Novel cellulases from an extremophilic filamentous fungi <Emphasis Type="Italic">Penicillium citrinum</Emphasis>: production and characterization

The enzymatic hydrolysis of cellulose has potential economical and environment-friendly applications. Therefore, discovery of new extremophilic cellulases is essential to meet the requirements of industry. Penicillium citrinum (MTCC 6489) that was previously isolated from soil in our laboratory, produced alkali tolerant and thermostable cellulases. Endoglucanase and filter paper activity hydrolase (FPAse) production of P. citrinum were studied using wheat bran substrate in solid state and submerged culture. Zymogram analysis of endoglucanase revealed the presence of two isoforms differing in molecular weight. One of them was 90 kDa and other one was 38 kDa. Partially purified endoglucanase showed two different peaks at pH 5.5 and 8.0, respectively, in its pH optima curve. But FPase showed only one peak (at pH 6.5) in its pH optima curve. Cellulase of P. citrinum is thermostable in nature. The present work reports for the first time, the alkali stable cellulase from alkali tolerant fungus Penicillium citrinum. Thermostable endoglucanase from P. citrinum may have potential effectiveness as additives to laundry detergents.     

An erythrocyte vesicle protein exported by the malaria parasite promotes tubovesicular lipid import from the host cell surface

Plasmodium falciparum is the protozoan parasite that causes the most virulent of human malarias. The blood stage parasites export several hundred proteins into their host erythrocyte that underlie modifications linked to major pathologies of the disease and parasite survival in the blood. Unfortunately, most are 'hypothetical' proteins of unknown function, and those that are essential for parasitization of the erythrocyte cannot be 'knocked out'. Here, we combined bioinformatics and genome-wide expression analyses with a new series of transgenic and cellular assays to show for the first time in malaria parasites that microarray read out from a chemical perturbation can have predictive value. We thereby identified and characterized an exported P. falciparum protein resident in a new vesicular compartment induced by the parasite in the erythrocyte. This protein, named Erythrocyte Vesicle Protein 1 (EVP1), shows novel dynamics of distribution in the parasite and intraerythrocytic membranes. Evidence is presented that its expression results in a change in TVN-mediated lipid import at the host membrane and that it is required for intracellular parasite growth, but not invasion. This exported protein appears to be needed for the maintenance of an essential tubovesicular nutrient import pathway induced by the pathogen in the host cell. Our approach may be generalized to the analysis of hundreds of 'hypothetical' P. falciparum proteins to understand their role in parasite entry and/or growth in erythrocytes as well as phenotypic contributions to either antigen export or tubovesicular import. By functionally validating these unknowns, one may identify new targets in host-microbial interactions for prophylaxis against this major human pathogen.     

Bayesian biomarker identification based on marker-expression proteomics data

We are studying variable selection in multiple regression models in which molecular markers and/or gene-expression measurements as well as intensity measurements from protein spectra serve as predictors for the outcome variable (i.e., trait or disease state). Finding genetic biomarkers and searching genetic–epidemiological factors can be formulated as a statistical problem of variable selection, in which, from a large set of candidates, a small number of trait-associated predictors are identified. We illustrate our approach by analyzing the data available for chronic fatigue syndrome (CFS). CFS is a complex disease from several aspects, e.g., it is difficult to diagnose and difficult to quantify. To identify biomarkers we used microarray data and SELDI-TOF-based proteomics data. We also analyzed genetic marker information for a large number of SNPs for an overlapping set of individuals. The objectives of the analyses were to identify markers specific to fatigue that are also possibly exclusive to CFS. The use of such models can be motivated, for example, by the search for new biomarkers for the diagnosis and prognosis of cancer and measures of response to therapy. Generally, for this we use Bayesian hierarchical modeling and Markov Chain Monte Carlo computation.     

Surface morphology of immunocompetent cells isolated from spleen of Bufo himalayanus (Günther)

Immunocompetent cells were isolated from spleen of B. himalayanus and studied surface morphology of the three different cell types--(i) plastic adherent; (ii) nylon wool adherent; and (iii) nylon wool non-adherent cells. As revealed by scanning electron microscopy, they resembled the macrophages, B and T cells, respectively. Presence of such cell types indicated that Bufo himalayanus possessed a well-organized immune system. Further work is needed to characterize the functional efficacy of these immunocompetent cells found in B. himalayanus.     

Discovery and structure–activity relationships of a novel series of benzopyran-based KATP openers for urge urinary incontinence

A novel series of benzopyran derivatives were synthesized and evaluated as K_ATP channel openers. Structure–activity relationships were investigated around 4-position of the benzopyran nucleus. Optimization of 4-substituent with some heterocyclic rings led to compound 13b bearing a benzo[d]isoxazol-3-one moiety as a potent and selective K_ATP channel opener in vitro. In two anesthetized rat models of myogenic bladder overactivity, compound 13b was found to inhibit spontaneous bladder contractions.     

In vivo protein tyrosine nitration in S. cerevisiae: Identification of tyrosine-nitrated proteins in mitochondria

Protein tyrosine nitration (PTN) is a selective post-translational modification often associated with pathophysiological conditions. Although yeast cells lack of mammalian nitric oxide synthase (NOS) orthologues, still it has been shown that they are capable of producing nitric oxide (NO). Our studies showed that NO or reactive nitrogen species (RNS) produced in flavohemoglobin mutant (Δyhb1) strain along with the wild type strain (Y190) of Saccharomyces cerevisiae can be visualized using specific probe 4,5-diaminofluorescein diacetate (DAF-2DA). Δyhb1 strain of S. cerevisiae showed bright fluorescence under confocal microscope that proves NO or RNS accumulation is more in absence of flavohemoglobin. We further investigated PTN profile of both cytosol and mitochondria of Y190 and Δyhb1 cells of S. cerevisiae using two-dimensional (2D) gel electrophoresis followed by western blot analysis. Surprisingly, we observed many immunopositive spots both in cytosol and in mitochondria from Y190 and Δyhb1 using monoclonal anti-3-nitrotyrosine antibody indicating a basal level of NO or nitrite or peroxynitrite is produced in yeast system. To identify proteins nitrated in vivo we analyzed mitochondrial proteins from Y190 strains of S. cerevisiae. Among the eight identified proteins, two target mitochondrial proteins are aconitase and isocitrate dehydrogenase that are involved directly in the citric acid cycle. This investigation is the first comprehensive study to identify mitochondrial proteins nitrated in vivo.     

Biogenic synthesis of <Emphasis Type="Italic">Marsilea quadrifolia</Emphasis> gold nanoparticles: a study of improved glucose utilization efficiency on 3T3-L1 adipocytes

This study aims mainly to provide an insight and understanding of the effect of glucose utilization efficiency of biogenic gold nanoparticles (GNPs) synthesized through the mediation of Marsilea quadrifolia (M. quadrifolia) methanol extract on 3T3-L1 adipocytes. The biosynthesized GNPs were characterized by UV visible spectrophotometry and FTIR. Simultaneously, the nature, stability, and morphological characteristics were analyzed by XRD, TG-DTA, SEM-EDS, HRTEM, and SAED. The results of characterization studies were used to assess the properties of GNPs. The in vitro cytotoxicity screening indicates that 100 μM of biogenic GNPs were displayed 71.23 ± 1.56% of cellular viability in 3T3-L1 adipocyte cells. Subsequently, increased glucose utilization of biosynthesized GNPs based on a dose-dependent manner on 3T3-L1 has also been demonstrated. The effect of GNPs (30 μg) on glucose uptake was higher than that of insulin and metformin. Moreover, the observed results clearly highlight that the biogenic GNPs have higher efficiency of glucose utilization and cellular viability in 3T3-L1 adipocytes with lower toxicity.