Metabolic profiles and phylogenetic diversity of microbial communities from chlorinated pesticides contaminated sites of different geographical habitats of India

Aims: To study the microbial communities in three sites contaminated with chlorinated pesticides and evaluation of dehydrodechlorinase (linA) gene variants involved in gamma‐hexachlorocyclohexane (γ‐HCH, lindane) degradation. Methods and Results: Using a culture‐independent method, 16S rRNA genes were amplified from microbial communities occurring in contaminated soils. From 375 clone libraries analysed, 55 different restriction fragment length polymorphism phylotypes were obtained. Dehydrodechlorinase (linA) gene, which initiates the γ‐HCH degradation, was directly amplified by PCR from the DNA extracted from soils. Deduced amino acid sequences of eight variant genotypes of linA showed few amino acid changes. All the variants of linA had mutations of F151L and S154T, and one of the genotype carried 12 amino acid changes when compared to a linA of Sphingomonas sp. reported from the same soil. Conclusions: The microbial communities displayed complex and diverse groups similar to bacteria involved in biodegradation. The presence of biodegradative genes like linA indicates the presence of communities with capacity to biodegrade the persistent pesticide HCH. Significance and Impact of the Study: This study provides insights to evaluate the presence of catabolic genes and assessing the bioremediation potential of the industrial soils contaminated by chlorinated pesticides.     

Multiblock reducible copolypeptides containing histidine-rich and nuclear localization sequences for gene delivery

Polyplexes sensitive to redox potential gradients represent promising gene delivery vectors. High molecular weight polypeptides containing disulfide bonds in the backbone were synthesized by an oxidative copolymerization of a histidine-rich peptide (HRP) and a nuclear localization sequence (NLS) peptide. The synthetic approach allowed an easy synthesis of reducible copolypeptides (rCPP) with different relative contents of the HRP and NLS sequences. Cytotoxicity and transfection activity of rCPP-based DNA polyplexes were evaluated in vitro. In comparison with control polyethylenimine (PEI), only minimum toxic effects of rCPPs were observed on the metabolic activity and membrane integrity of human endothelial cells. These findings are predominantly ascribed to favorable structural features like lower charge density and higher chain rigidity of the rCPPs compared to PEI and also to a reductive intracellular and plasma membrane degradation. Transfection activity in all tested cell lines increased with increasing content of the HRP sequence in the rCPPs, while no clearly measurable effect of the NLS sequence was found.     

Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis

Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth, angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate, a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous, intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 +/- 2 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase. Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics. Specifically, ADI with either 12 kDa or 20 kDa PEG attachments on 33% of the primary amines retained about 60% or 48% of enzyme activity, respectively; the Km and pH profiles were nearly unchanged; IC50 values were diminished by less than 30%; while stability studies demonstrated full retention of activity at 4 degrees C for 5 months. A comparison of the enzymatic properties of a second ADI from Pseudomonas putida illustrated the superior characteristics of the M. arthritidis ADI enzyme.     

Isolation of pyrene degrading <Emphasis Type="Italic">Achromobacter xylooxidans</Emphasis> and characterization of metabolic product

Polycyclic aromatic hydrocarbons (PAHs) are common ubiquitous pollutants existing in nature with high recalcitrance and toxicity. In this study a bacterium capable of aerobic degradation of high molecular weight PAHs (with special reference to pyrene) was isolated by selective enrichment culture technique from oil refinery effluent sludge. The isolate was characterized as Achromobacter xylooxidans by 16S rRNA gene sequence analysis technique. For the first time it is hereby reported a bacterium capable of effectively degrading pyrene (up to 80%), as evident by reverse phase high performance liquid chromatographic analysis (RP-HPLC). After incubation of Achromobacter xylooxidans in minimal salt medium (MSM) containing pyrene, at concentration of 200 mg/L, as sole source of carbon and energy, there was decrease in pyrene concentration concomitant with increase in bacterial cell protein concentration. RP-HPLC analysis revealed that pyrene was degraded into three metabolites viz. I, II and III. The RP-HPLC eluent fraction were collected from 2.5 to 32.0 min by repeated injection of degraded sample, concentrated and analyzed on gas chromatography mass spectroscopy (GC-MS) for metabolite identification. The fraction shows 1-hydroxypyrene, 1-hydroxy-6-methoxypyrene and 1,6dimethoxypyrene as metabolic product of pyrene degradation, on the basis of their m/z values. On contrary to the reported PAH degradation with reference to pyrene by different isolates till date; the efficient degradation, as evident by RP-HPLC, by this isolate holds a promising potential for planning of bioremediation strategies of contaminated sites.     

Gomphia barberi,a new species of Ochnaceae from Tirunelveli Hills,Western Ghats of India

Gomphia barberi sp. nov. (Ochnaceae) is described with an illustration from the Tirunelveli Hills of southern Western Ghats of Tamil Nadu, India.     

Genome analysis of alginate synthesizing Pseudomonas aeruginosa strain SW1 isolated from degraded seaweeds

Antonie van Leeuwenhoek - Pseudomonas aeruginosa strain SW1 is an aerobic, motile, Gram-negative, and rod-shaped bacterium isolated from degraded seaweeds. Based on the 16S rRNA gene sequence and...     

Inhibition of complement alternative pathway suppresses experimental autoimmune anterior uveitis by modulating T cell responses

The objective of the current study was to delineate the pathway of complement activation that is crucial for the induction of experimental autoimmune anterior uveitis (EAAU). We studied the development of EAAU in melanin-associated antigen (MAA)-sensitized Lewis rats treated with antibody against C4 or factor B. Control animals received isotype IgG control. Antibody against C4 had no effect on EAAU, and all of the animals developed EAAU similar to those injected with control IgG. In contrast, EAAU was completely inhibited in all MAA-sensitized Lewis rats injected with factor B antibody. Treatment with anti-factor B antibody resulted in suppression of ocular complement activation. Adoptive transfer of T lymphocytes harvested from draining lymph nodes of donor animals treated with anti-factor B did not transfer EAAU to naïve syngenic rats. Anti-factor B antibody inhibited the ability of MAA-specific CD4⁺ T cells to proliferate (in vitro) in response to MAA in a dose-dependent manner. Level of TNF-α and IFN-γ decreased in the presence of anti-factor B. Collectively, our results provide the novel finding that complement activation via the alternative pathway contributes to intraocular inflammation in EAAU, and anti-factor B-mediated inhibition of EAAU is due to diminished antigen-specific CD4⁺ T cell responses to MAA. Our findings explain the interactions between the complement system and T cells that are critical for the induction of EAAU and may lead to the development of therapy for idiopathic anterior uveitis based on selective blockade of the alternative pathway.     

Presence of 46 kDa Gelatinase on the Outer Membrane of <Emphasis Type="Italic">Leptospira</Emphasis>

Leptospira infection involves the adhesion of the bacteria followed by invasion of the host crossing the extracellular matrix barrier. In an effort to understand the molecular mechanism of this process, the possibility of occurrence of matrix degrading enzymes from Leptospira was investigated. Zymographic analysis showed that the outer membrane of Leptospires contains a gelatinase of average molecular size of 46 kDa. The gelatinase exhibited maximum activity at neutral pH and was inhibited by metal chelators such as EGTA, EDTA, and Orthophenanthroline and was activated by calcium, magnesium, zinc, and copper, suggesting that it is a membrane-associated neutral matrix metalloproteinase. Analysis of the production of the enzyme by various serovars showed that the pathogenic serovars expressed significant amount of this enzyme while nonpathogenic forms either did not express or showed only very low activity, suggesting that this enzyme may be associated with pathogenesis of leptospirosis.