Effect of Sorbitol on Alpha-Crystallin Structure and Function.

Loss of eye lens transparency due to cataract is the leading cause of blindness all over the world. While aggregation of lens crystallins is the most common endpoint in various types of cataracts, chaperone-like activity (CLA) of α-crystallin preventing protein aggregation is considered to be important for maintaining the eye lens transparency. Osmotic stress due to increased accumulation of sorbitol under hyperglycemic conditions is believed to be one of the mechanisms for diabetic cataract. In addition, compromised CLA of α-crystallin in diabetic cataract has been reported. However, the effect of sorbitol on the structure and function of α-crystallin has not been elucidated yet. Hence, in the present exploratory study, we described the effect of varying concentrations of sorbitol on the structure and function of α-crystallin. Alpha-crystallin purified from the rat lens was incubated with varying concentrations of sorbitol in the dark under sterile conditions for up to 5 days. At the end of incubation, structural properties and CLA were evaluated by spectroscopic methods. Interestingly, different concentrations of sorbitol showed contrasting results: at lower concentrations (5 and 50 mM) there was a decrease in CLA and subtle alterations in secondary and tertiary structure but not at higher concentrations (500 mM). Though, these results shed a light on the effect of sorbitol on α-crystallin structure-function, further studies are required to understand the mechanism of the observed effects and their implication to cataractogenesis.

Effect of vitamin B12 supplementation on retinal lesions in diabetic rats.

Purpose: Diabetic retinopathy (DR) is the most common complication of diabetes involving microvasculature and neuronal alterations in the retina. Previously, we reported that vitamin B12 deficiency could be an independent risk factor for DR in humans. However, the effect of vitamin B12 supplementation in experimental DR is unknown. Thus, in this study, we investigated the impact of dietary supplementation of vitamin B12 on retinal changes in diabetic rats. Methods: Diabetes was induced in 2-month-old Sprague-Dawley rats and maintained for 4 months. One group of diabetic rats were fed normal levels of vitamin B12, and one group double the quantity of vitamin B12 (50 µg/kg diet). Vitamin B12 and homocysteine levels in the plasma were analyzed with radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC), respectively. At the end of 4 months of experimentation, the eyeballs were collected. Retinal changes were analyzed with hematoxylin and eosin (H&E) staining, immunoblotting, and immunofluorescence methods. Results: Dietary supplementation of vitamin B12 had no effect on food intake, bodyweight, fasting blood glucose, and plasma homocysteine levels in the diabetic rats. However, vitamin B12 supplementation prevented loss of rhodopsin, and overexpression of VEGF, and completely prevented overexpression of HIF1α, GFAP, and endoplasmic reticulum (ER) stress markers (GRP78, ATF6α, XBP1, CHOP, and caspase 12) in the diabetic rat retina. Further, vitamin B12 ameliorated apoptosis in the retina as shown with terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and prevented retinal thinning. Conclusions: Vitamin B12 supplementation of diabetic rats appeared to be beneficial by circumventing retinal hypoxia, VEGF overexpression, and ER stress-mediated cell death in the retina. The present study adds another potential therapeutic strategy of vitamin B12 in diabetes.

Role of sorbitol-mediated cellular stress response in obesity-associated retinal degeneration.

PURPOSE: Obesity is a global health problem associated with several diseases including ocular complications. Earlier we reported progressive retinal degeneration because of obesity in a spontaneous obese rat (WNIN/Ob) model. In the current study, we examined the molecular mechanisms leading to retinal degeneration in WNIN/Ob rat. METHODS: Sorbitol was estimated by the fluorometric method in the retina of WNIN/Ob rats at different age (3-, 6- and 12- months), along with their respective lean rats. Immunoblotting was performed in the retina to assess the status of the insulin signaling pathway, ER stress and cellular stress (p38MAPK and ERK1/2). Human SK-N-SH cells were treated with 0.5 and 1.0 M sorbitol for 30 min to study insulin signaling, ER stress, and cellular stress. TUNEL assay was done to measure apoptosis. The retinal function in the rats was determined by electroretinogram. RESULTS: A gradual but significantly higher intracellular sorbitol accumulation was observed in the retina of obese rats from 3- to 12-months. The cellular osmotic stress has activated the insulin signaling mechanism without activating AKT and also triggered ER stress. Both the stresses activated the ERK and p38MAPK signaling causing apoptosis in the retina leading to retinal degeneration. Retinal dysfunction was confirmed by altered scotopic and photopic electroretinogram responses. These in vivo results were mimicked in SK-N-SH cells when exposed to sorbitol in vitro. CONCLUSIONS: These results suggest cellular stress due to sorbitol accumulation impairing the ER function, thereby leading to progressive retinal degeneration under obese conditions.

Hyperglycemia induced expression, phosphorylation, and translocation of αB-crystallin in rat skeletal muscle.

αB-Crystallin (αBC) is a member of the small heat shock protein family that responds to a variety of stress and prevents the aggregation of partially unfolded proteins. Chronic hyperglycemia created during diabetes results in skeletal muscle atrophy and leads to diabetic myopathy. The aim of this study was to investigate the role of αBC under chronic hyperglycemia in rat skeletal muscle. Diabetes was induced in Wistar rats by a single i.p injection of streptozotocin and maintained for a period of 12 weeks at the end of which the animals were sacrificed and the muscle was collected. The protein levels of αBC and its phosphorylation status in gastrocnemius muscle were analyzed by immunoblotting. The translocation of phosphorylated αBC was analyzed by detergent solubility assay, co-immunoprecipitation (Co-IP), and immunohistochemistry. The cell death was analyzed by TUNEL assay and by apoptotic markers. The interaction of αBC with Bax was analyzed by Co-IP. Chronic hyperglycemia significantly increased the protein levels of αBC and its phosphorylation at S59 by activation of p38 mitogen-activated protein kinase (p38MAPK) and at S45 by activation of the extracellular regulated protein kinase 1/2 (ERK1/2). Further, phosphorylated αBC translocated and interacted with desmin indicating that phosphorylated αBC forms might be involved in protection of sarcomere structures from disruption in chronic hyperglycemia. Further, Co-IP studies showed an impaired interaction of αBC with Bax which could be one of the possible factors for increased cell death as evidenced by TUNEL assay in diabetic muscle. These results suggest that an increased expression, phosphorylation, translocation of αBC, and its involvement in apoptosis might play a significant role in maintenance of cytoskeletal architecture and protection of cells from apoptosis in diabetic skeletal muscle.

Total activity of glutathione-S-transferase (GST) and polymorphisms of GSTM1 and GSTT1 genes conferring risk for the development of age related cataracts.

The pathogenesis of cataract is influenced by a number of factors including oxidative stress. Glutathione-S-transferase (GST) catalyses the nucleophilic addition of the thiol of GSH to electrophilic acceptors. It is important for detoxification of xenobiotics in order to protect tissues from oxidative damage. In humans, GSTT1 and GSTM1 deletion genotypes are associated with a variety of pathological conditions including certain ophthalmic diseases. In the present study, it is aimed to determine the risk of genetic polymorphisms of GSTM1 and GSTT1 isoforms of GST for developing of age related cataracts (ARCs). We compared the prevalence of GSTT1 and GSTM1 deletion genotypes, which were determined by multiplex polymerase chain reaction, in 455 patients with ARCs (108 with nuclear (NC), 105 with cortical (CC), 96 with posterior subcapsular, (PSC) and 146 with mixed type (MT)) and 205 age and sex matched controls. The GST activity in erythrocytes (RBC) and cataractous lenses was measured spectrophotometrically using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The frequency of GSTM1 positive individuals was significantly higher in MT cataracts followed by NC, CC and PSC types with corresponding decrease in the GSTM1 null genotypes as compared to controls. Considering the GSTT1 locus, GSTT1 null genotypes showed high frequency in patients in general as compared to controls with corresponding reduction in the GSTT1 positive genotype. The activity of GST in RBC was higher in all the types of cataracts as compared to that in controls and in cataractous lenses the mean values were slightly higher in cases of NC cataracts as compared to CC, PSC and MT. The data suggests that GSTM1 positive, GSTT1 null and double null (GSTM1 null and GSTT1 null) genotypes may confer risk for the development of ARC. The increased activity of GST found in the present study could be due to a compensatory mechanism operating in response to increased oxidative stress.

INDOOR RADON AND THORON IN THE VICINITY OF PROPOSED URANIUM MINING SITE: A CASE STUDY AT DASARLAPALLY VILLAGE, TELANGANA STATE, INDIA.

Studies are being conducted for the past few decades in and around the uranium mining sites across the globe to identify environmental nuclear radiation risk to the common public. The area near Dasarlapally village was identified for uranium exploration by the AMDER, Hyderabad. The present study was carried out to measure the indoor radon and thoron activity concentrations in the dwellings of Dasarlapally village. For this purpose different types of dwellings were chosen randomly across the village. The measured annual average concentration of radon and thoron in dwellings were found to be 141 ± 42 and 139 ± 77 Bqm-3, respectively, and the calculated annual effective inhalation dose due to radon was determined to be 3.5 mSv. Seasonal variation and diurnal variation of radon and thoron activity concentration were investigated. The variation of radon and thoron activity concentration in different types of dwellings was also studied, and the variation was found to be statistically insignificant. The uncertainty propagated in the effective inhalation dose due to thoron was discussed.

Prevention of non-enzymic glycation of proteins by dietary agents: prospects for alleviating diabetic complications.

The accumulation of advanced glycation endproducts (AGE) due to non-enzymic glycation of proteins has been implicated in several pathophysiologies associated with ageing and diabetes. The formation of AGE is accelerated in hyperglycaemic conditions, which alter the structure and function of long-lived proteins. Thus inhibition of the formation of AGE is believed to play a role in the prevention of diabetic complications. In the present study we evaluated the antiglycating effect of aqueous extracts of various plant-based foods. The effect of aqueous extracts of these agents in terms of their ability to prevent the accumulation of AGE due to fructose-mediated in vitro glycation of eye lens soluble proteins was investigated. The degree of protein glycation in the absence and presence of dietary extracts was assessed by different complementary methods, i.e. non-tryptophan AGE fluorescence, AGE-induced cross-linking by SDS-PAGE and glyco-oxidative damage by carbonyl assay. Five out of the seventeen agents tested showed significant inhibitory potential against in vitro protein glycation in a dose-dependent manner. Prominent among them were ginger, cumin, cinnamon, black pepper and green tea, which inhibited in vitro AGE formation to lens proteins 40-90 % at 1.0 mg/ml concentration. Assessing their potential to reduce the amount of glycated protein using boronate affinity chromatography and also their ability to prevent the formation of specific antigenic-AGE structures by immunodetection further substantiated the importance of ginger, cumin and cinnamon in reducing AGE burden. These findings indicate the potential of some dietary components to prevent and/or inhibit protein glycation. Thus these dietary agents may be able to be exploited for controlling AGE-mediated diabetic pathological conditions in vivo.

Significance of alpha-crystallin heteropolymer with a 3:1 alphaA/alphaB ratio: chaperone-like activity, structure and hydrophobicity.

The small heat-shock protein alpha-crystallin isolated from the eye lens exists as a large (700 kDa) heteropolymer composed of two subunits, alphaA and alphaB, of 20 kDa each. Although trace amounts of alphaA-crystallin are found in other tissues, non-lenticular distribution of alpha-crystallin is dominated by the alphaB homopolymer. In most vertebrate lens, the molar ratio of alphaA to alphaB is generally 3:1. However, the importance of this ratio in the eye lens is not known. In the present study, we have investigated the physiological significance of the 3:1 ratio by determining the secondary/tertiary structure, hydrophobicity and chaperone-like activity of alphaA- and alphaB-homopolymers and heteropolymers with different ratios of alphaA to alphaB subunits. Although, under physiologically relevant conditions, the alphaB-homopolymer (37-40 degrees C) has shown relatively higher activity, the alphaA-homopolymer or the heteropolymer with a higher alphaA proportion (3:1 ratio) has shown greater chaperone-like activity at elevated temperatures (>50 degrees C) and also upon structural perturbation. Furthermore, higher chaperone activity at elevated temperatures as well as upon structural perturbation is mainly mediated through increased hydrophobicity of alphaA. Although homopolymers and heteropolymers of alpha-crystallin did not differ in their secondary structure, changes in tertiary structure due to structural perturbations upon pre-heating are mediated predominantly by alphaA. Interestingly, the heteropolymer with higher alphaA proportion (3:1) or the alphaA-homopolymer seems to be better chaperones in protecting lens beta- and gamma-crystallins at both normal and elevated temperatures. Thus lens might have favoured a combination of these qualities to achieve optimal protection under both native and stress (perturbed) conditions for which the heteropolymer with alphaA to alphaB in the 3:1 ratio appears to be better suited.

Prevalence of vitamin deficiencies in an apparently healthy urban adult population: Assessed by subclinical status and dietary intakes.

OBJECTIVES: Studies in children and pregnant women consistently showed pandemic proportions of micronutrient deficiencies in the Indian subcontinent. However, vitamin deficiencies in apparently healthy adults are seldom recognized, hence the aim of this exploratory study was to assess their subclinical vitamin status and dietary intakes. METHODS: In all, 270 apparently healthy urban adults 30 to 70 y of age, from Hyderabad city, India participated in this study. Blood levels of vitamins (A, B1, B2, B6, total and active B12, D, and folate) and homocysteine were assessed. Anthropometric parameters were measured; dietary intake was obtained by food frequency questionnaire, and probability of adequacy (PA) was calculated by the estimated average requirement. RESULTS: Among the study population, the overall prevalence of deficiency of vitamin B2 was strikingly high (50%) followed by the vitamins B6 (46%), active B12 (46%), total B12 (37%), folate (32%), D (29%), B1 (11%), and A (6%). Hyperhomocysteinemia (HHcys) was widely prevalent (52%) in the study participants. In case of dietary intakes, PA was lowest for vitamin B12 (4%) and folate (9%) followed by vitamins A (22%), B2 (33%), B6 (30%), and B1 (59%). The mean PA of these vitamins was noticeably low (28%). The unadjusted logistic regression analysis found men and those with a deficiency of folate and total and active B12 to be at higher risk for HHcys. In the adjusted model, the risk for active B12 deficiency almost doubled. CONCLUSION: The study demonstrated a high prevalence of multiple subclinical vitamin deficiencies, dietary inadequacies, and HHcys, which are possible risk factors for disease burden among apparently healthy adults.

Alzheimer's and Danish dementia peptides induce cataract and perturb retinal architecture in rats.

Familial Danish dementias (FDDs) are autosomal dominant neurodegenerative disorders that are associated with visual defects. In some aspects, FDD is similar to Alzheimer's disease (AD)- the amyloid deposits in FDD and AD are made of short peptides: amyloid ß (Aß) in AD and ADan in FDD. Previously, we demonstrated an interaction between the dementia peptides and α-crystallin leading to lens opacification in organ culture due to impaired chaperone activity of α-crystallin. Herein, we report the in vivo effects of ADan and Aß on the eye. ADan [reduced (ADan-red) and oxidized (ADan-oxi)] and Aß (Aß1-40 and Aß1-42) were injected intravitreally in rats. The onset of cataract was seen after injection of all the peptides, but the cataract matured by 2 weeks in the case of ADan-red, 5 weeks for ADan-oxi and 6 weeks for Aß1-40, while Aß1-42 had minimal effect on cataract progression. The severity of cataract is associated with insolubilization and alterations in crystallins and loss of chaperone activity of α-crystallin. Further, disruption of the architecture of the retina was evident from a loss of rhodopsin, increased gliosis, and the thinning of the retina. These results provide a basis for the dominant heredo-otoophthalmo-encephalopathy (HOOE)/FDD syndrome and indicate that ADan peptides are more potent than Aßpeptides in inflicting visual impairment.

Alphab-crystallin-assisted reactivation of glucose-6-phosphate dehydrogenase upon refolding.

Alphab-crystallin, a small heat-shock protein has been shown to prevent the aggregation of other proteins under various stress conditions. We have investigated the role of alphaB-crystallin in the reactivation of denaturant [GdmCl (guanidinium chloride)]-inactivated G6PD (glucose-6-phosphate dehydrogenase). Studies indicate that unfolding and inactivation of G6PD by GdmCl proceeds via formation of a molten globule-like state at low concentrations of GdmCl, which was characterized by having maximum surface hydrophobicity and no catalytic activity. At high concentrations of GdmCl, G6PD was completely unfolded, which upon dilution-induced refolding yielding 35% of original activity. In contrast, no activity was recovered when G6PD was refolded from a molten globule-like state. Interestingly, refolding of completely unfolded G6PD in the presence of alphaB-crystallin resulted in 70% gain of the original activity, indicating that alphaB-crystallin assisted in enhanced refolding of G6PD. Intriguingly, alphaB-crystallin was unable to reactivate G6PD from a molten globule-like state. Size-exclusion chromatography data indicate that alphaB-crystallin-assisted reactivation of completely unfolded G6PD is concomitant with the restoration of the native structure of G6PD. Nonetheless, alphaB-crystallin failed to reactivate G6PD from preformed aggregates. Moreover, methylglyoxal-modified alpha-crystallin, which occurs in aged and diabetic cataract lenses, was less efficient in the reactivation of denaturant inactivated G6PD. Diminished chaperone-like activity of alpha-crystallin due to post-translational modifications may thus result in the accumulation of aggregated/inactivated proteins.

Amelioration of neuronal cell death in a spontaneous obese rat model by dietary restriction through modulation of ubiquitin proteasome system.

Dietary restriction (DR) has been shown to increase longevity, delay onset of aging, reduce DNA damage and oxidative stress and prevent age-related decline of neuronal activity. We previously reported the role of altered ubiquitin proteasome system (UPS) in the neuronal cell death in a spontaneous obese rat model (WNIN/Ob rat). In this study, we investigated the effect of DR on obesity-induced neuronal cell death in a rat model. Two groups of 40-day-old WNIN/Ob rats were either fed ad libitum (Ob) or pair-fed with lean. The lean phenotype of WNIN/Ob rats served as ad libitum control. These animals were maintained for 6.5months on their respective diet regime. At the end of the study, cerebral cortex was collected and markers of UPS, endoplasmic reticulum (ER) stress and autophagy were analyzed by quantitative real-time polymerase chain reaction, immunoblotting and immunohistochemistry. Chymotrypsin-like activity of proteasome was assayed by the fluorimetric method. Apoptotic cells were analyzed by TUNEL assay. DR improved metabolic abnormalities in obese rats. Alterations in UPS (up-regulation of UCHL1, down-regulation of UCHL5, declined proteasomal activity), increased ER stress, declined autophagy and increased expression of α-synuclein, p53 and BAX were observed in obese rats and DR alleviated these changes in obese rats. Further, DR decreased TUNEL-positive cells. In conclusion, DR in obese rats could not only restore the metabolic abnormalities but also preserved neuronal health in the cerebral cortex by preventing alterations in the UPS.

Modulation of alpha-crystallin chaperone activity in diabetic rat lens by curcumin.

PURPOSE: A decline in the chaperone-like activity of eye lens alpha-crystallin in diabetic conditions has been reported. In this study, we investigated whether curcumin, a dietary antioxidant, can manipulate the chaperone-like activity of alpha-crystallin in diabetic rat lens. METHODS: A group of rats received ip injection of streptozotocin (STZ; 35 mg/kg body weight in buffer) to induce hyperglycemia, while another group of rats received only buffer as vehicle and served as control. STZ-treated rats were assigned to 3 groups and fed either no curcumin or 0.002% or 0.01% curcumin, respectively. Cataract progression due to hyperglycemia was monitored with a slit lamp biomicroscope. At the end of 8 weeks animals were sacrificed and lenses were collected. alphaH- and alphaL-crystallins from a set of pooled lenses in each group were isolated by gel filtration. Chaperone activity, hydrophobicity, and secondary and tertiary structure of alphaH- and alphaL-crystallins were assessed by light scattering/spectroscopic methods. RESULTS: A decrease in chaperone-like activity of alphaH- and alphaL-crystallins was observed in STZ-treated diabetic rats. The declined chaperone-like activity due to hyperglycemia was associated with reduced hydrophobicity and altered secondary and tertiary structure of alphaH- and alphaL-crystallins. Interestingly, alphaH- and alphaL-crystallins isolated from curcumin fed diabetic rat lenses had shown improved chaperone-like activity as compared to alphaH- and alphaL-crystallins from untreated diabetic rat lens. Feeding of curcumin prevented the alterations in hydrophobicity and structural changes due to STZ-induced hyperglycemia. Modulation of functional and structural properties by curcumin was found to be greater with the alphaL-crystallin than alphaH-crystallin. Loss of chaperone activity of alpha-crystallin, particularly alphaL-crystallin, in diabetic rat lens could be attributed at least partly to increased oxidative stress. Being an antioxidant, curcumin feeding has prevented the loss of alpha-crystallin chaperone activity and delayed the progression and maturation of diabetic cataract. CONCLUSIONS: We demonstrate that curcumin, at the levels close to dietary consumption, prevented the loss of chaperone-like activity of alpha-crystallin vis-a-vis cataractogenesis due to diabetes in rat lens.

Effect of dicarbonyl-induced browning on alpha-crystallin chaperone-like activity: physiological significance and caveats of in vitro aggregation assays.

Alpha-crystallin is a member of the small heat-shock protein family and functions like a molecular chaperone, and may thus help in maintaining the transparency of the eye lens by protecting the lens proteins from various stress conditions. Non-enzymic glycation of long-lived proteins has been implicated in several age- and diabetes-related complications, including cataract. Dicarbonyl compounds such as methylglyoxal and glyoxal have been identified as the predominant source for the formation of advanced glycation end-products in various tissues including the lens. We have investigated the effect of non-enzymic browning of alpha-crystallin by reactive dicarbonyls on its molecular chaperone-like function. Non-enzymic browning of bovine alpha-crystallin in vitro caused, along with altered secondary and tertiary structures, cross-linking and high-molecular-mass aggregation. Notwithstanding these structural changes, methylglyoxal- and glyoxal-modified alpha-crystallin showed enhanced anti-aggregation activity in various in vitro aggregation assays. Paradoxically, increased chaperone-like activity of modified alpha-crystallin was not associated with increased surface hydrophobicity and rather showed less 8-anilinonaphthalene-l-sulphonic acid binding. In contrast, the chaperone-like function of modified alpha-crystallin was found to be reduced in assays that monitor the prevention of enzyme inactivation by UV-B and heat. Moreover, incubation of bovine lens with methylglyoxal in organ culture resulted in cataract formation with accumulation of advanced glycation end-products and recovery of alpha-crystallin in high proportions in the insoluble fraction. Furthermore, soluble alpha-crystallin from methylglyoxal-treated lenses showed decreased chaperone-like activity. Thus, in addition to describing the effects of methylglyoxal and glyoxal on structure and chaperone-like activity, our studies also bring out an important caveat of aggregation assays in the context of the chaperone function of alpha-crystallin.

Suppression of DTT-induced aggregation of abrin by alphaA- and alphaB-crystallins: a model aggregation assay for alpha-crystallin chaperone activity in vitro.

The eye lens small heat shock proteins (sHSP), alphaA- and alphaB-crystallins, have been shown to function like molecular chaperones, both in vitro and in vivo. It is essential to assess the protective effect of alphaA- and alphaB-crystallins under native conditions to extrapolate the results to in vivo conditions. Insulin and alpha-lactalbumin have widely been used to investigate the chaperone mechanism of alpha-crystallin under native conditions. Due to its smaller size, insulin B-chain may not represent the binding of putative physiological substrate proteins. As it stands, the aggregation of alpha-lactalbumin and binding of alpha-crystallin to it varies under different experimental conditions. Abrin, a ribosome inactivating protein isolated from the seeds of Abrus precatorius, consists of a 30 kDa A-chain and a lectin-like B-chain of 33 kDa joined by a single disulfide bond. Reduction of the disulfide link between the two chains of abrin leads to the aggregation of the B-chain. In this study, we demonstrate that dithiothreitol (DTT)-induced aggregation of abrin B-chain could be monitored by light scattering similar to that of insulin. Moreso, this process could be suppressed by recombinant human alphaA- and alphaB-crystallins in a concentration dependent manner, notably by binding to aggregation prone abrin B-chain. SDS-PAGE and HPLC gel filtration analysis indicate that there is a soluble complex formation between alpha-crystallin and abrin B-chain. Interestingly, in contrast to insulin, there is no significant difference between alphaA- and alphaB-crystallin in suppressing the aggregation of abrin B-chain at two different temperatures (25 and 37 degrees C). HSP26, an another small heat shock/alpha-crystallin family protein, was also able to prevent the DTT-induced aggregation of abrin. These results suggest that due to relatively larger size of its B-chain (33 kDa), compared to insulin B-chain (about 3 kDa), abrin may serve as a better model substrate for in vitro chaperone studies of alpha-crystallin and as well as other sHSP.

Effect of long-term dietary manipulation on the aggregation of rat lens crystallins: role of alpha-crystallin chaperone function.

PURPOSE: To investigate the effect of food, protein, and vitamin restriction on the susceptibility of lens crystallins to aggregation and chaperone activity of alpha-crystallin. METHODS: Thirty day old Wistar/NIN rats were maintained on regular rodent diet (C), 50% food restriction (FR), 75% protein restriction (PR), and 50% vitamin restriction (VR) diet for 20 weeks. At the end, alpha-, beta-, and gamma-crystallins were isolated from the lenses of these animals and subjected to in vitro aggregation induced by oxidation, UV irradiation and heat. Aggregation and chaperone activity was assessed by light scattering methods. RESULTS: Dietary restriction has been shown to extend the mean and maximum life span and retard age-related diseases, including cataract. In this study, we demonstrate that while beta- and gamma-crystallins isolated from FR and PR groups were less susceptible to in vitro induced aggregation, beta- and gamma-crystallins from the VR group were more susceptible, compared to controls. Alpha-crystallin from any of the groups did not shown a considerable amount of aggregation. On the other hand, the chaperone activity of alpha-crystallin from FR and PR groups was not significantly different from controls. However, alpha-crystallin from the VR group demonstrated substantially higher chaperone activity than controls. CONCLUSIONS: These results indicate that while food and protein restriction appear to lower the susceptibility of beta- and gamma-crystallins towards aggregation, vitamin restriction tends to increase the aggregation. Chaperone activity of alpha-crystallin is affected (improved) by only vitamin restriction.

Antioxidant defense system and lipid peroxidation in patients with skeletal fluorosis and in fluoride-intoxicated rabbits.

Fluorosis is a serious public health problem in many parts of the world where drinking water contains more than 1 ppm of fluoride. The main manifestations of skeletal fluorosis are crippling bone deformities, spinal compressions, and restricted movements of joints. Although fluorosis is irreversible, it could be prevented by appropriate and timely intervention through understanding the process at biochemical and molecular levels. As in the case of many chronic degenerative diseases, increased production of reactive oxygen species (ROS) and lipid peroxidation has been considered to play an important role, even in the pathogenesis of chronic fluoride toxicity. However, there is inconclusive proof for an altered oxidative stress and antioxidant balance in fluorosis, and the existing data are not only conflicting but also contradictory. In the present communication we have evaluated the antioxidant defense system (both enzymatic and nonenzymatic) and lipid peroxidation in both humans from an endemic fluorosis area (5 ppm fluoride in the drinking water) and in rabbits receiving water with 150 ppm of fluoride for six months. There was no significant difference in lipid peroxidation, glutathione, and vitamin C in the blood of human fluorotic patients and fluoride-intoxicated rabbits as compared to respective controls. Neither were there any changes in the activities of catalase, superoxide dismutase, glutathione peroxidase, or glutathione S-transferase in the blood due to fluoride intoxication (of rabbits) or fluorosis in humans. The results together do not subscribe to oxidative stress theory in fluorosis. Thus, in the absence of clear proof of oxidative damage and to counter toxic effects of fluoride through supplementation of antioxidants, extensive investigations are needed to conclusively prove the role of oxidative stress in skeletal fluorosis.

Concurrence of Danish dementia and cataract: insights from the interactions of dementia associated peptides with eye lens alpha-crystallin.

Familial Danish Dementia (FDD) is an autosomal disease, which is distinguished by gradual loss of vision, deafness, progressive ataxia and dementia. Cataract is the first manifestation of the disease. In this article, we demonstrate a specific correlation between the poisoning of the chaperone activity of the rat eye lens alpha-crystallins, loss of lens transparency in organ culture by the pathogenic form of the Danish dementia peptide, i.e. the reduced Danish dementia peptide (redADan peptide), by a combination of ex vivo, in vitro, biophysical and biochemical techniques. The interaction of redADan peptide and lens crystallins are very specific when compared with another chaperone, HSP-70, underscoring the specificity of the pathogenic form of Danish dementia peptide, redADan, for the early onset of cataract in this disease.