibitors had no effect. After 7 months, only NC-1 and NC-2 induced a 50 % reduction in pentosidine (n.s, p=0.035 respectively). NC-1 also decreased carboxymethyllysine (CML) (p=0.032) and carboxyethyllysine (CEL) (p= n.s). Fluorescent crosslink K2P was decreased by NC-1, NC-2, AG and PM (p= n.s).
Pharmacologically blocking protein ascorbylation with absorbable guanidino compounds is feasible and may represent a new strategy for the delay of age-related nuclear sclerosis of the lens.
PMCID: PMC2576497  PMID: 18421088
2.  Role of lipid peroxidation in the pathogenesis of myopic and senile cataract. 
AIMS/BACKGROUND: Increased production of free radicals, consumption of antioxidant, and oxidation of unsaturated lipids have been observed recently in cataractous lenses and active participation of the retina in human cataractogenesis has been proposed. To verify this hypothesis, the total (GSH) and oxidised (GSSG) glutathione concentrations were assayed in the lens and the malondialdehyde (MDA) levels assayed in the vitreous and in the lens of normal controls and patients with senile or myopic cataract. METHODS: The study was conducted on 34 lenses (nucleus and epinucleus) (nine clear lenses, 14 lenses with idiopathic senile cataract, and 11 lenses affected by severe myopic cataract) and vitreous of 19 (seven non-myopic, seven myopic, and five control) subjects. Glutathione determination was performed following the method of Reed, while malondialdehyde was assayed using a modification of the method of Dahle. RESULTS: Cataractous lenses showed a decreased content of GSH and increased concentration of GSSG compared with clear lenses. A higher oxidative consumption of GSH was found in myopic cataracts compared with senile ones. Also, increased levels of MDA were observed both in cataractous lenses and in the vitreous of myopic patients compared with the control and the senile ones. CONCLUSION: The observed alterations strongly suggest that retinal lipid peroxidation might play a key role in human cataractogenesis, especially in the myopic type.
PMCID: PMC505624  PMID: 8942384
3.  Glycation by Ascorbic Acid Oxidation Products Leads to the Aggregation of Lens Proteins 
Biochimica et biophysica acta  2007;1782(1):22-34.
Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 × 106 up to 3.0 × 108 Da. This compared to proteins ranging from 1.8 × 106 up to 3.6 × 108 Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-14C] ascorbate was incorporated into the aggregate fraction and because CNBH3, which reduces the initial Schiff base, prevented any protein aggregation.
Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of α-crystallin, significant aggregation of βH-crystallin, but rapid precipitation of purified βL- and γ-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, α-crystallin prevented the precipitation of the purified βL- and γ-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with βL-, or γ-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of α- and βL-, or γ-crystallins showed similar molar masses (up to 108 Da) and hydrodynamic diameter (up to 80 nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.
PMCID: PMC2274899  PMID: 18023423
ascorbic acid; glycation; lens proteins; protein aggregation; light scattering
4.  Spectroscopic and biochemical correlations during the course of human lens aging 
BMC Ophthalmology  2006;6:10.
With age, the human lens accumulates variety of substances that absorbs and fluorescence, which explains the color of yellow, brunescent and nigrescent cataract in terms of aging. The aim of this study was to assess lens fluorophores with properties comparable to those of advanced glycated end products (AGEs) in relation to age in human lenses. These fluorescent compounds are believed to be involved in the development of cataract.
Spectroscopic (UV-Vis-NIR) and fluorescence photography (CCD-Digital based image analysis) studies were carried out in randomly selected intact human lenses (2–85 years). AGE-like fluorophores were also measured in water soluble and insoluble (alkali soluble) fractions of human lenses (20–80 years).
Our experimental findings suggest that there was a progressive shift in the absorbance characteristic of intact lens in the range of λ210 nm-λ470 nm. A relative increase in the absorptivity at λ(511–520 nm), with age, was also observed. In addition, the ratio of absorptivity at λ(511–520 nm) versus the maximum absorbance recorded at blue-end cut-off (210–470 nm) was also found to increase, with age. The fluorescent intensity in the intact lens at both UV-B (λEx312 nm) and UV-A (λEx365 nm) were found to be positively correlated (r2 = 0.91 & 0.94, respectively; Confidence interval 95%) upto 50 years of age. In addition, a concomitant changes in AGE- like fluorophores were also observed in the processed lens samples (soluble and insoluble fractions) along the age. A significant increase in the concentration of AGE- like fluorophores, both in intact and processed lens was observed during the period of 40 – 50 years.
Based on the present investigation, it was concluded that significant changes do occur in the AGE-like fluorophores of human lenses during the period of 40–50 years.
PMCID: PMC1450316  PMID: 16519820
5.  Perinuclear lens retrodots: a role for ascorbate in cataractogenesis. 
Lens retrodots are round, oblong, or oval features in the perinuclear zone of the adult lens after the fifth decade of life and associated with cataract. Retrodots were found in 47 out of 121 eyes with cataract (39%) in the present series. They show birefringence in vivo and in vitro, and chemical studies suggest that they contain calcium oxalate. It is proposed that ascorbic acid, which is abundant in the normal human lens, is the most likely source for this oxalate. Ascorbic acid is thought to have a protective role against oxidative stress in the lens and other parts of the eye, and its level is known to be reduced in senile cataract. The presence of the retrodots may identify lenses which have been exposed to oxidative stress and are less capable of resisting oxidative damage.
PMCID: PMC1041096  PMID: 3828268
Free radical biology & medicine  2010;49(5):847-856.
The effects of anaerobic (lens) vs aerobic (skin) environment on carbonyl and oxidant stress are compared using de novo and existing data on advanced glycation and oxidation products in human crystallins and collagen. Almost all modifications increase with age. Methylglyoxal hydroimidazolones (MG-H1), carboxymethyl-lysine (CML), and carboxyethyl-lysine (CEL) are several folds higher in lens than skin, and markedly increase upon incubation of lens crystallins with 5 mM ascorbic acid. Vice-versa, fructose-lysine, glucosepane crosslinks, glyoxal hydroimidazolones (G-H1), metal catalyzed oxidation (allysine) and H2O2 dependent modifications (2-aminoapidic acid and methionine sulfoxide) are markedly elevated in skin, but relatively suppressed in the aging lens. In both tissues ornithine is the dominant modification, implicating arginine residues as the principal target of the Maillard reaction in vivo. Diabetes (here mostly type 2 studied) increases significantly fructose-lysine and glucosepane in both tissues (P<0.001) but has surprisingly little effect on the absolute level of most other advanced glycation end products (AGEs) . However, diabetes strengthens the Spearman correlation coefficients for age-related accumulation of hydrogen peroxide mediated modifications in the lens. Overall, the data suggest oxoaldehyde stress involving methylglyoxal from either glucose or ascorbate is predominant in the aging non-cataractous lens, while aging skin collagen undergoes combined attack by non-oxidative glucose mediated modifications, as well as those from metal catalyzed oxidation and H2O2.
PMCID: PMC2910832  PMID: 20541005
crystallins; collagen; glycation; oxidative stress; methylglyoxal; metals
7.  Nonenzymatic glycation of human lens crystallin. Effect of aging and diabetes mellitus. 
Journal of Clinical Investigation  1984;74(5):1742-1749.
We have examined the nonenzymatic glycation of human lens crystallin, an extremely long-lived protein, from 16 normal human ocular lenses 0.2-99 yr of age, and from 11 diabetic lenses 52-82-yr-old. The glucitol-lysine (Glc-Lys) content of soluble and insoluble crystallin was determined after reduction with H-borohydride followed by acid hydrolysis, boronic acid affinity chromatography, and high pressure cation exchange chromatography. Normal lens crystallin, soluble and insoluble, had 0.028 +/- 0.011 nanomoles Glc-Lys per nanomole crystallin monomer. Soluble and insoluble crystallins had equivalent levels of glycation. The content of Glc-Lys in normal lens crystallin increased with age in a linear fashion. Thus, the nonenzymatic glycation of nondiabetic lens crystallin may be regarded as a biological clock. The diabetic lens crystallin samples (n = 11) had a higher content of Glc-Lys (0.070 +/- 0.034 nmol/nmol monomer). Over an age range comparable to that of the control samples, the diabetic crystallin samples contained about twice as much Glc-Lys. The Glc-Lys content of the diabetic lens crystallin samples did not increase with lens age.
PMCID: PMC425353  PMID: 6438156
8.  Clinical and experimental advances in congenital and paediatric cataracts 
Cataracts (opacities of the lens) are frequent in the elderly, but rare in paediatric practice. Congenital cataracts (in industrialized countries) are mainly caused by mutations affecting lens development. Much of our knowledge about the underlying mechanisms of cataractogenesis has come from the genetic analysis of affected families: there are contributions from genes coding for transcription factors (such as FoxE3, Maf, Pitx3) and structural proteins such as crystallins or connexins. In addition, there are contributions from enzymes affecting sugar pathways (particularly the galactose pathway) and from a quite unexpected area: axon guidance molecules like ephrins and their receptors. Cataractous mouse lenses can be identified easily by visual inspection, and a remarkable number of mutant lines have now been characterized. Generally, most of the mouse mutants show a similar phenotype to their human counterparts; however, there are some remarkable differences. It should be noted that many mutations affect genes that are expressed not only in the lens, but also in tissues and organs outside the eye. There is increasing evidence for pleiotropic effects of these genes, and increasing consideration that cataracts may act as early and readily detectable biomarkers for a number of systemic syndromes.
PMCID: PMC3061104  PMID: 21402583
paediatric; cataracts; human; mouse; genetics
9.  Green Tea Extract Suppresses the Age-Related Increase in Collagen Crosslinking and Fluorescent Products in C57BL/6 Mice 
Collagen crosslinking during aging in part results from Maillard reaction endproducts of glucose and oxoaldehydes. Because of the tight link between oxidative and carbonyl stress, we hypothesized that natural antioxidants and “nutriceuticals” could block collagen aging in C57BL/6 mice. Six groups of young and adult mice received vitamin C, vitamin E, vitamin C&E, blueberry, green tea extract (GTE), or no treatment for a period of 14 weeks. Body weights and collagen glycation were unaltered by the treatment. However, GTE or vitamin C&E combined blocked tendon crosslinking at 10 months of age (p < 0.05, adult group). GTE also blocked fluorescent products at 385 and 440 nm (p = 0.052 and < 0.05, respectively) and tended to decrease skin pentosidine levels. These results suggest that green tea is able to delay collagen aging by an antioxidant mechanism that is in part duplicated by the combination of vitamin C and E.
PMCID: PMC3561737  PMID: 14743550
Glucose; polyphenols; oxidant stress; vitamin C; vitamin E
10.  Docking studies of Vitamin C, Vitamin E, Damnacanthal and Scopoletin with human lens gamma D-crystalline 
Bioinformation  2013;9(14):721-724.
Vitamin C, Vitamin E, scopoletin and damnacanthal are the major constituents of Noni (Morinda citrifolia). These compounds are known to have good medicinal properties and they are known to act as antioxidants. Loss of vision in elderly is due to opaqueness of the lens proteins such as gamma-D-crystallin during oxidative stress conditions. Therefore, it is of importance to find the potential interaction of Vitamin C, Vitamin E, Scopoletin and Damnacanthal with the lens protein gamma-D-crystallin. Hence, their physical binding to gamma-D crystallin (PDB ID: 2G98) was evaluated using molecular and structural docking procedures. Results show the potential binding of all the above anti-oxidants to gamma-D-crystalline with equal affinity. Thus, the role of cumulative anti-oxidant effect in Noni fruit juice through their potential yet predicted interaction with the lens protein gamma-D-crystallin is implied for cataract treatment.
PMCID: PMC3746095  PMID: 23976828
Cataract formation; Oxidative stress; gamma-D-crystallin; Anti-oxidants; Docking scores
11.  Phototransformations of Advanced Glycation End Products in the Human Eye Lens due to Ultraviolet A Light Irradiation 
Previous studies from this laboratory have shown that ultraviolet A (UVA) light can bleach the yellow advanced glycation end products (AGEs) of aged and cataractous human lenses. The AGEs OP-lysine and argpyrimidine are two UVA-absorbing posttranslational modifications that are abundant in the eye lens. The purpose of this study was to outline the changes in these two AGEs due to UVA irradiation. The changes of OP-lysine, OP-phenethylamine (a phenethylamine analogue of OP-lysine), and argpyrimidine due to irradiation with UVA light in the presence or absence of air and ascorbic acid were followed by different spectral methods. Aged human lenses were similarly irradiated in artificial aqueous humor. The amounts of OP-lysine in the irradiated lenses and in the corresponding dark controls were determined by HPLC. Both OP-lysine and argpyrimidine decreased 20% when irradiated with UVA light in the absence of ascorbic acid. Under the same conditions, OP-lysine was bleached 80% in the presence of ascorbic acid during irradiation experiments. In contrast, argpyrimidine UVA light bleaching was not affected by the presence of ascorbic acid. Interestingly the major product of OP-phenethylamine after UVA irradiation in the presence of ascorbic acid was phenethylamine, which indicates that the entire heterocycle of this AGE was cleaved and the initial amino group was restored. Some AGEs in the human eye lens can be transformed by UVA light.
PMCID: PMC1564128  PMID: 16037236
ascorbic acid; OP-lysine; UVA light; eye lens; glycation
12.  Antioxidant Capacity of Lenses with Age-Related Cataract 
The immediate cause of the occurrence of cataract is unknown, but oxidative damage and effects of reactive oxygen species are considered important in its etiopathogenesis. Our research was aimed at testing the nonenzyme antioxidant power of corticonuclear lens blocks, with different types and different maturity of age-related cataract. Clinical and biochemical researches were carried out in 101 patients with age-related cataract. In corticonuclear lens blocks of the patient, the concentration of nonprotein and total-SH groups and the concentration of total vitamin C and dehydroascorbic acid (DHA) were determined; the current redox balance of dehydroascorbate/ascorbate and total antioxidant power measured by ferric-reducing ability were examined. In corticonuclear lens blocks with incipient cataract a significantly higher concentration of GSH, total SH groups, concentration of total vitamin C and ascorbic acid (AA), and ferric-reducing ability were measured. The measured concentration of DHA is higher than the concentration of AA in the lenses with the incipient and mature cataract. The concentration ratio of redox couple DHA/AA is higher in lenses with mature cataract, where the measured concentration of AA was lower than in the incipient cataract. Timely removal of DHA from the lens is important because of its potential toxicity as an oxidant. An increase of the current concentration of DHA/AA redox balance can be an indicator of oxidative stress.
PMCID: PMC3272861  PMID: 22363833
13.  Advanced glycation end products in diabetic and non-diabetic human subjects suffering from cataract 
Age  2010;33(3):377-384.
Advanced glycation end products (AGEs) play a pivotal role in loss of lens transparency, i.e., cataract. AGEs formation occurs as a result of sequential glycation and oxidation reaction between reducing sugars and protein. AGEs production takes place throughout the normal aging process but its accumulation is found to be more rapid in diabetic patients. In this study, we quantified AGEs and N-(carboxyethyl) lysine (CEL) in human cataractous lenses from non-diabetic (n = 50) and diabetic patients (n = 50) using ELISA. We observed significantly higher (p < 0.001) levels of lens AGEs and CEL in diabetic patients with cataract as compared with their respective controls. The presence of AGEs and CEL was also determined by western blotting and immuno-histochemical analysis. Furthermore, isolated β-crystallin from cataractous lenses of non-diabetic and diabetic patients was incubated with different sugars to evaluate the extent of glycation in a time dependent manner. Our data indicated more pronounced glycation in patients suffering from diabetes as compared to non-diabetics subjects demonstrating the need to focus on developing normoglycemic approaches. Such studies may provide an insight in developing therapeutic strategies and may have clinical implications.
PMCID: PMC3168597  PMID: 20842534
AGEs; Cataract; Diabetes; CEL; Non-enzymatic glycation
14.  Protein-Protein Interactions and Lens Transparency 
Experimental eye research  2008;87(6):496-501.
Past studies have identified posttranslational modifications of human lens proteins occurring during cataract formation, and have also demonstrated that protein-protein interactions exist between different lens crystallins. Based upon current theories of lens transparency, these posttranslational modifications and their possible effects upon crystallin interactions may be the key to understanding why the lens is able to transmit light, and why transmission is decreased during cataractogenesis. This review will summarize current knowledge of posttranslational modifications during human cataractogenesis, and will propose their possible role in protein-protein interactions that are thought to be necessary for lens transparency. Based upon this premise, model systems will be described that will test the validity of the theory.
PMCID: PMC2666974  PMID: 18835387
15.  Lens Aging: Effects of Crystallins 
Biochimica et biophysica acta  2009;1790(10):1095-1108.
The primary function of the eye lens is to focus light on the retina. The major proteins in the lens—a, b, and g-crystallins—are constantly subjected to age-related changes such as oxidation, deamidation, truncation, glycation, and methylation. Such age-related modifications are cumulative and affect crystallin structure and function. With time, the modified crystallins aggregate, causing the lens to increasingly scatter light on the retina instead of focusing light on it and causing the lens to lose its transparency gradually and become opaque. Age-related lens opacity, or cataract, is the major cause of blindness worldwide. We review deamidation, and glycation that occur in the lenses during aging keeping in mind the structural and functional changes that these modifications bring about in the proteins. In addition, we review proteolysis and discuss recent observations on how crystallin fragments generated in vivo, through their anti-chaperone activity may cause crystallin aggregation in aging lenses. We also review hyperbaric oxygen treatment induced guinea pig and ‘humanized’ ascorbate transporting mouse models as suitable options for studies on age-related changes in lens proteins.
PMCID: PMC2743770  PMID: 19463898
lens crystallins; aging; lens opacity; chaperones; deamidation; glycation; oxidation; peptides
16.  Accumulation of Maillard reaction products in skin collagen in diabetes and aging. 
Journal of Clinical Investigation  1993;91(6):2463-2469.
To investigate the contribution of glycation and oxidation reactions to the modification of insoluble collagen in aging and diabetes, Maillard reaction products were measured in skin collagen from 39 type 1 diabetic patients and 52 nondiabetic control subjects. Compounds studied included fructoselysine (FL), the initial glycation product, and the glycoxidation products, N epsilon-(carboxymethyl) lysine (CML) and pentosidine, formed during later Maillard reactions. Collagen-linked fluorescence was also studied. In nondiabetic subjects, glycation of collagen (FL content) increased only 33% between 20 and 85 yr of age. In contrast, CML, pentosidine and fluorescence increased five-fold, correlating strongly with age. In diabetic patients, collagen FL was increased threefold compared with nondiabetic subjects, correlating strongly with glycated hemoglobin but not with age. Collagen CML, pentosidine and fluorescence were increased up to twofold in diabetic compared with control patients: this could be explained by the increase in glycation alone, without invoking increased oxidative stress. There were strong correlations among CML, pentosidine and fluorescence in both groups, providing evidence for age-dependent chemical modification of collagen via the Maillard reaction, and acceleration of this process in diabetes. These results support the description of diabetes as a disease characterized by accelerated chemical aging of long-lived tissue proteins.
PMCID: PMC443306  PMID: 8514858
17.  Drevogenin D prevents selenite-induced oxidative stress and calpain activation in cultured rat lens 
Molecular Vision  2007;13:1121-1129.
Selenite-induced cataractogenesis is mediated by oxidative stress, accumulation of calcium and activation of lenticular calpains. Calpains are a super family of Ca2+ dependent proteases, which are involved in lens protein proteolysis and insolubilization. Many inhibitors could prevent calpain-induced proteolysis of α- and β-crystallins in rodent cataracts. Evaluating natural sources with antioxidant property and subsequent prevention of calpain activation may lead to the development of safer and more effective agents against cataractogenesis. There are no reports on the protective role of bioactive components against calpain-mediated proteolysis and subsequent cataractogenesis. The purpose of the study was to evaluate the role of Drevogenin D, a triterpenoid aglycone, isolated from Dregea volubilis in preventing selenite-induced, calcium-activated, calpain-mediated proteolysis in cultured rat lenses.
Lenses were extracted from Sprague-Dawley strain rats at the age of one month and were organ cultured in M-199 medium with HEPES buffer. The lenses were divided into three groups with eight lenses in each group as follows: lenses cultured in a normal medium (GI), lenses cultured in a sodium selenite supplemented medium (GII), and lenses cultured in a medium supplemented with sodium selenite and Drevogenin D-treated (GIII). Changes to transparency and opacity formation of lenses were monitored under microscopic observation. At the end of the experiment, biochemical parameters such as activity of lens superoxide dismutase (SOD), lens Ca2+ ATPase, concentration of Ca2+, levels of sulfhydryl content, and thiobarbituric acid reacting substances (TBARS) were determined. Changes to casein zymography for calpains, immunoblot for Lp82, and SDS-PAGE of lens water soluble protein fraction (WSF) were also done.
Microscopic evaluation of lens morphology showed that Drevogenin D prevented the opacification in G-III. Drevogenin D inhibited the accumulation of calcium, the activation of calpain system, and lipid peroxidation. Activity of Ca2+ATPase, SOD, and SDS-PAGE profile of water soluble proteins was normalized following treatment with Drevogenin D.
Selenite-induced cataractogenesis is mediated by oxidative stress leading to a decrease in the activity of Ca2+ ATPase, resulting in the accumulation of calcium and the subsequent activation of lenticular calpains. The results obtained indicated that Drevogenin D treatment was effective in protecting the lens proteins by controlling stress-induced protein oxidation, maintenance of Ca2+ ATPase activity, calcium accumulation, lipid peroxidation, and prevention of calpain activation. Hence, Drevogenin D can be used as a potential therapeutic agent against oxidative stress-induced cataract.
PMCID: PMC2779145  PMID: 17653057
18.  Lens-specific promoter activity of a mouse gamma-crystallin gene. 
Molecular and Cellular Biology  1985;5(9):2221-2230.
Crystallins are the major water-soluble proteins in vertebrate eye lenses. These lens-specific proteins are encoded by several gene families, and their expression is differentially regulated during lens cell differentiation. Here we show that a cloned mouse gamma-crystallin promoter is active in lens explants derived from 14-day-old chicken embryos but inactive in a variety of cells of non-lens origin. We also show that sequences required for proper utilization of this promoter are contained between nucleotide positions -392 and +47 relative to the transcription initiation site; deletion of sequences from positions -392 to -171 completely abolishes promoter activity. Since chickens do not have gamma-crystallin genes, the expression of a mouse gamma-crystallin promoter in chicken lens cells suggests that different classes of crystallin genes may be regulated by common lens tissue-specific mechanism(s) independent of species.
PMCID: PMC366947  PMID: 3837188
19.  Mechanism of Insolubilization by a Single point Mutation in αA-Crystallin Linked with Hereditary Human Cataract 
Biochemistry  2008;47(36):9697-9706.
αA-Crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The single point mutation R49C in αA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the αA-R49C mutant show nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90° light scattering, viscosity, refractive index and bis-ANS fluorescence of lens proteins isolated from the αA-R49C mouse lenses and find that the concentration of total water-soluble proteins showed a pronounced decrease in αA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high molecular weight aggregated material in the void volume which still remains soluble in AA-R49C homozygous lens homogenates. Increased binding of β-and γ-crystallin to the α-crystallin fraction was observed in αA-R49C heterozygous and homozygous lenses but not in wild type. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to α-crystallin in αA-R49C lenses as compared with the wild type protein. These results suggest that the decrease in solubility of the αA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of αA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the knock-in mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts.
PMCID: PMC2642957  PMID: 18700785
Small heat shock protein; mutation; cataract; mechanism; hydrophobicity
20.  Modulation of Advanced Glycation Endproduct Synthesis by Kynurenines in Human Lens Proteins 
Biochimica et biophysica acta  2009;1804(4):829-838.
Human lens proteins (HLP) become chemically modified by kynurenines and advanced glycation end products (AGEs) during aging and cataractogenesis. We investigated the effects of kynurenines on AGE synthesis in HLP. We found that incubation with 5 mM ribose or 5 mM ascorbate produced significant quantities of pentosidine, and this was further enhanced in the presence of two different kynurenines (200–500 µM): N-formylkynurenine (Nfk) and kynurenine (Kyn). Another related compound, 3-hydroxykynurenine (3OH-Kyn), had disparate effects; low concentrations (10–200 µM) promoted pentosidine synthesis, but high concentrations (200–500 µM) inhibited it. 3OH-Kyn showed similar effects on pentosidine synthesis from Amadori-enriched HLP or ribated lysine. Chelex-100 treatment of phosphate buffer reduced pentosidine synthesis from Amadori-enriched HLP by ~90%, but it did not inhibit the stimulating effect of 3OH-Kyn and EDTA. 3OH-Kyn (100–500 µM) spontaneously produced copious amounts of H2O2 (10–25 µM), but externally added H2O2 had only a mild stimulating effect on pentosidine but had no effect on Nε-carboxymethyl lysine (CML) synthesis in HLP from ribose and ascorbate. Further, human lens epithelial cells incubated with ribose and 3OH-Kyn showed higher intracellular pentosidine than cells incubated with ribose alone. CML synthesis from glycating agents was inhibited 30 to 50% by 3OH-Kyn at concentrations of 100–500 µM. Argpyrimidine synthesis from 5 mM methylglyoxal was slightly inhibited by all kynurenines at concentrations of 100–500 µM. These results suggest that AGE synthesis in HLP is modulated by kynurenines, and such effects indicate a mode of interplay between kynurenines and carbohydrates important for AGE formation during lens aging and cataract formation.
PMCID: PMC2829369  PMID: 20026434
Kynurenines; Glycation; Lens proteins; Cataract
21.  Effect of vitamin C and E activity on surgically removed cataractous human lens epithelium cells 
Oxidative stress has been proposed as a common underlying mechanism of cataractogenesis. Experimental and observational data suggest that micronutrients like vitamin C and vitamin E with antioxidant capabilities may retard the development of age-related cataract. Effect of these factors on lens epithelium cells, center of lens metabolic activities, is not completely elucidated. The aim of present study was to examine the effect of vitamin C and E on surgically removed lens epithelium cells of patients with cataract. Capsulorhexis samples were collected from 170 patients, admitted for cataract surgery. Catalase specific activity was estimated in lens epithelium cells with and without vitamin (C or E) treatment at different concentration for different time duration. Student’s t-test was employed for data analysis. We observed that in ex-vivo condition, a) both vitamin C and E bring about a decrease in catalase activity in lens epithelial cells. b) vitamin C showed toxic effect at high concentration. c) 100μM was the optimum concentration at which both vitamins showed maximum antioxidant activity. It was concluded that both vitamin C and E has direct effect on lens epithelium cells. At optimum concentration, they can reduce oxidative stress in these cells thus can support to prevent or delay cataract development.
PMCID: PMC3453048  PMID: 23105864
Cataract; Lens epithelium; Vitamin C; Vitamin E.
22.  Profiling of lens protease involved in generation of αA-66-80 crystallin peptide using an internally quenched protease substrate 
Experimental eye research  2013;109:51-59.
Proteins of lens fiber cells are prone to accumulate extensive post-translational modifications because of very little protein turnover. Lens proteins are degraded via the lens proteolytic systems into peptides, which are subsequently hydrolyzed by downstream aminopeptidases. Inefficient degradation can lead to accumulation of protein fragments and subsequent aggregation. Previously we showed that αA-66-80 peptide and its truncated products accumulate in aging and cataract human lenses. These peptides interact with crystallins, causing crystallin aggregation and precipitation. N- and C-terminal-blocked peptides that have the cleavage sites to generate the αA-66-80 fragment were used to test lens extracts for sequence-specific proteases in lens extracts. An internally quenched fluorogenic peptide substrate containing the sequence-specific site for a lens protease to generate αA-66-80 peptide was designed, synthesized and used to characterize protease(s) that are capable of generating this peptide in bovine and human lenses. We show that proteases with the potential to generate αA-66-80 peptide are present in bovine and human lenses. We also show that the αA-66-80 peptides are resistant to hydrolysis by aminopeptidases present in the lenses and they can suppress the degradation of other peptides. Failure of complete hydrolysis of these peptides in vivo can lead to their accumulation in the lens and subsequent lens protein aggregation, which may ultimately lead to the formation of cataract.
PMCID: PMC3619423  PMID: 23410823
αA-66-80 peptide; crystallin; cataract; lens; protease; peptidase; hydrolysis
23.  Immunochemical detection of glycated lens crystallins and their circulating autoantibodies in human serum during aging 
Molecular Vision  2008;14:2056-2066.
The aim of this investigation was to exploit lens-specific glycated crystallins as an immunogen to detect human glycated crystallins and their circulating autoantibodies in human serum during aging in relation to the development of cataract.
Polyclonal antibodies were produced against human total lens proteins (40–80 years) in rabbits. The specificity of the antibodies produced were determined by antibody capture assay using purified human lens crystallins (high molecular weight fraction [HMW]+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) as antigens. The cross-reactivity of these lens specific antibodies against rat β-, β-glycated, γ-, and γ-glycated lens crystallins was also analyzed. A non-competitive enzyme linked immunosorbent assay (ELISA) methodology was developed for the detection of circulating lens crystallins in human sera using HMW+α, HMW+α-glycated, β-, and β-glycated crystallins from humans and γ- and γ-glycated crystallins from rats as immobilized antigens. Circulating autoantibodies were also detected in human sera by antibody capture assay. The methodology was validated by evaluating 60 human serum samples collected from cataract patients and 30 human serum samples from apparently normal subjects belonging to the same age group.
The polyclonal antibodies raised against human total lens proteins showed 90% and 65% cross-reactivity with rat γ- and β-crystallins, respectively, by ELISA. Further, these polyclonal antibodies were capable of detecting both native and in vitro synthesized glycated crystallins. Their IC50 values were observed to be (i) human total lens proteins (55 ng), (ii) human HMW+α (16.45 ng), (iii) human HMW+α-glycated (273 ng), (iv) human β- (37.82 ng), (v) human β-glycated (260 ng), (vi) rat γ- (105.34 ng), and (vii) rat γ-glycated (313 ng). The immunochemical analysis of human serum indicated a significant change (p<0.001) in the levels of circulating β-glycated and γ-glycated crystallins in the age group of 40–80 years with respect to their control groups. However, there was no statistically significant change in the levels of HMW+α-glycated crystallins in the age group of 40–80 years as compared to their age-matched controls. Notably, the levels of serum γ-glycated crystallins were found to be threefold higher than that of HMW+α-glycated and β-glycated crystallins in the age group of 70–80 years. Circulating autoantibodies to HMW+α-glycated, β-glycated, and γ-glycated crystallins were detected in the serum of both apparently normal and cataract patients in the age group of 40–80 years by antibody capture assay. The levels of these autoantibodies were significantly higher at every time point compared to their respective controls. Autoantibodies to γ-glycated crystallins were found to be twofold and 3.2 fold higher as compared to the levels of autoantibodies to β-glycated and HMW+α-glycated crystallins, respectively. Western blot and immunohistochemical analysis substantiated the observations made in non-competitive ELISA.
During the course of aging, leakage of lens crystallins (HMW+α, HMW+α-glycated, β, β-glycated, γ, and γ-glycated) elicit an immune response resulting in the formation of autoantibodies in cataract patients (40–80 years) as compared to age matched controls. This is the first experimental report where polyclonal antibodies raised against lens-specific glycated crystallins were capable of detecting the early leakage of glycated crystallins in human subjects. This immunochemical approach has implications in the early detection of senile cataract.
PMCID: PMC2584771  PMID: 19023447
24.  Diverse Roles of Eph/ephrin Signaling in the Mouse Lens 
PLoS ONE  2011;6(11):e28147.
Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout (-/-) mice often display anterior polar cataracts while EphA2(-/-) lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5(-/-) lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5(-/-) lenses. Moreover, the disruption of membrane-associated β-catenin and E-cadherin junctions is observed in ephrin-A5(-/-) lens central epithelial cells. In contrast, EphA2(-/-) lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates β-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells.
PMCID: PMC3226676  PMID: 22140528
25.  Relationship between proteins encoded by three human gamma-crystallin genes and distinct polypeptides in the eye lens. 
Molecular and Cellular Biology  1987;7(9):3320-3323.
Although individual gamma-crystallins from the human eye lens have not been successfully purified and sequenced, most of the genes coding for these lens-specific structural proteins have been cloned and characterized. To investigate the relationship between these genes and the gamma-crystallins of the human lens, we made use of mouse cell lines which contain stably integrated copies of the coding sequences for three of the human gamma-crystallin genes coupled to the human metallothionein IIA promoter. The proteins produced by these hybrid genes in cell culture were detected immunologically and compared by physical characteristics with the gamma-crystallins from the human lens. The protein encoded by the G3 gene showed properties identical to those of the 21,000-molecular-weight gamma-crystallin from 11-month-old lens. The protein isolated from the cells expressing the G4 gene was similar to a 19,000-molecular-weight lens gamma-crystallin, while gene G5 encodes a highly basic gamma-crystallin which may be synthesized in only limited amounts in the human lens. These correlations provide a basis for future investigations on the relationship between putative mutations in human gamma-crystallin genes and altered proteins in hereditary lens cataracts.
PMCID: PMC367970  PMID: 3313014

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