A computational docking study for prediction of binding mode of diospyrin and derivatives: Inhibitors of human and leishmanial DNA topoisomerase-I

A computational approach was utilized to study the relative binding modes of diospyrin (bisnaphthoquinonoid) with the crystal structure of human DNA-TopoI and the recently reported Leishmania donavani DNA-TopoI. Additionally, the binding site interactions of amino derivatives of diospyrin with human TopoI were studied extensively. Based on the docking results, binding modes of diospyrin with the human and leishmanial TopoI catalytic core were predicted. The parallel use of two efficient and predictive docking programs, GOLD and Ligandfit, allowed mutual validation of the predicted binding poses. A reasonably good correlation coefficient between the calculated docking scores and the experimentally determined cytotoxicity helped in validating the docking method. Furthermore, a structure-based pharmacophore model was developed for L. donavani DNA-TopoI inhibition which helped in elucidating the topological and spatial requirements of the ligand-receptor interactions. This study provides an understanding of the structural basis of ligand binding to the topoisomerase receptor, which may be used for the structure-based design of potent and novel ligands for anticancer and antileishmanial therapy. To our knowledge, this is the first report of a binding mode exploration study for diospyrin and its derivatives as inhibitors of the leishmanial and human TopoI enzymes.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

The immunomodulatory Pseudomonas aeruginosa signalling molecule N-(3-oxododecanoyl)-L-homoserine lactone enters mammalian cells in an unregulated fashion

The Pseudomonas aeruginosa quorum-sensing signal molecule N-3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been reported to affect the function of a wide range of mammalian cell types, including cells of the immune system. In T cells, it has been reported to inhibit the production of most cytokines, and it has been reported to inhibit the function of antigen-presenting cells. The intracellular target of OdDHL in these cells remains to be identified, although the lipophilic nature of the molecule suggested that the target could be membrane associated. We explored the association of radiolabelled OdDHL with the membrane and cytoplasm of Jurkat T-cell lines and of primary murine T cells and dendritic cells. We found that not only did 3H-OdDHL enter the cytoplasm of Jurkat cells without disproportionate association with the cell membrane, it also reached maximum levels in the cytoplasm very quickly, and that the intracellular concentration was proportional to the extracellular concentration. Similar results were obtained when 3H-OdDHL was incubated with primary murine T cells or cultured dendritic cells. In addition, we show that the cellular distribution of OdDHL does not significantly alter after stimulation of Jurkat cells or primary murine CD4 T cells with immobilized anti-CD3, with little activity being associated with nuclear fractions. Together, these data strongly suggest that OdDHL enters mammalian cells by passive mechanisms, and that it does not preferentially associate with the membrane or nucleus upon T-cell receptor ligation.     

RAL-1 controls multivesicular body biogenesis and exosome secretion

Exosomes are secreted vesicles arising from the fusion of multivesicular bodies (MVBs) with the plasma membrane. Despite their importance in various processes, the molecular mechanisms controlling their formation and release remain unclear. Using nematodes and mammary tumor cells, we show that Ral GTPases are involved in exosome biogenesis. In Caenorhabditis elegans, RAL-1 localizes at the surface of secretory MVBs. A quantitative electron microscopy analysis of RAL-1–deficient animals revealed that RAL-1 is involved in both MVB formation and their fusion with the plasma membrane. These functions do not involve the exocyst complex, a common Ral guanosine triphosphatase (GTPase) effector. Furthermore, we show that the target membrane SNARE protein SYX-5 colocalizes with a constitutively active form of RAL-1 at the plasma membrane, and MVBs accumulate under the plasma membrane when SYX-5 is absent. In mammals, RalA and RalB are both required for the secretion of exosome-like vesicles in cultured cells. Therefore, Ral GTPases represent new regulators of MVB formation and exosome release.     

Deploying QTL-seq for rapid delineation of a potential candidate gene underlying major trait-associated QTL in chickpea

A rapid high-resolution genome-wide strategy for molecular mapping of major QTL(s)/gene(s) regulating important agronomic traits is vital for in-depth dissection of complex quantitative traits and genetic enhancement in chickpea. The present study for the first time employed a NGS-based whole-genome QTL-seq strategy to identify one major genomic region harbouring a robust 100-seed weight QTL using an intra-specific 221 chickpea mapping population (desi cv. ICC 7184 × desi cv. ICC 15061). The QTL-seq-derived major SW QTL (CaqSW1.1) was further validated by single-nucleotide polymorphism (SNP) and simple sequence repeat (SSR) marker-based traditional QTL mapping (47.6% R² at higher LOD >19). This reflects the reliability and efficacy of QTL-seq as a strategy for rapid genome-wide scanning and fine mapping of major trait regulatory QTLs in chickpea. The use of QTL-seq and classical QTL mapping in combination narrowed down the 1.37 Mb (comprising 177 genes) major SW QTL (CaqSW1.1) region into a 35 kb genomic interval on desi chickpea chromosome 1 containing six genes. One coding SNP (G/A)-carrying constitutive photomorphogenic9 (COP9) signalosome complex subunit 8 (CSN8) gene of these exhibited seed-specific expression, including pronounced differential up-/down-regulation in low and high seed weight mapping parents and homozygous individuals during seed development. The coding SNP mined in this potential seed weight-governing candidate CSN8 gene was found to be present exclusively in all cultivated species/genotypes, but not in any wild species/genotypes of primary, secondary and tertiary gene pools. This indicates the effect of strong artificial and/or natural selection pressure on target SW locus during chickpea domestication. The proposed QTL-seq-driven integrated genome-wide strategy has potential to delineate major candidate gene(s) harbouring a robust trait regulatory QTL rapidly with optimal use of resources. This will further assist us to extrapolate the molecular mechanism underlying complex quantitative traits at a genome-wide scale leading to fast-paced marker-assisted genetic improvement in diverse crop plants, including chickpea.     

Evidence for cytochrome P450 2B1/2B2 isoenzymes in freshly prepared peripheral blood lymphocytes

Cytochrome P450 2B1 and 2B2, the major hepatic drug metabolizing enzymes belonging to CYP2 family and associated constitutive androstane receptor (CAR) were found to be expressed in peripheral blood lymphocytes (PBL) isolated from rats. As observed in liver, pretreatment of phenobarbital (PB) or phenytoin were found to increase the expression of CYP2B1, CYP2B2 and associated enzyme activity in PBL. Like in liver, blood lymphocyte CYP2B1/2B2 catalyzed the activity of 7-pentoxyresorufin O-dealkylase (PROD). The present data, demonstrating similarities in the regulation of blood lymphocyte CYP2B-isoenzymes with the liver enzymes, suggests that blood lymphocyte CYP2B-isoenzymes could be used as a biomarker to monitor tissue levels.     

Identification of haptoglobin and apolipoprotein A-I as biomarkers for high altitude pulmonary edema

We have investigated the plasma proteome using 2D gel electrophoresis and matrix-assisted laser desorption/ionization tandem time of flight from patients with high altitude pulmonary edema (HAPE). A complete proteomic analysis was performed on 20 patients with HAPE and ten healthy sea level controls. In total, we have identified 25 protein spots in human plasma and found that 14 of them showed altered changes in HAPE patients, which mainly were acute phase proteins (APPs), compliment components, and apolipoproteins among others. Among the APPs, haptoglobin α2 chain, haptoglobin β chain, transthyretin, and plasma retinol binding precursor showed overexpression in HAPE patients as compared to controls. To validate the result of proteomic analysis, two proteins were selected for enzyme-linked immunosorbent assay and Western blotting analysis. Our data conclusively shows that two proteins, haptoglobin and apolipoprotein A-I are upregulated in plasma of HAPE patients. These proteins may provide a fast and effective control of inflammatory damage until the subsequent mechanisms can begin to operate. Taken together, our findings further support the hypothesis that inflammatory response system is linked to the pathophysiology of HAPE.