Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0086543 (cataract)
 29,165 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The proteins isolated from aged human lenses and brunescent cataracts exhibit extensive disulfide bond formation. Diabetic rat lenses similarly contain disulfide-bonded protein aggregates. These observations are consistent with the known link between diabetes, glycation and oxidative damage, and suggest a role for reactive oxygen species (ROS) in this process. To assess whether the glycation-related modifications in human lens proteins spontaneously generate ROS, superoxide anion formation was measured using both cataractous lens proteins and calf lens proteins glycated in vitro with ascorbic acid (ascorbylated). The water-insoluble fraction from aged normal human lenses generated 0.3-0.6 nmol superoxide h(-1)mg protein(-1), whereas the activity increased to 0.5-1.8 nmol h(-1)mg protein(-1)with the WI fraction from brunescent cataracts, and 2.3 nmol h(-1)mg protein(-1)with calf lens proteins ascorbylated for 4 weeks in vitro. The activity in the human lens proteins was observed in both the water-soluble and water-insoluble fractions, and was completely dependent upon the presence of oxygen. The pH optimum curve for superoxide formation increased from pH 6.5 to 10 with both the cataract and ascorbylated proteins. The superoxide-generating activity in human lens was completely bound to a boronate affinity column, but only partially bound with the ascorbylated proteins. The superoxide anion produced by a 5 m m solution of purified N(epsilon)-fructosyl-lysine was barely detectable, and therefore, could not account for the superoxide formed by any of the lens protein preparations. Also, superoxide formation increased 10-fold at pH 8.8 with fructosyl-lysine, but only 1.3-1.8-fold with human lens proteins. The addition of copper-stimulated superoxide formation with glycated bovine serum albumin, but no stimulation was seen with cataractous proteins. Assays of specific compounds showed that catechol, hydroquinone, 3-OH kynurenine and 3-OH anthranylic acid exhibited the greatest activity for superoxide generation, but had a very short halflife. 2,3-Dihydroxypyridine and 4,5 dihydroxynaphthalene were one and two orders of magnitude less reactive. In long-term incubations at 37 degrees, cataractous proteins retained the potential to produce superoxide anion, losing only half of the initial activity after 6-7 days. Therefore, the water-insoluble fraction from aged human lenses and dark brown cataracts are potentially capable of generating >100 nmol mg protein(-1)and >170 nmol mg protein(-1)of superoxide anion respectively, likely due to the presence of advanced glycation endproducts in human lens proteins. This spontaneous generation of superoxide anion in vivo could account for a major portion of the oxidation of sulfur amino acids seen during aging and cataract formation.
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PMID:Spontaneous generation of superoxide anion by human lens proteins and by calf lens proteins ascorbylated in vitro. 1043 59

In humans, the crystallin proteins of the ocular lens become yellow-coloured and fluorescent with ageing. With the development of senile nuclear cataract, the crystallins become brown and additional fluorophores are formed. The mechanism underlying crystallin colouration is not known but may involve interaction with kynurenine-derived UV filter compounds. We have recently identified a sulphur-linked glutathionyl-3-hydroxykynurenine glucoside adduct in the lens and speculated that kynurenine may also form adducts with GSH and possibly with nucleophilic amino acids of the crystallins (e.g. Cys). Here we show that kynurenine modifies calf lens crystallins non-oxidatively to yield coloured (365 nm absorbing), fluorescent (Ex 380 nm/Em 450-490 nm) protein adducts. Carboxymethylation and succinylation of crystallins inhibited kynurenine-mediated modification by approx. 90%, suggesting that Cys, Lys and possibly His residues may be involved. This was confirmed by showing that kynurenine formed adducts with GSH as well as with poly-His and poly-Lys. NMR studies revealed that the novel poly-Lys-kynurenine covalent linkage was via the epsilon-amino group of the Lys side chain and the betaC of the kynurenine side chain. Analysis of tryptic peptides of kynurenine-modified crystallins revealed that all of the coloured peptides contained either His, Cys or an internal Lys residue. We propose a novel mechanism of kynurenine-mediated crystallin modification which does not require UV light or oxidative conditions as catalysts. Rather, we suggest that the side chain of kynurenine-derived lens UV filters becomes deaminated to yield an alpha,beta-unsaturated carbonyl which is highly susceptible to attack by nucleophilic amino acid residues of the crystallins. The inability of the lens fibre cells to metabolise their constituent proteins results in the accumulation of coloured/fluorescent crystallins with age.
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PMID:Non-oxidative modification of lens crystallins by kynurenine: a novel post-translational protein modification with possible relevance to ageing and cataract. 1066 91

Understanding aqueous protein-protein interactions is crucial for the development of a molecular-thermodynamic model for salt-induced protein precipitation. In addition, protein interactions are important in many disease states, including cataract formation and alpha-amyloid diseases. Fluorescence anisotropy provides a means to measure intermolecular interactions. In this work, monomer-dimer equilibrium of the peptide T4 LYS(11-36) was studied by fluorescence anisotropy over the pH range 4-7 and the NaCl concentration range 0.0-1.0 M, in a 25 mM sodium phosphate buffer. This 26 amino-acid peptide is derived from the beta-sheet region of the T4 lysozyme molecule and has the potential to form amyloid fibrils. The association constant for dimerization increases with rising pH and ionic strength. The potential of mean force for peptide-peptide interactions was calculated from these association constants. Circular-dichroism measurements show that the peptide becomes more structured as the pH rises, possibly contributing to increased association.
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PMID:Interactions of proteins in aqueous electrolyte solutions from fluorescence anisotropy and circular-dichroism measurements. 1079 32

Aggregation and covalent cross-linking of the crystallins, the major structural proteins of the eye lens, increase light scattering by the lens leading to opacification and cataract. Disturbance of calcium homeostasis in the tissue is one of the factors implicated in cataractogenesis. Calcium-activated transglutaminase (TG)-catalyzed cross-linking of some lens proteins has been reported earlier. We show here that alpha-crystallin, a major structural protein in the lens and a member of the small heat shock protein family, is also a substrate for TG-mediated cross-linking, indicating the presence of donor Lys and acceptor Gln residues in the protein. Upon TG-catalyzed dimerization, the secondary and tertiary structures of the protein are altered, and its surface hydrophobicity reduced. The chaperone-like property of the protein, suspected to be one of its functions in situ, is substantially reduced upon such cross-linking. These results, taken together with earlier ones on lens beta-crystallins and vimentin, suggest that TG-mediated events might compromise lens function. Also, since alpha-crystallin occurs not only in the lens but in other tissues as well, such TG-catalyzed cross-linking and the associated alterations in its structure and activity would be of general pathological interest.
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PMID:Transglutaminase-mediated cross-linking of alpha-crystallin: structural and functional consequences. 1142 25

Glycation of proteins leads to the formation of early glycation adducts (fructosamine derivatives) and advanced glycation endproducts (AGEs). Formation of AGEs has been linked to the development of cataract, diabetic complications, uraemia, Alzheimer's disease and other disorders. AGEs are a group of compounds of diverse molecular structure and biological function. To characterize AGE-modified proteins used in studies of structural and functional effects of glycation, an assay was developed that surveys the content of early and advanced glycation adducts in proteins. The assay procedure involved enzymic hydrolysis of protein substrate, derivatization of the hydrolysate with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and HPLC of the resulting adducts with fluorimetric detection. Structural isomers of methylglyoxal-derived hydroimidazolone, glyoxal-derived hydroimidazolone, 3-deoxyglucosone-derived hydroimidazolone and N(delta)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)-ornithine (THP) were determined for the first time. AGEs with intrinsic fluorescence (argpyrimidine, pentosidine) were assayed without derivatization. Limits of detection were 2-17 pmol and levels of recovery were 50-99%, depending on the analyte. The AQC assay resolved structural and epimeric isomers of methylglyoxal-derived hydroimidazolones and THP. Hydroimidazolones, THP and argpyrimidine were AGEs of short-to-intermediate stability under physiological conditions, with half-lives of 1-2 weeks. Their measurement provides further insight into the glycation process. The assay was applied to the characterization of human serum albumin minimally and highly modified by N(epsilon)-carboxymethyl-lysine and N(epsilon)-(1-carboxyethyl)-lysine.
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PMID:Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to Nepsilon-carboxymethyl-lysine- and Nepsilon-(1-carboxyethyl)lysine-modified albumin. 1198 70

An autosomal dominant congenital cataract associated with a missense mutation, Arg-116 to Cys (R116C), in the coding sequence of human alphaA-crystallin has been reported. Subsequent study of this mutant, generated by site-directed mutagenesis, showed significant changes in secondary and tertiary structures, partial loss of chaperone activity, and substantially increased oligomeric size. The study presented here aims to show whether these changes are due to the loss of a positive charge at this position or due to the presence of an extra Cys. To show this, Arg-116 in alphaA-crystallin was mutated to Lys (R116K), Cys (R116C), Gly (R116G), and Asp (R116D) and expressed in Escherichia coli cells. The wild-type (alphaA-wt) and mutant proteins were purified by size exclusion chromatography and characterized by measurements of circular dichroism, intrinsic tryptophan fluorescence, and TNS fluorescence and by determination of molecular masses and chaperone function which was assessed as the ability to suppress target protein aggregation or enhance target protein refolding. Mutation of Arg-116 to a Cys or Gly showed very similar changes in structure, oligomerization, and chaperone function which suggest that the presence of this Cys per se is not the cause of the changes. The R116K mutant, on the other hand, had nearly the same structure, oligomeric size, and chaperone function as alphaA-wt, whereas the mutant with an acidic amino acid in this position, R116D, showed drastic changes in protein structure. Thus, a positive charge must be preserved at this position for the structural and functional integrity of alphaA-crystallin.
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PMID:A positive charge preservation at position 116 of alpha A-crystallin is critical for its structural and functional integrity. 1236 32

Several mechanisms have been proposed for the way in which glucose and its metabolites cause cataract, retinopathy and other complications of diabetes, the most convincing being glycation. Glycation, the reaction of sugars with free amino groups of proteins, is one of a variety of non-enzymic post-translational modifications. The aim of the present study was to identify some of the most reactive proteins in the lens when incubated under physiological conditions. Fresh intact bovine lenses were incubated with [14C]glucose in a conventional tissue-culture medium with added antibiotics. After 3 and 6 days of incubation, the water-soluble proteins were separated by size-exclusion chromatography. Glycated proteins from the water-soluble fractions were separated by using a sugar affinity column (Affi-Gel 601). Then the radioactive fractions were identified on SDS/polyacrylamide gels. In addition, the whole bovine lenses were incubated with 10 mM fructose and glucose for 3 and 6 days. The glycated proteins from the water-soluble fractions in parallel with the radioactive fractions were separated by affinity chromatography, and were identified further by amino-acid sequencing. A progressive uptake of radioactive label showed that the majority of proteins incorporating both glucose and fructose were water-soluble fractions. Chromatography and SDS/polyacrylamide gel results showed that alpha- and gamma-crystallin and some proteins of a mean molecular mass of 36-37 kDa incorporated sugars early during incubation. After 6 days of incubation, more crystallins were glycated compared with 3 days, in particular beta-crystallin. Affinity-chromatography results indicated that proteins with subunit masses of 36 kDa and 20 kDa were possibly radiolabelled at an early stage. The purified glycated proteins following incubation with both glucose and fructose, which corresponded to 20 kDa and 36 kDa bands on SDS/polyacrylamide gels, were sequenced by Edman degradation. N-terminal sequences of both 20 kDa bands were Gly-Lys-Ile-Thr, characteristic of gamma-crystallins, but the N-termini of both 36 kDa bands were blocked. Further sequencing after digestion of 36 kDa bands with trypsin and running on HPLC revealed that the glucose sample gave the peptide sequences as Gly-Glu-Tyr-Pro-Asp-Tyr-Gln-Gln and Tyr-Glu-Leu-Pro-Asn-Tyr-Arg, which match with bovine gammaIIIb-crystallin. The peptide sequence Tyr-Glu-Leu-Pro-Asn-Tyr-Arg is only present in the published sequence of bovine gammaIIIb-crystallin and not in any other type of gamma-crystallin. The fructose sample gave the peptide sequences Ile-Thr-Phe-Tyr-Glu-Asp-Arg, Arg-Gly-Asp-Tyr-Pro-Asp-Tyr-Gln-Gln-Trp, Gln-Tyr-Leu-Leu-Arg and Val-Val-Asp-Leu-Tyr, which all matched with bovine gammaIIIa-crystallin. The sequence Val-Val-Asp-Leu-Tyr only appears in the sequence of bovine gammaIIIa-crystallin. gammaIII-Crystallin is the most susceptible lens protein to glycation. The primary target of glucose is gammaIIIb-crystallin, whereas that of fructose is gammaIIIa-crystallin. The early glycation of gammaIII-crystallin by glucose and fructose could result in structural alterations, leading to aggregation of crystallin and eventually cataract formation.
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PMID:Gamma III-crystallin is the primary target of glycation in the bovine lens incubated under physiological conditions. 1280 41

Post-translational modifications of proteins take place during the aging of human lens. The present study describes a newly isolated glycation product of lysine, which was found in the human lens. Cataractous and aged human lenses were hydrolyzed and fractionated using reverse-phase and ion-exchange high performance liquid chromatography (HPLC). One of the nonproteinogenic amino acid components of the hydrolysates was identified as a 3-hydroxypyridinium derivative of lysine, 2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine). The compound was synthesized independently from 3-hydroxypyridine and methyl 2-[(tert-butoxycarbonyl)amino]-6-iodohexanoate. The spectral and chromatographic properties of the synthetic OP-lysine and the substance isolated from hydrolyzed lenses were identical. HPLC analysis showed that the amounts of OP-lysine were higher in water-insoluble compared with water-soluble proteins and was higher in a pool of cataractous lenses compared with normal aged lenses, reaching 500 pmol/mg protein. The model incubations showed that an anaerobic reaction mixture of Nalpha-tert-butoxycarbonyllysine, glycolaldehyde, and glyceraldehyde could produce the Nalpha-t-butoxycarbonyl derivative of OP-lysine. The irradiation of OP-lysine with UVA under anaerobic conditions in the presence of ascorbate led to a photochemical bleaching of this compound. Our results argue that OP-lysine is a newly identified glycation product of lysine in the lens. It is a marker of aging and pathology of the lens, and its formation could be considered as a potential cataract risk-factor based on its concentration and its photochemical properties.
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PMID:2-ammonio-6-(3-oxidopyridinium-1-yl)hexanoate (OP-lysine) is a newly identified advanced glycation end product in cataractous and aged human lenses. 1463 19

Mice heterozygous for the Sod2 gene (Sod2+/- mice) have been used to study the phenotype of life-long reduced Mn-superoxide dismutase (MnSOD) activity. The Sod2+/- mice have reduced MnSOD activity (50%) in all tissues throughout life. The Sod2+/- mice have increased oxidative damage as demonstrated by significantly elevated levels of 8-oxo-2-deoxyguanosine (8oxodG) in nuclear DNA in all tissues of Sod2+/- mice studied. The levels of 8oxodG in nuclear DNA increased with age in all tissues of Sod2+/- and wild-type (WT) mice, and at 26 mo of age, the levels of 8oxodG in nuclear DNA were significantly higher (from 15% in heart to over 60% in liver) in the Sod2+/- mice compared with WT mice. The level of 8oxodG was also higher in mitochondrial DNA isolated from liver and brain in Sod2+/- mice compared with WT mice. The increased oxidative damage to DNA in the Sod2+/- mice is associated with a 100% increase in tumor incidence (the number of mice with tumors) in old Sod2+/- mice compared with the old WT mice. However, the life spans (mean and maximum survival) of the Sod2+/- and WT mice were identical. In addition, biomarkers of aging, such as cataract formation, immune response, and formation of glycoxidation products carboxymethyl lysine and pentosidine in skin collagen changed with age to the same extent in both WT and Sod2+/- mice. Thus life-long reduction of MnSOD activity leads to increased levels of oxidative damage to DNA and increased cancer incidence but does not appear to affect aging.
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PMID:Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging. 1467 99

The aim of the present work was to evaluate the effect of bendazac lysine on the human lens epithelial cell line HLE-B3 adhesion to polymethylmethacrylate (PMMA) intraocular lenses (IOLs). After adherence to IOLs, cells were incubated in the presence of the drug for 24 h. The number of cells contained in a 6-mm(2) area was then counted with an inverted phase microscope and adherent cells were distinguished from detached floating cells by focusing through the medium. Results obtained show that bendazac is able to induce a linear dose-dependent inhibition of HLE-B3 adhesiveness to PMMA IOLs. In particular, treatment with bendazac 33, 100 and 300 microM resulted in a 15, 32 and 54% inhibition, respectively. Statistical analysis shows that this effect is significant at 100 microM (p < 0.05) and 300 microM (p < 0.01). The analysis of the effects of bendazac on the viability and on the proliferative capacity of HLE-B3 cells did not show any drug-related toxicity up to the concentration of 400 microM. The present study demonstrates that bendazac lysine is able to inhibit adhesion of lens epithelial cells to PMMA IOLs and suggests the potential beneficial use of this drug in preventing secondary cataract development.
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PMID:Bendazac lysine inhibition of human lens epithelial cell adhesion to polymethylmethacrylate intraocular lenses. 1510 5


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