Acquisition of ionic copper by the bacterial outer membrane protein OprC through a novel binding site

Copper, while toxic in excess, is an essential micronutrient in all kingdoms of life due to its essential role in the structure and function of many proteins. Proteins mediating ionic copper import have been characterised in detail for eukaryotes, but much less so for prokaryotes. In particular, it is still unclear whether and how gram-negative bacteria acquire ionic copper. Here, we show that Pseudomonas aeruginosa OprC is an outer membrane, TonB-dependent transporter that is conserved in many Proteobacteria and which mediates acquisition of both reduced and oxidised ionic copper via an unprecedented CxxxM-HxM metal binding site. Crystal structures of wild-type and mutant OprC variants with silver and copper suggest that acquisition of Cu(I) occurs via a surface-exposed “methionine track” leading towards the principal metal binding site. Together with whole-cell copper quantitation and quantitative proteomics in a murine lung infection model, our data identify OprC as an abundant component of bacterial copper biology that may enable copper acquisition under a wide range of conditions.

How do Gram-negative bacteria acquire copper? This study shows that the outer membrane protein OprC from Pseudomonas aeruginosa is abundant during infection and mediates highly selective acquisition of both copper redox states via an extracellular "methionine track" and an unprecedented near-irreversible binding site.     

Quercetin derivatives as non-nucleoside inhibitors for dengue polymerase: molecular docking,molecular dynamics simulation,and binding free energy calculation

Dengue is an important public health problem in tropical and subtropical regions of the world. Neither vaccine nor an antiviral medication is available to treat dengue. This insists the need of drug discovery for dengue. In order to find a potent lead molecule, RNA-dependent RNA polymerase which is essential for dengue viral replication is chosen as a drug target. As Quercetin showed antiviral activity against several viruses, quercetin derivatives developed by combinatorial library synthesis and mined from PubChem databases were screened for a potent anti-dengue viral agent. Our study predicted Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a dengue polymerase inhibitor. The results were validated by molecular dynamics simulation studies which reveal water bridges and hydrogen bonds as major contributors for the stability of the polymerase-lead complex. Interactions formed by this compound with residues Trp795, Arg792 and Glu351 are found to be essential for the stability of the polymerase-lead complex. Our study demonstrates Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a potent non-nucleoside inhibitor for dengue polymerase.     

Isolation and partial characterization of collagen from outer skin of Sepia pharaonis (Ehrenberg, 1831) from Puducherry coast

Type I collagen from outer skin of Sepia pharaonis was extracted and partially characterized. Yield of Acid Soluble Collagen (ASC) and Pepsin Soluble Collagen (PSC) were calculated as 1.66% and 3.93% and the total protein content of ASC and PSC were found as 18.4% and 48.6%. FT-IR spectrum of ASC and PSC recorded 12 and 14 peaks, respectively. ¹H NMR spectrum of ASC showed singlets at 1.23 ppm, 3.1 ppm, 3.55 ppm and 3.7 ppm and PSC at 1.23 ppm and 2.08 ppm. The molecular weight for ASC was calculated as 102 kDa and for PSC as 110, 108 and 102 kDa through SDS-PAGE. Differential Scanning Calorimetry (DSC) results supported that PSC withstand high thermal stability (82.85 °C) than ASC (73.13 °C). Higher denaturation temperature with high molecular weight well support the property of type I collagen from skin of S. pharaonis and it could be used as another potent source for the extraction of collagen.     

Critical role of bicarbonate and bicarbonate transporters in cardiac function

Bicarbonate is one of the major anions in mammalian tissues and extracellular fluids. Along with accompanying H+, HCO3- is generated from CO2 and H2 O, either spontaneously or via the catalytic activity of carbonic anhydrase. It serves as a component of the major buffer system, thereby playing a critical role in pH homeostasis. Bicarbonate can also be utilized by a variety of ion transporters, often working in coupled systems, to transport other ions and organic substrates across cell membranes. The functions of HCO3- and HCO3--transporters in epithelial tissues have been studied extensively, but their functions in heart are less well understood. Here we review studies of the identities and physiological functions of Cl-/HCO3- exchangers and Na+/HCO3-cotransporters of the SLC4 A and SLC26 A families in heart. We also present RNA Seq analysis of their cardiac mRNA expression levels. These studies indicate that slc4a3(AE3) is the major Cl-/HCO3- exchanger and plays a protective role in heart failure, and that Slc4a4(NBCe1) is the major Na+/HCO3- cotransporter and affects action potential duration. In addition, previous studies show that HCO3- has a positive inotropic effect in the perfused heart that is largely independent of effects on intracellular Ca2+. The importance of HCO3- in the regulation of contractility is supported by experiments showing that isolated cardiomyocytes exhibit sharply enhanced contractility, with no change in Ca2+ transients, when switched from Hepes-buffered to HCO3-- buffered solutions. These studies demonstrate that HCO3- and HCO3--handling proteins play important roles in the regulation of cardiac function.     

Description of a novel indole-oxidizing bacterium Pseudomonas indoloxydans sp. nov., isolated from a pesticide-contaminated site

A Gram-negative, deep brown-pigmented Gammaproteobacteria, strain IPL-1(T), capable of oxidizing indole was isolated from a lindane-contaminated site and subjected to a polyphasic taxonomic study. Most of the physiological and biochemical properties, major fatty acids (C(18:1)omega7c, C(16:1)omega7c/iso C(15:0) 2OH and C(16:0)), estimated DNA G+C content (67.2mol%) and 16S rRNA gene sequence analysis showed that strain IPL-1(T) belonged to the genus Pseudomonas. Strain IPL-1(T) exhibited highest 16S rRNA gene sequence similarity with Pseudomonas pseudoalcaligenes (99.0%), followed by Pseudomonas alcaliphila (98.7%), Pseudomonas oleovorans (98.3%), Pseudomonas nitroreducens (98.0%), Pseudomonas mendocina (97.6%) and Pseudomonas stutzeri (97.4%). However, the DNA-DNA relatedness values between strain IPL-1(T) and the closely related taxa were between 22% and 61%. On the basis of differential phenotypic characteristics and genotypic distinctiveness, strain IPL-1(T) should be classified within the genus Pseudomonas as a novel species, for which the name Pseudomonas indoloxydans is proposed. The type strain is IPL-1(T) (=MTCC 8062(T)=JCM 14246(T)).     

Towards genetic improvement of commercially important microalga Haematococcus pluvialis for biotech applications

Haematococcus pluvialis is a unicellular green alga which produces a ketocarotenoid, astaxanthin which has pharmaceutical and nutraceutical applications owing to its high antioxidant activity. Biotechnological approaches such as genetic transformation methods (Agrobacterium-mediated) and cloning strategies, are essential to improve/regulate this ketocarotenoid in Haematococcus. For this studies are necessary to improve Haematococcus through biotechnological means. In this connection, a suitable cocultivation medium for Haematococcus and Agrobacterium tumefaciens was standardized and different antibiotics were screened. Among the cocultivation media viz Z₈, Bold’s Basal Medium, Tris Acetate Phosphate Z₈ with 0.5% mannitol and BBM and Z₈ with half strength LB TAP medium was found suitable for growth of both the alga Haematococcus and Agrobacterium. For the different antibiotics screened to identify the sensitivity of algae, hygromycin at more than 2 mg L⁻¹ showed lethal effect which can be used as a selectable marker gene in genetic transformation, whereas cefotaxime and augmentin showed no effect up to 2,000 mg L⁻¹. The growth of algae was higher in solid selection medium when compared to the liquid selection medium.     

Simple and rapid methods to evaluate methane potential and biomass yield for a range of mixed solid wastes

This paper describes rapid techniques to evaluate the methane potential and biomass yield of solid wastes. A number of solid wastes were mixed to provide a range of C:N ratios. Empirical formulae were calculated for each waste based on the results of chemical analysis and these formulae were used to estimate the COD equivalent and stoichiometric methane potential (SMP). The actual COD and biochemical methane potential (BMP) were determined experimentally for each waste and for both parameters there was a good agreement between the empirical and experimental values. The potential of adenosine triphosphate (ATP) to act as an indicator of biomass yield (mg VSS mg(-1) COD removed) was determined during the anaerobic digestion process. The biomass yield determined from ATP analysis was in the range 0.01-0.25mg VSS mg(-1) COD removed which corroborated well with previously reported studies. Empirical formula based SMP together with ATP measurement were shown to provide rapid methods to replace or augment the traditional BMP and VSS measurements and are useful for evaluating the bioenergy and biomass potential of solid wastes for anaerobic digestion.     

Effect of H4R antagonist N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamides and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole on histamine and 4-methylhistamine-induced mast cell response

Context: The histamine plays a decisive role in acute and chronic inflammatory responses and is regulated through its four types of distinct receptors designated from H1 to H4. Recently histamine 4 receptor (H4R) antagonists have been reported to possess various pharmacological effects against various allergic diseases.

Objective: To investigate the inhibitory effect of N-(2-aminoethyl)-5-chloro-1H-indol-2-carboxamide (Compound A) and 5-chloro-2-(piperazin-1-ylmethyl)-1H-benzimidazole (Compound L) on H4R-mediated calcium mobilization, cytokine IL-13 production, ERK1/2, Akt and NF-κB activation in human mastocytoma cells-1 (HMC-1).

Materials and methods: Compounds A and L were synthesized chemically and their inhibitory effect on intracellular calcium release was analyzed by Fluo-4 calcium assay, cytokine measurement through ELISA and activation of signaling molecules by western blot.

Results: Pre-treatment with compounds A and L significantly reduced the H4R-mediated intracellular calcium release. Histamine and 4-methylhistamine (4-MH) induced Th2 cytokine IL-13 production in HMC-1 cells, was inhibited by compound A (77.61%, 74.25% at 1?μM concentration) and compound L (79.63%, 81.70% at 1?μM concentration). Furthermore, histamine induced the phosphorylation of ERK1/2, Akt and NF-κB was suppressed by compounds A and L at varying levels, ERK1/2 (88%, 86%), Akt (88%, 89%) and NF-κB (89%, 87%) in HMC-1 cells.

Discussion and conclusions: Taken together these data demonstrate that compound A and compound L may block H4R-mediated downstream signaling events.     

Genome-wide characterization and stress-responsive expression profiling of <Emphasis Type="Italic">MCM</Emphasis> genes in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Brassica rapa</Emphasis>

The Minichromosome maintenance protein [MCM (2-7)] complex is associated with helicase activity for replication fork formation during DNA replication. We identified and characterized each 12 putative MCM genes from Brassica oleracea and Brassica rapa. MCM genes were classified into nine groups according to their evolutionary relationships. A high number of syntenic regions were present on chromosomes C03 and A03 in B. oleracea and B. rapa, respectively, compared to the other chromosomes. Expression analysis showed that most of the MCM(2-7) helicase-subunit genes and their coregulating MCM genes were upregulated during hydroxyurea (HU) induced stress in B. oleracea. In B. rapa, MCM(2-7) helicase genes BrMCM2_2, BrMCM7_1, BrMCM7_2 and their co-regulating genes were upregulated during replication stress. During cold stress, BoMCM6 in B. oleracea and BrMCM5 in B. rapa were remarkably upregulated. During salt stress, BoMCM6_2, BoMCM7_1, BoMCM8, BoMCM9, and BoMCM10 were markedly upregulated in B. oleracea. Hence, our study identified the candidate MCM family genes those possess abiotic stress-responsive behavior and DNA replication stress tolerance. As the first genome-wide analysis of MCM genes in B. oleracea and B. rapa, this work provides a foundation to develop stress responsive plants. Further functional and molecular studies on MCM genes will be helpful to enhance stress tolerance in plants.