Inhibition of Methylglyoxal-Mediated Protein Modification in Glyoxalase I Overexpressing Mouse Lenses

Objective. Here we tested the role of Glo I in the prevention of advanced glycation end product (AGE) formation in transgenic mouse lenses. Methods. A transgenic animal line that expressed high levels of human Glo I in the lens was developed from the C57B6 mouse strain. The role of Glo I in the inhibition of MGO-AGE formation was tested in organ-cultured lenses. Results. Organ culture of Wt and Glo I lenses with 5 mM D, L-glyceraldehyde (GLD) enhanced MGO by 29-fold and 17-fold in Wt lenses and Glo I lenses, respectively. Argpyrimidine levels were 192 ± 73 pmoles/mg protein, and hydroimidazolone levels were 22 ± 0.7 units/μg protein in GLD-incubated Wt lenses. In Glo I lenses, formation of AGEs was significantly inhibited; the argpyrimidine levels were 82 ± 18 pmoles/mg protein, and the HI levels were 2.6 ± 2.3 units/μg protein. Incubation of Wt lens proteins with 5 mM ribose for 7 days resulted in the formation of pentosidine. However, the levels were substantially higher in Glo I lens proteins incubated with ribose. Conclusion. Our study provides direct evidence that Glo I activity plays an important role in the regulation of AGE synthesis in the lens; while Glo I activity blocks the formation of MGO-AGEs, it might promote the formation of sugar-derived AGEs

Topical ocular application of aggrelyte-2A reduces lens stiffness in mice

Presbyopia is the progressive loss of the ability of the lens to focus on nearby objects due to its increased stiffness. It occurs in the mid-40s and continues to worsen until the mid-60s. The age-associated increase in protein cross-linking in the lens leads to protein aggregation and water insolubility, especially in the nuclear region, contributing to lens stiffness. This study reports the development of aggrelyte-2A (methyl S-acetyl-N-(3,3-dimethylbutanoyl) cysteinate, a derivative of our previously reported aggrelyte-2) for reversing the stiffness of aged lenses. Aggrelyte-2A showed minimal toxicity in cultured mouse lens epithelial cells (up to 2000 µM) and human lens epithelial cells (up to 250 µM). Lenses from aged mice (age: 24-25 months) treated with 1 mM aggrelyte-2A for 24 h, and human lenses (age: 47-67 years) treated with 250 µM aggrelyte-2A for 48 h showed 11-14% reductions in stiffness, accompanied by an increase in acetyllysine in lens proteins, and free-thiols in the lens. Topical application of aggrelyte-2A (40 mM, 5 µl twice daily for 4 weeks) on mouse eyes significantly reduced lens stiffness. The topical application showed no toxicity to the lens, cornea, or retina, as revealed by morphological examination, H&E staining, and optical coherence tomography. These data suggest that aggrelyte-2A could be developed as a presbyopia-reversing therapeutic

AGEs in human lens capsule promote the TGFβ2-mediated EMT of lens epithelial cells:Implications for age-associated fibrosis

Proteins in basement membrane (BM) are long-lived and accumulate chemical modifications during aging; advanced glycation end-product (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial-to-mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. Wefound age-dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC-MS/MS. The TGFβ2-mediated upregulation of the mRNA levels (by qPCR) of EMT-associated proteins was significantly enhanced in cells cultured on AGE-modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2-mediated a-smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2-mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes-associated fibrosis

A study of complementary feeding practices among higher educated mothers in north-western part of Rajasthan

Background: Infants and young children are at an increased risk of malnutrition from 6 months of age onward when breast milkalone is no longer sufficient to meet all their nutritional requirements and complementary feeding should be started. Objective: Thisstudy was undertaken to assess the practices of complementary feeding and knowledge of infant milk substitute (IMS) act in highereducated mothers. Materials and Methods: This hospital-based cross-sectional descriptive study was conducted in a medicalcollege of Rajasthan from January to December 2015. Totally, 300 mothers with graduate/post-graduate degree whose children werebetween 9 and 15 months of age,were included in the study. All the mothers were interviewed as per the standardized pro formaregarding the complementary feeding practices. Results: Maximum numbers of mothers were homemakers (67.3%) as comparedto employed (32.7%). In the present study, 38.98% mothers practiced exclusive breastfeeding till 6 months of age, and 69.49%infants were exclusive breastfed till 4 months of age. Out of total 300 mothers, 116 (38.6%) introduced complementary feed before6 months of age while 142 (47.3%) introduced at 6 months of age. 140 (46.6%) educated mothers started complementary feedingwith homemade preparations while 115 (38.4%) started with market preparations. 33 (39.2%) mothers stopped breastfeeding at6-9 months of age, and 33 (39.2%) mothers stopped breast feeding between 10 and 12+ months of age. A significant portion274 (91.3%) of educated mothers did not have knowledge of IMS act. Conclusion: In the present study, majority of the mothersconsidered milk supplementation as complementary feeding and employed mothers preferred market preparations. Knowledgeregarding IMS act was not satisfactory

A clinical study of foreign bodies in air passages

Background: Foreign body in the airways is a serious event and fatal if not managed properly in time. Accident resulting from the inhalation of the foreign body continues to take formidable toll of lives every year.Objecti ve: This study was undertaken to find out the incidence of foreign bodies in air passages in this part of the country and types of foreign bodies, to establish the role of radiological investigations, and to find out the effective measures which are valuable in decreasing the post endoscopic complications and morbidity. Methods: This retrospective study was conducted in all suspected cases of foreign body in the air passages attending as well as referred to the Department of Otolaryngology of a Medical College of Rajasthan from January 2014 to December 2015. In each case, a procedure like direct laryngoscopy or bronchoscopy was done depending on the site of the foreign body. Results: Out of 100 cases, 58 were males and 42 were females. 88% of the patients were children below the age of 12 years. The most common foreign body extracted was betel nut in 47% of the cases. Commonest sign observed was decreased air entry in the lung (83%). Commonest radiological finding was obstructive collapse (37%). 48% of the total foreign bodies were present in right bronchus while 32% were lodged in the left bronchus and 3% at the carina. Conclusion: A diagnostic bronchoscopy should always be done in refractory cases of chronic respiratory infection, allergy or pneumonia. A negative radiological examination does not exclude a foreign body

Targeted Deletion of the Murine Lgr4 Gene Decreases Lens Epithelial Cell Resistance to Oxidative Stress and Induces Age-Related Cataract Formation

Oxidative stress contributes to the formation of cataracts. The leucine rich repeat containing G protein-coupled receptor 4 (LGR4, also known as GPR48), is important in many developmental processes. Since deletion of Lgr4 has previously been shown to lead to cataract formation in mice, we sought to determine the specific role that Lgr4 plays in the formation of cataracts. Initially, the lens opacities of Lgr4−/− mice at different ages without ocular anterior segment dysgenesis (ASD) were evaluated with slit-lamp biomicroscopy. Lenses from both Lgr4−/− and wild-type mice were subjected to oxidation induced protein denaturation to assess the ability of the lens to withstand oxidation. The expression of antioxidant enzymes was evaluated with real-time quantitative PCR. Phenotypically, Lgr4−/− mice showed earlier onset of lens opacification and higher incidence of cataract formation compared with wild-type mice of similar age. In addition, Lgr4−/− mice demonstrated increased sensitivity to environmental oxidative damage, as evidenced by altered protein expression. Real-time quantitative PCR showed that two prominent antioxidant defense enzymes, catalase (CAT) and superoxidase dismutase-1 (SOD1), were significantly decreased in the lens epithelial cells of Lgr4−/− mice. Our results suggest that the deletion of Lgr4 can lead to premature cataract formation, as well as progressive deterioration with aging. Oxidative stress and altered expression of several antioxidant defense enzymes contribute to the formation of cataracts

Effect of pyridoxamine on chemical modification of proteins by carbonyls in diabetic rats: characterization of a major product from the reaction of pyridoxamine and methylglyoxal

Abstract Advanced glycation end products (AGEs) from the Maillard reaction contribute to protein aging and the pathogenesis of ageand diabetes-associated complications. The a-dicarbonyl compound methylglyoxal (MG) is an important intermediate in AGE synthesis. Recent studies suggest that pyridoxamine inhibits formation of advanced glycation and lipoxidation products. We wanted to determine if pyridoxamine could inhibit MG-mediated Maillard reactions and thereby prevent AGE formation. When lens proteins were incubated with MG at 37°C, pH 7.4, we found that pyridoxamine inhibits formation of methylglyoxal-derived AGEs concentration dependently. Pyridoxamine reduces MG levels in red blood cells and plasma and blocks formation of methylglyoxallysine dimer in plasma proteins from diabetic rats and it prevents pentosidine (an AGE derived from sugars) from forming in plasma proteins. Pyridoxamine also decreases formation of protein carbonyls and thiobarbituric-acid-reactive substances in plasma proteins from diabetic rats. Pyridoxamine treatment did not restore erythrocyte glutathione (which was reduced by almost half) in diabetic animals, but it enhanced erythrocyte glyoxalase I activity. We isolated a major product of the reaction between MG and pyridoxamine and identified it as methylglyoxal-pyridoxamine dimer. Our studies show that pyridoxamine reduces oxidative stress and AGE formation. We suspect that a direct interaction of pyridoxamine with MG partly accounts for AGE inhibition. Ó 2002 Elsevier Science (USA). All rights reserved

Aspirin Inhibits TGFβ2-Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells:Selective acetylation of K56 and K122 in histone H3

Posterior capsule opacification (PCO) is a complication after cataract surgery that can disrupt vision. The epithelial to mesenchymal transition (EMT) of lens epithelial cells (LECs) in response to transforming growth factor β2 (TGFβ2) has been considered an obligatory mechanism for PCO. In this study, we tested the efficacy of aspirin in inhibiting the TGFβ2-mediated EMT of human LECs, LECs in human lens capsular bags, and lensectomized mice. In human LECs, the levels of the EMT markers α-smooth muscle actin (α-SMA) and fibronectin were drastically reduced by treatment with 2 mM aspirin. Aspirin also halted the EMT response of TGFβ2 when introduced after EMT initiation. In human capsular bags, treatment with 2 mM aspirin significantly suppressed posterior capsule wrinkling and the expression α-SMA in capsule-adherent LECs. The inhibition of TGFβ2-mediated EMT in human LECs was not dependent on Smad phosphorylation or MAPK and AKT-mediated signaling. We found that aspirin significantly increased the acetylation of K56 and K122 in histone H3 of human LECs. Chromatin immunoprecipitation assays using acetyl-H3K56 or acetyl-H3K122 antibody revealed that aspirin blocked the TGFβ2-induced acetylation of H3K56 and H3K122 at the promoter regions of ACTA2 and COL1A1. After lensectomy in mice, we observed an increase in the proliferation and α-SMA expression of the capsule-adherent LECs, which was ameliorated by aspirin administration through drinking water. Taken together, our results showed that aspirin inhibits TGFβ2-mediated EMT of LECs, possibly from epigenetic down-regulation of EMT-related genes