Influence of kynurenines in pathogenesis of cataract formation in tryptophan-deficient regimen in Wistar rats.

L-Tryptophan (Trp) is an essential amino acid and its deficiency is involved in various pathologies. In this present investigation an attempt was made to study the role of tryptophan and its metabolites in cataract formation in wistar rats. Rats were divided and maintained in 3 groups, Group A--control; Group B--marginal-tryptophan and Group C--Tryptophan-deficient diet for 3 months. Slit lamp microscope observations indicated lenticular opacities in Group-C (tryptophan-deficient) rats. In the rats that were maintained on tryptophan deficient diet, a decrease in protein content, kynurenines, reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione-s-tranferase (GSTs) and tryptophan-fluorescence intensities and an increase in lipid peroxidation indicative of oxidative stress have been observed. The above changes were normalized in the rats on supplementation of 0.05% tryptophan (Group-B) in their diets. These results suggest that tryptophan-deficiency in the diet leads to an overall significant decrease in kynurenines and levels of antioxidant enzymes (except SOD) in ocular tissue with a concomitant lenticular opacification. The results suggest that diet with adequate tryptophan has protective influence and is of immense benefit in mitigating the changes that may otherwise contribute to the lenticular opacities.

Cloning and sequencing of complete tau-crystallin cDNA from embryonic lens of Crocodylus palustris.

tau-Crystallin is a taxon-specific structural protein found in eye lenses. We present here the cloning and sequencing of complete tau-crystallin cDNA from the embryonic lens of Crocodylus palustris and establish it to be identical to the a-enolase gene from non-lenticular tissues. Quantitatively, the tau-crystallin was found to be the least abundant crystallin of the crocodilian embryonic lenses. Crocodile tau-crystallin cDNA was isolated by RT-PCR using primers designed from the only other reported sequence from duck and completed by 5'- and 3'-rapid amplification of cDNA ends (RACE) using crocodile gene specific primers designed in the study. The complete tau-crystallin cDNA of crocodile comprises 1305 bp long ORF and 92 and 409 bp long untranslated 5'- and 3'-ends respectively. Further, it was found to be identical to its putative counterpart enzyme a-enolase, from brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of tau-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the gene lineage reconstruction analysis helps our understanding of the evolution of crocodilians and avian species.

Development of an immunoanalytical method for the detection of beta- and gamma-crystallins and anti-crystallin antibodies. A molecular biomarker for cataract.

PURPOSE: To develop and evaluate an immunoanalytical method for the detection of beta- and gamma-crystallins and anti-crystallin antibodies. MATERIALS AND METHODS: Beta and gamma-crystallins isolated from rat lens were used as immunogens to raise polyclonal antibodies in rabbits. Antibody capture assay and western blot analysis showed that the antibodies to beta- and gamma-crystallins were specific. An indirect competitive enzyme linked immunosorbent assay (ELISA) developed to quantitate beta- and gamma-crystallin showed an IC50 value of 70 ng and 65 ng, respectively, based on regression analysis. Spiking studies with purified beta-crystallin antibodies showed that 33 ng of the purified antibody gave an absorbance of 1.1 at 450 nm, indicating the sensitivity of the method. RESULTS: Antibodies to beta- and gamma-crystallins were not detected in serum samples of the cataractous CFY/NIN rats (used as an animal model for induction of experimental cataract by feeding high galactose diet). However, the cataractous rat serum samples effectively displaced beta- and gamma-crystallin antibodies, indicating that these crystallins leak during cataract formation. The concentration of beta- and gamma-crystallins in the rat serum, as analysed by indirect competitive ELISA, was found to be in the range of 17.6-81.6 micrograms/ml [corrected] and 12.4-19.6 micrograms/ml, respectively. CONCLUSIONS: The methodology developed in the present study may find application as a biochemical tool in molecular epidemiology of cataract.

Flavin nucleotides in human lens: regional distribution in brunescent cataracts.

The biochemical mechanism(s) underlying brunescent cataracts remain unclear. Oxidative stress due to reactive oxygen species may have a role in the pigmentation process in eye lens. We have analysed human cataractous lenses for flavins by high-performance liquid chromatography (HPLC), since flavins are light sensitive and act as endogenous sensitizers generating reactive oxygen species in the eye. The most significant observation in this study is that higher levels of flavin nucleotides occur in brown lens compared to yellow lens. The concentration of flavin nucleotides (flavin monouncleotide, FMN + flavin adenine dinucleotide, FAD) was highest in the nuclear region of the lens followed by the cortical and capsule-epithelial regions. However, the ratio of FAD/FMN was lowest in the nuclear region of the lens followed by other regions. On the other hand, riboflavin was not detected in any of the lens (cataractous) regions. These results suggest that the observed increase in flavin nucleotides in the ocular tissue could contribute towards deepening of lens pigmentation.

Near ultraviolet radiation induced changes in goat lens.

The possibility that the ultraviolet radiation from sunlight and other ambient sources as a major causative factor for the onset of cataract processes and photolytic changes of the eye lens constituents was studied. Normal goat lenses exposed in vitro to near UV radiation in the region of 315-400 nm (UV-A) revealed distinct morphological changes in the ultrastructure, increase in the inorganic elements; C, H, N, and a sharp shift in the intrinsic fluorescence spectra. UV exposure resulted in an alteration in the lambda max of the excitation spectra, a red shift in the emission absorption maxima and also an increase in the absolute fluorescence intensity. Scanning electron microscopic study showed a significant increase in the interfibrillar distances of the lens structural proteins. It is argued that the UV light induced covalent modifications of the lens proteins and their aggregations might have occurred due to the generation of photolytic products which then lead to oxidative damages.

Light scattering for young human and cataractous lenses.

The Rayleigh ratio for young human and cataractous lenses, having spherical particles with constant diameter embedded in a medium with a different refractive index, has been calculated as a function of concentration (volume fraction) using the theory of the small-q behaviour of the static structure factor, S(q). It involves treating the long-range forces between particles in the gel in the random phase approximation, dividing the pairwise interaction potential into a reference part and a perturbed part based on WCA model, and applying the perturbation approach of WCA to lenses obeying van der Waals' potential as a perturbative attraction over the Percus-Yevick (PY) hard sphere model. The calculated Rayleigh ratios are found in excellent agreement with experimental values, thereby showing that the present model works well for young human and cataractous lenses.

Melanins and lens pigments: a comparative study

Based on previous findings that lens pigments and melanins share many physicochemical properties, human lens pigments and natural (hair) and synthetic melanins were submitted to oxidation with permanganate under strong acidic conditions. This procedure has been utilized for the characterization of melanins and results in the well defined products, thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA), which can be quantitated by high performance liquid chromatography (HPLC). PTCA is regarded as a marker of black eumelanins and was therefore a main component of synthetic DOPA-eumelanin and dark hair. Its identity was established by synthesis from 5-hydroxyindole-2-carboxylic acid. TDCA derives from pheomelanins and was therefore an important component of red hair and synthetic GSH-pheomelanin. TDCA was identified by its retention time relative to PTCA. The analysis of a series of cataract digests of increasing pigmentation (type I < type IV < type V) and a purified fraction of lens pigments (DE52 pigment) revealed the presence in these preparations of both PTCA and TDCA. The concentration of TDCA significantly increased with the degree of pigmentation of the digests and reached a maximum in the DE52 pigment. The TDCA/PTCA ratio was high in the lens preparations and comparable to that given by hair pheomelanin. These findings support that pheomelanin is an integral part of lens pigments. By comparing the yields of TDCA in GSH-pheomelanin and in the purified lens pigment, a 9 per cent contribution of pheomelanin to the lens pigment was estimated

Chromatographic studies on experimental candida keratitis

Chromatographic analysis and soluble protein contents of aqueous humour and crystalline lens were done in induced ocular infection of rabbit's eyes by Candida albicans in both treated and non-treated inflamed eyes as well as their normal controls. The results showed a significant decrease in the soluble protein contents and molecular weights in aqueous humour and crystalline lens of non-treated eyes. The topical treatment showed an important response which made the treated eyes to be slightly affected by the Candida albicans infections. We conclude from this study that early treatment prevents severe ocular complications and column chromatography is a sensitive technique for protein analysis of crystalline lens and aqueous humour

Isolation, purification and preliminary studies on fish eye lens glycoprotein.

Glycoprotein from the eye lens of fish, Mystus cavasius, was isolated by extraction with 1% Triton X-100 in saline. The crude extract which was found to be electrophoretically heterogeneous was fractionated on a DEAE-cellulose column. One of the fractions obtained in major amount was further resolved by column chromatography using Sephadex G-150 into two homogeneous fractions (GP-1 and GP-2]. GP-1 contained carbohydrates (11.2%) and protein (77.5%). The constituent sugars were D-glucose, D-mannose, D-galactosamine and N-acetyl neuraminic acid. The principal amino acids were aspartic acid, serine, glutamic acid, glycine and alanine. The proportions of these residues were determined.