Genetic variants of TNFα, IL10, IL1β, CTLA4 and TGFβ1 modulate the indices of alcohol-induced liver injury in East Indian population

Alcohol induced liver disease or alcoholic liver disease (ALD), a complex trait, encompasses a gamut of pathophysiological alterations in the liver due to continuous exposure to a toxic amount of alcohol (more than 80g per day). Of all chronic heavy drinkers, only 15-20% develops hepatitis or cirrhosis concomitantly or in succession. Several studies revealed that inter-individual as well as inter-ethnic genetic variation is one of the major factors that predispose to ALD. The role of genetic factors in ALD has long been sought for in ethnically distinct population groups. ALD is fast emerging as an important cause of chronic liver disease in India; even in populations such as "Bengalis" who were "culturally immune" earlier. While the genetic involvement in the pathogenesis of ALD is being sought for in different races, the complex pathophysiology of ALD as well as the knowledge of population level diversity of the relevant alcohol metabolizing and inflammatory pathways mandates the need for well designed studies of genetic factors in ethnically distinct population groups. An array of cytokines plays a critical role as mediators of injury, inflammation, fibrosis and cirrhosis in ALD. We, therefore, studied the association of polymorphisms in five relevant cytokine genes with "clinically significant" ALD in an ethnic "Bengali" population in Eastern India. Compared with "alcoholic" controls without liver disease (n=110), TNFα -238AA genotype, IL1β -511CC genotype, TGFβ1 -509CC genotype and IL10 -592AA genotype were significantly overrepresented in ALD patients (n=181; OR=2.4 and 95% CI 1.2-5.5, P(genotype)=0.042, P(allelic)=0.008; OR=2.7 and 95% CI 1.2-5.9, P(genotype)=0.018, P(allelic)=0.023; OR=4.7 and 95% CI 1.7-13.1, P(genotype)=0.003, P(allelic)=0.014; and OR=2.2 and 95% CI 1.1-4.8, P(genotype)=0.04, P(allelic)=0.039 respectively). Moreover a cumulative genetic risk analysis revealed a significant trend for developing ALD with an increase in the number of risk alleles on IL10 and TGFβ1 loci among alcoholics. The risk genotype of IL1β and TGFβ1 also influences the total bilirubin, albumin and alanine aminotransferase levels among alcoholic "Bengalis". The present study is the first case-control study from Eastern India that comprehensively identified polymorphic markers in TNFα, IL10, IL1β and TGFβ1 genes to be associated with ALD in the Bengali population, accentuating the significance of genetic factors in clinical expressions of ALD.     

Discovery of betamethasone 17alpha-carbamates as dissociated glucocorticoid receptor modulators in the rat

A series of betamethasone 17alpha-carbamates were designed, synthesized, and evaluated for their ability to dissociate the two main functions of the glucocorticoid receptor, that is, transactivation and transrepression, in rat cell lines. A number of alkyl substituted betamethasone 17alpha-carbamates were identified with excellent affinity for the glucocorticoid receptor (e.g., 7, GR IC(50) 5.1 nM) and indicated dissociated profiles in functional assays of transactivation (rat tyrosine aminotransferase, TAT, and rat glutamine synthetase, GS) and transrepression (human A549 cells, MMP-1 assay). Gratifyingly, the in-vivo profile of these compounds, for example, 7, also indicated potent anti-inflammatory activity with impaired effects on glucose, insulin, triglycerides, and body weight. Taken together, these results indicate that dissociated glucocorticoid receptor modulators can be identified in rodents.     

Insights into the Photoprotective Switch of the Major Light-harvesting Complex II (LHCII): A PRESERVED CORE OF ARGININE-GLUTAMATE INTERLOCKED HELICES COMPLEMENTED BY ADJUSTABLE LOOPS*

Light-harvesting antennae of the LHC family form transmembrane three-helix bundles of which two helices are interlocked by conserved arginine-glutamate (Arg-Glu) ion pairs that form ligation sites for chlorophylls. The antenna proteins of photosystem II have an intriguing dual function. In excess light, they can switch their conformation from a light-harvesting into a photoprotective state, in which the excess and harmful excitation energies are safely dissipated as heat. Here we applied magic angle spinning NMR and selective Arg isotope enrichment as a noninvasive method to analyze the Arg structures of the major light-harvesting complex II (LHCII). The conformations of the Arg residues that interlock helix A and B appear to be preserved in the light-harvesting and photoprotective state. Several Arg residues have very downfield-shifted proton NMR responses, indicating that they stabilize the complex by strong hydrogen bonds. For the Arg C_α chemical shifts, differences are observed between LHCII in the active, light-harvesting and in the photoprotective, quenched state. These differences are attributed to a conformational change of the Arg residue in the stromal loop region. We conclude that the interlocked helices of LHCII form a rigid core. Consequently, the LHCII conformational switch does not involve changes in A/B helix tilting but likely involves rearrangements of the loops and helical segments close to the stromal and lumenal ends.     

Significance of location of enzymes on their release during microbial cell disruption.

The release kinetics of the enzyme invertase and alcohol dehydrogenase from yeast and penicillin acylase from E. coli during disruption using various techniques has been investigated. The disruption techniques used were sonication, high-pressure homogenization, and hydrodynamic cavitation. The first-order-release kinetics was applied for the determination of release rate of these enzymes and total soluble proteins. Location factor (LF) values were calculated using these release rates. The location of the enzymes as given by the values of location factor coincided well with those reported in the literature. Varying values of location factor for the same enzyme by different disruption techniques gave some indications about the selectivity of release of a target enzyme by different disruption techniques. Varying values of location factor for the same enzyme with the use of a particular equipment or disruption technique at different conditions reveals the degree to which the cell is disrupted. Few plausible applications of this location factor concept have been predicted and these speculations have been examined. This location factor concept has been used for monitoring the heat-induced translocation of ADH and location of penicillin acylase during the growth period of E. coli cells.     

High-resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe grisea

In order to understand the molecular mechanisms involved in the gene-for-gene type of pathogen resistance, high-resolution genetic and physical mapping of resistance loci is required to facilitate map-based cloning of resistance genes. Here, we report the molecular mapping and cloning of a dominant gene (Pi-k ^h ) present in the rice line Tetep, which is associated with resistance to rice blast disease caused by Magnaporthe grisea. This gene is effective against M. grisea populations prevalent in the Northwestern Himalayan region of India. Using 178 sequence tagged microsatellite, sequence-tagged site, expressed sequence tag and simple sequence repeat (SSR) markers to genotype a population of 208 F₂ individuals, we mapped the Pi-k ^h gene between two SSR markers (TRS26 and TRS33) which are 0.7 and 0.5 cM away, respectively, and can be used in marker-assisted-selection for blast-resistant rice cultivars. We used the markers to identify the homologous region in the genomic sequence of Oryza sativa cv. Nipponbare, and a physical map consisting of two overlapping bacterial artificial chromosome and P1 artificial chromosome clones was assembled, spanning a region of 143,537 bp on the long arm of chromosome 11. Using bioinformatic analyses, we then identified a candidate blast-resistance gene in the region, and cloned the homologous sequence from Tetep. The putative Pi-k ^h gene cloned from Tetep is 1.5 kbp long with a single ORF, and belongs to the nucleotide binding site-leucine rich repeat class of disease resistance genes. Structural and expression analysis of the Pi-k ^h gene revealed that its expression is pathogen inducible.     

Mapping Human Telomere Regions with YAC and P1 Clones: Chromosome-Specific Markers for 27 Telomeres Including 149 STSs and 24 Polymorphisms for 14 Proterminal Regions

A YAC library enriched for telomere clones was constructed and screened for the human telomere-specific repeat sequence (TTAGGG). Altogether 196 TYAC library clones were studied: 189 new TYAC clones were isolated, 149 STSs were developed for 132 different TYACs, and 39 P1 clones were identified using 19 STSs from 16 of the TYACs. A combination of mapping methods including fluorescencein situhybridization, somatic cell hybrid panels, clamped homogeneous electric fields, meiotic linkage, and BLASTN sequence analysis was utilized to characterize the resource. Forty-five of the TYACs map to 31 specific telomere regions. Twenty-four linkage markers were developed and mapped within 14 proterminal regions (12 telomeres and 2 terminal bands). The polymorphic markers include 12 microsatellites for 10 telomeres (1q, 2p, 6q, 7q, 10p, 10q, 13q, 14q, 18p, 22q) and the terminal bands of 11q and 12p. Twelve RFLP markers were identified and meiotically mapped to the telomeres of 2q, 7q, 8p, and 14q. Chromosome-specific STSs for 27 telomeres were identified from the 196 TYACs. More than 30,000 nucleotides derived from the TYAC vector-insert junction regions or from regions flanking TYAC microsatellites were compared to reported sequences using BLASTN. In addition to identifying homology with previously reported telomere sequences and human repeat elements, gene sequences and a number of ESTs were found to be highly homologous to the TYAC sequences. These genes include human coagulation factor V (F5), Wee1 protein tyrosine kinase (WEE1), neurotropic protein tyrosine kinase type 2 (NTRK2), glutathioneS-transferase (GST1), and β tubulin (TUBB). The TYAC/P1 resource, derivative STSs, and polymorphisms constitute an enabling resource to further studies of telomere structure and function and a means for physical and genetic map integration and closure.     

Miconazole nitrate bearing ultraflexible liposomes for the treatment of fungal infection

Miconazole nitrate is a widely used antifungal agent, but its use in topical formulations is not efficacious because deep seated fungal infections are difficult to treat with conventional topical formulation. Miconazole nitrate loaded ultraflexible liposomes have been prepared and their topical performance has been compared with conventional liposomes containing miconazole nitrate. Various ultraflexible liposomal formulations were prepared and extensively characterized for vesicular shape, size, entrapment efficiency, degree of deformability and in-vitro skin permeation through rat skin. Higher rate of drug transfer across the skin with ultraflexible liposomal formulations of miconazole nitrate suggests that the drug in its lipo-solubilized state might have gained facilitated entry into the tough barrier consisting of subcutaneous. In-vivo study showed better antifungal activity as compared to traditional liposomes and plain drug solution. This was confirmed through fluoroscence microscopy. It is concluded that prepared ultraflexible liposomes can facilitate improved and localized drug action in the skin, thus providing a better option to deal with deep seated skin problems.     

Climate-Induced Elevational Range Shifts and Increase in Plant Species Richness in a Himalayan Biodiversity Epicentre

Global average temperature increase during the last century has induced species geographic range shifts and extinctions. Montane floras, in particular, are highly sensitive to climate change and mountains serve as suitable observation sites for tracing climate-induced biological response. The Himalaya constitute an important global biodiversity hotspot, yet studies on species’ response to climate change from this region are lacking. Here we use historical (1849–50) and the recent (2007–2010) data on temperature and endemic species’ elevational ranges to perform a correlative study in the two alpine valleys of Sikkim. We show that the ongoing warming in the alpine Sikkim Himalaya has transformed the plant assemblages. This study lends support to the hypothesis that changing climate is causing species distribution changes. We provide first evidence of warmer winters in the region compared to the last two centuries, with mean temperatures of the warmest and the coldest months may have increased by 0.76±0.25°C and 3.65±2°C, respectively. Warming-driven geographical range shifts were recorded in 87% of 124 endemic plant species studied in the region; upper range extensions of species have resulted in increased species richness in the upper alpine zone, compared to the 19^th century. We recorded a shift of 23–998 m in species’ upper elevation limit and a mean upward displacement rate of 27.53±22.04 m/decade in the present study. We infer that the present-day plant assemblages and community structure in the Himalaya is substantially different from the last century and is, therefore, in a state of flux under the impact of warming. The continued trend of warming is likely to result in ongoing elevational range contractions and eventually, species extinctions, particularly at mountaintops.     

Control of ornithine decarboxylase activity in jute seeds by antizyme

The control of ornithine decarboxylase activity by antizyme was studied during early germination of jute seeds(Corchorus olitorius). When 2 mM of putrescine and spermidine were applied to the germinating medium, the enzyme activity was markedly inhibited (1.7-fold) during 16 h imbibition. This inhibition could be attributed to the formation of an inhibitory protein termed antizyme. The antizyme was partially purified from jute and barley seedlings. The activity of jute ornithine decarboxylase antizyme was weaker than that of barley.     

Mixed bilayer containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine: lipid complexation, ion binding, and electrostatics

Two mixed bilayers containing dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine at a ratio of 5:1 are simulated in NaCl electrolyte solutions of different concentration using the molecular dynamics technique. Direct NH.O and CH.O hydrogen bonding between lipids was observed to serve as the basis of interlipid complexation. It is deduced from our results and previous studies that dipalmitoylphosphatidylcholine alone is less likely to form interlipid complexes than in the presence of bound ions or other bilayer "impurities" such as dipalmitoylphosphatidylserine. The binding of counterions is observed and quantitated. Based upon the calculated ion binding constants, the Gouy-Chapman surface potential (theta) is calculated. In addition we calculated the electrostatic potential profile (Phi) by twice integrating the system charge distribution. A large discrepancy between and the value of Phi at the membrane surface is observed. However, at "larger" distance from the bilayer surface, a qualitative similarity in the z-profiles of Phi and psi(GC) is seen. The discrepancy between the two potential profiles near the bilayer surface is attributed to the discrete and nonbulk-like nature of water in the interfacial region and to the complex geometry of this region.