A 32 kb critical region excluding Y402H in CFH mediates risk for age-related macular degeneration

Complement factor H shows very strong association with Age-related Macular Degeneration (AMD), and recent data suggest that multiple causal variants are associated with disease. To refine the location of the disease associated variants, we characterized in detail the structural variation at CFH and its paralogs, including two copy number polymorphisms (CNP), CNP147 and CNP148, and several rare deletions and duplications. Examination of 34 AMD-enriched extended families (N = 293) and AMD cases (White N = 4210 Indian = 134; Malay = 140) and controls (White N = 3229; Indian = 117; Malay = 2390) demonstrated that deletion CNP148 was protective against AMD, independent of SNPs at CFH. Regression analysis of seven common haplotypes showed three haplotypes, H1, H6 and H7, as conferring risk for AMD development. Being the most common haplotype H1 confers the greatest risk by increasing the odds of AMD by 2.75-fold (95% CI = [2.51, 3.01]; p = 8.31×10⁻¹⁰⁹); Caucasian (H6) and Indian-specific (H7) recombinant haplotypes increase the odds of AMD by 1.85-fold (p = 3.52×10⁻⁹) and by 15.57-fold (P = 0.007), respectively. We identified a 32-kb region downstream of Y402H (rs1061170), shared by all three risk haplotypes, suggesting that this region may be critical for AMD development. Further analysis showed that two SNPs within the 32 kb block, rs1329428 and rs203687, optimally explain disease association. rs1329428 resides in 20 kb unique sequence block, but rs203687 resides in a 12 kb block that is 89% similar to a noncoding region contained in ΔCNP148. We conclude that causal variation in this region potentially encompasses both regulatory effects at single markers and copy number.     

Exploring the evolutionary relationship of insulin receptor substrate family using computational biology

Insulin receptor substrate (IRS) harbors proteins such as IRS1, IRS2, IRS3, IRS4, IRS5 and IRS6. These key proteins act as vital downstream regulators in the insulin signaling pathway. However, little is known about the evolutionary relationship among the IRS family members. This study explores the potential to depict the evolutionary relationship among the IRS family using bioinformatics, algorithm analysis and mathematical models.     

Profiling the phyto-constituents of Punica granatum fruits peel extract and accessing its in-vitro antioxidant,anti-diabetic,anti-obesity,and angiotensin-converting enzyme inhibitory properties

This context was investigated to assess the in vitro antioxidant, anti-diabetic, anti-obesity, and angiotensin-converting enzyme (ACE) inhibition traits of Punica granatum fruits peel extract. Initially, among various extracts tested, aqueous and ethanolic peel extracts depicted the presence of diverse phytoconstituents. In vitro antioxidative properties of peel extracts were determined using standard methodologies. Results showed that aqueous and ethanolic extracts had IC₅₀ values of 471.7 and 509.16 μg/mL, respectively in terms of 1,1,diphenyl 2,2,picrylhydrazyl scavenging. Likewise, IC₅₀ values of aqueous and ethanol extract were obtained as 488.76 and 478.47 μg/mL towards the degradation of hydrogen peroxide. The ethanolic extract exhibited the highest inhibition of α-glucosidase by showing activity of 53.34 ± 2.0 to 15.18 ± 1.4 U/L in a dose dependent manner (100–1000 µg/mL). Ethanolic extract was reported as the most active inhibitor of lipase with an IC₅₀ value of 603.50 µg/mL. Ethanolic extract showed increased inhibition of ACE in a concentration dependent manner (100–1000 µg/mL) with IC₅₀ value of 519.45 µg/mL. Fourier transform-infrared spectrum revealed the availability of various functional groups in the ethanolic extract of peel. Gas chromatography-mass spectrometry chromatogram of peel extract illustrated 23 diversified chemical constituents including 1,2,3,4-butanetetrol, Dimethyl sulfone, 9-octadecenamide, and Pentadecanoic acid as predominant compounds. In summary, P. granatum fruits peel extract revealed promising antioxidant, anti-diabetic, anti-obesity, and anti-hypertensive properties.     

Effect of glucose and phytohaemagglutinin (PHA) rich Phaseolus vulgaris extract on growth and protein synthesis of pharmaceutically important cyanobacteria Nostoc ellipsosporum NCIM 2786

Nostoc ellipsosporum is a highly potent cyanobacterium for production of pharmaceutically important chemicals. In this study, an effort has been made to determine the effect of glucose and phytohaemagglutinin (PHA) rich Phaseolus vulgaris extract on N. ellipsosporum growth and protein production. Maximum growth was observed in Fog’s medium supplemented with glucose. SEM analysis showed that the regular and well developed heterocysts were observed in Fog’s media supplemented with glucose. Significant medium components were evaluated by Plackett–Burman (PB) design and PHA extract was found to be the most significant in growth medium. Results of this study showed that both glucose and PHA rich P. vulgaris extract have positive effects and enhance the growth and protein synthesis.     

The Modified Selenenyl Amide,M-hydroxy Ebselen,Attenuates Diabetic Nephropathy and Diabetes-Associated Atherosclerosis in ApoE/GPx1 Double Knockout Mice

Seleno-organic glutathione peroxidase (GPx) mimetics, including ebselen (Eb), have been tested in in vitro studies for their ability to scavenge reactive oxygen and nitrogen species, including hydrogen peroxide and peroxynitrite. In this study, we investigated the efficacies of two Eb analogues, m-hydroxy ebselen (ME) and ethanol-ebselen (EtE) and compared these with Eb in cell based assays. We found that ME is superior in attenuating the activation of hydrogen peroxide-induced pro-inflammatory mediators, ERK and P38 in human aortic endothelial cells. Consequently, we investigated the effects of ME in an in vivo model of diabetes, the ApoE/GPx1 double knockout (dKO) mouse. We found that ME attenuates plaque formation in the aorta and lesion deposition within the aortic sinus of diabetic dKO mice. Oxidative stress as assessed by 8-OHdG in urine and nitrotyrosine immunostaining in the aortic sinus and kidney tubules, was reduced by ME in diabetic dKO mice. ME also attenuated diabetes-associated renal injury which included tubulointerstitial fibrosis and glomerulosclerosis. Furthermore, the bioactivity of the pro-fibrotic cytokine transforming growth factor-β (TGF-β) as assessed by phospho-Smad2/3 immunostaining was attenuated after treatment with ME. TGF-β-stimulated increases in collagen I and IV gene expression and protein levels were attenuated by ME in rat kidney tubular cells. However, in contrast to the superior activity of ME in in vitro and cell based assays, ME did not further augment the attenuation of diabetes-associated atherosclerosis and renal injury in our in vivo model when compared with Eb. In conclusion, this study strengthens the notion that bolstering GPx-like activity using synthetic mimetics may be a useful therapeutic strategy in lessening the burden of diabetic complications. However, these studies highlight the importance of in vivo analyses to test the efficacies of novel Eb analogues, as in vitro and cell based assays are only partly predictive of the in vivo situation.     

Electron paramagnetic resonance oximetry as a quantitative method to measure cellular respiration: a consideration of oxygen diffusion interference

Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO(2)-independent respiration at higher pO(2) ranges, pO(2)-dependent respiration at low pO(2) ranges, and a static equilibrium with no change in pO(2) at very low pO(2) values. The approach here enables one to comprehensively analyze all of the three zones together-where the progression of O(2) diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO(2) data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry.     

Endurance training of older men: responses to submaximal exercise.

The purpose of this study was to quantify the exercise response of older subjects on a time-to-fatigue (TTF) submaximal performance test before and after a training program. Eight older men (67.4 +/- 4.8 yr) performed two maximal treadmill tests to determine maximum oxygen uptake (VO2max) and ventilation threshold (TVE) and a constant-load submaximal exercise treadmill test that required an oxygen uptake (VO2) between TVE and VO2max. The submaximal test, performed at the same absolute work rate before and after the training program, was performed to volitional fatigue to measure endurance time. The men trained under supervision at an individualized pace representing approximately 70% of VO2max (80% maximum heart rate) for 1 h, four times per week for 9 wk. Significant increases were demonstrated for VO2max (ml.kg-1.min-1; 10.6%); maximal ventilation (VE, l/min; 11.6%), and TVE (l/min; 9.8%). Weight decreased 2.1%. Performance time on the TTF test increased by 180% (7.3 +/- 3.0 to 20.4 +/- 13.5 min). The similar end points for VO2, VE, and heart rate during the TTF and maximal treadmill tests established that the TTF test was stopped because of physiological limitations. The increase in performance time among the subjects was significantly correlated with improvements in VO2max and TVE, with the submaximal work rate representing a VO2 above TVE by 88% of the difference between TVE and VO2max pretraining and 73% of this difference on posttraining values.     

Homology model of a novel thermostable xylanase from Bacillus subtilis-AK1

Xylanases are glycosyl hydrolases that catalyze the hydrolysis of internal beta-1,4 glycosidic bonds of xylan, the major hemi-cellulose component of the plant cell wall. Enzymes such as xylanases are used considerably in industries. Their industrial usage is especially attractive since they can replace some of the environmental pollutants. We have earlier isolated a family 11-xylanase gene from Bacillus subtilis-AK1, which is active at high temperature as well as at alkaline pH. In order to understand the factors liable for the adaptation of this enzyme, three dimensional model of B. subtilis-AK1 xylanase has now been obtained by homology modeling. Modeling was carried out using Molecular Operating Environment (MOE) software developed by Chemical Computing Group Inc., running on Pentium IV workstation. The model showed that B. subtilis-AK1 xylanase having molecular weight around 20 kDa contains in its fold an alpha-helix and two beta-sheets packed against each other forming a beta-sandwich. The conserved active site amino acids E78R, Y80L were mutated in this novel B. subtilis-AK1 strain, but the protein folding and the function was maintained with high thermal stability. Several minor modifications appeared to be responsible for the increased thermo stability of AK1. Docking studies of the substrate xylan with -AK1 shows the possibility of the Arg 78 acting as the nucleophile instead of Glu 78.     

Speeding up proton transfer in a fast enzyme: kinetic and crystallographic studies on the effect of hydrophobic amino acid substitutions in the active site of human carbonic anhydrase II

Catalysis of the hydration of CO2 by human carbonic anhydrase isozyme II (HCA II) is sustained at a maximal catalytic turnover of 1 mus-1 by proton transfer between a zinc-bound solvent and bulk solution. This mechanism of proton transfer is facilitated via the side chain of His64, which is located 7.5 A from the zinc, and mediated via intervening water molecules in the active-site cavity. Three hydrophilic residues that have previously been shown to contribute to the stabilization of these intervening waters were replaced with hydrophobic residues (Y7F, N62L, and N67L) to determine their effects on proton transfer. The structures of all three mutants were determined by X-ray crystallography, with crystals equilibrated from pH 6.0 to 10.0. A range of changes were observed in the ordered solvent and the conformation of the side chain of His64. Correlating these structural variants with kinetic studies suggests that the very efficient proton transfer (approximately 7 micros-1) observed for Y7F HCA II in the dehydration direction, compared with the wild type and other mutants of this study, is due to a combination of three features. First, in this mutant, the side chain of His64 showed an appreciable inward orientation pointing toward the active-site zinc. Second, in the structure of Y7F HCA II, there is an unbranched chain of hydrogen-bonded waters linking the proton donor His64 and acceptor zinc-bound hydroxide. Finally, the difference in pKa of the donor and acceptor appears favorable for proton transfer. The data suggest roles for residues 7, 62, and 67 in fine-tuning the properties of His64 for optimal proton transfer in catalysis.