Rapid purification of tRNA(Lys) from rat liver

Fast protein liquid chromatography (FPLC) system using Mono Q (HR 5/5) anion-exchange column chromatography followed by highly cross-linked urea-polyacrylamide gel electrophoresis (urea-PAGE) was used for the purification of lysine-specific tRNA (tRNA(Lys)) from rat liver. Crude tRNA from rat liver was fractionated with a linear gradient of NaCl (0.3-0.8 M) in triethanolamine-HCl buffer, pH 4.5, and the activity of tRNA(Lys) was found to elute between 0.51 and 0.57 M NaCl. Using this concentration range of NaCl, tRNA(Lys) was refractionated on the same column with a shallow gradient, where a single peak of tRNA(Lys) activity was obtained. tRNA(Lys)-rich fractions recovered from the second run were electrophoretically separated on 16% polyacrylamide-7 M urea gel into one major band and three minor bands. The major band showed a specific activity of 997 pmols/A260 U for tRNALys with a 43-fold purification and approximately 17% recovery. The minor bands displayed negligible or no activity for lysine. tRNA(Lys) obtained by this method was found to be homogeneous by competitive aminoacylation. The advantages of FPLC followed by urea-PAGE in the purification of an amino acid-specific tRNA over conventional column chromatography are discussed.

Utilizing an ionic liquid for synthesizing a soft matter polymer ``gel'' electrolyte for high rate capability lithium-ion batteries

A cross-linked polymer ``gel'' electrolyte obtained from free radical polymerization of a vinyl monomer (acrylonitrile; AN) in a room temperature ionic liquid electrolyte (N,N-methyl butyl pyrrolidinium-bis (trifluoromethanesulphonyl)imide-lithium bis(trifluoromethanesulphonyl) imide;LiTFSI-[Py(1,4)-TFSI]) for application in high rate capability rechargeable lithium-ion batteries is discussed here. This is a novel alternative compared to the often employed approach of using a molecular liquid as the medium for performing the polymerization reaction. The polymer ``gel'' electrolytes (AN:Py(1,4)-TFSI = 0.16-0.18, w/w) showed remarkable compliable mechanical strength and higher thermal stability compared to LiTFSI-[Py(1,4)-TFSI]. Despite two orders increase in magnitude of viscosity of polymer ``gels'', the room temperature ionic conductivity of the ``gels'' (1.1 x 10(-3)-1.7 x 10(-3) Omega(-1) cm(-1)) were nearly identical to that of the ionic liquid (1.8 x 10(-3) Omega(-1) cm(-1)). The present ``gel'' electrolytes did not exhibit any ageing effects on ionic conductivity similar to the conventional polymer gel electrolytes (e.g. high molecular weight polymer + salt + high dielectric constant molecular solvent). The disorder (ionic liquid) to a relative order (cross-linked polymer electrolyte) transformation does not at all influence the concentration of conducting species. The polymer framework is still able to provide efficient pathways for fast ion transport. Unlike the ionic liquid which is impossible to assemble without a conventional separator in a cell, the polymer ``gel'' electrolyte could be conveniently assembled without a separator in a Li vertical bar lithium iron phosphate (LiFePO(4)) cell. Compared to the ionic liquid, the ``gel'' electrolyte showed exceptional cyclability and rate capability (current density: 35-760 mA g(-1) with LiFePO(4) electronically wired with carbon (amorphous or multiwalled nanotube [MWCNT]).

Chitosan-polyvinyl alcohol-sulfonated polyethersulfone mixed-matrix membranes as methanol-barrier electrolytes for DMFCs

Chitosan (CS)-polyvinyl alcohol (PVA) cross-linked with sulfosuccinic acid (SSA) and modified with sulfonated polyethersulfone (SPES) mixed-matrix membranes are reported for their application in direct methanol fuel cells (DMFCs). Polyethersulfone (PES) is sulfonated by chlorosulfonic acid and factors affecting the sulfonation reaction, such as time and temperature, are studied. The ion-exchange capacity, degree of sulfonation, sorption, and proton conductivity for the mixed-matrix membranes are investigated. The mixed-matrix membranes are also characterised for their mechanical and thermal properties. The methanol-crossover flux across the mixed-matrix membranes is studied by measuring the mass balance of methanol using the density meter. The methanol cross-over for these membranes is found to be about 33% lower in relation to Nafion-117 membrane. The DMFC employing CS-PVA-SPES mixed-matrix membrane with an optimum content of 25 wt % SPES delivers a peak power-density of 5.5 mW cm-2 at a load current-density of 25 mA cm-2 while operating at 70 degrees C. (C) 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Corticosterone Treatment Results in Enhanced Release of Peptidergic Vesicles in Astrocytes via Cytoskeletal Rearrangements

While the effect of stress on neuronal physiology is widely studied, its effect on the functionality of astrocytes is not well understood. We studied the effect of high doses of stress hormone corticosterone, on two physiological properties of astrocytes, i.e., gliotransmission and interastrocytic calcium waves. To study the release of peptidergic vesicles from astrocytes, hippocampal astrocyte cultures were transfected with a plasmid to express pro-atrial natriuretic peptide (ANP) fused with the emerald green fluorescent protein (ANP.emd). The rate of decrease in fluorescence of ANP.emd on application of ionomycin, a calcium ionophore was monitored. Significant increase in the rate of calcium-dependent exocytosis of ANP.emd was observed with the 100 nM and 1 M corticosterone treatments for 3 h, which depended on the activation of the glucocorticoid receptor. ANP.emd tagged vesicles exhibited increased mobility in astrocyte culture upon corticosterone treatment. Increasing corticosterone concentrations also resulted in concomitant increase in the calcium wave propagation velocity, initiated by focal ATP application. Corticosterone treatment also resulted in increased GFAP expression and F-actin rearrangements. FITC-Phalloidin immunostaining revealed increased formation of cross linked F-actin networks with the 100 nM and 1 M corticosterone treatment. Alternatively, blockade of actin polymerization and disruption of microtubules prevented the corticosterone-mediated increase in ANP.emd release kinetics. This study reports for the first time the effect of corticosterone on gliotransmission via modulation of cytoskeletal elements. As ANP acts on both neurons and blood vessels, modulation of its release could have functional implications in neurovascular coupling under pathophysiological conditions of stress.

Molecular dynamic simulations of elastomer structure and its influence on anisotropic stress under time-varying strain

We investigate the evolution of polymer structure and its influence on uniaxial anisotropic stress under time-varying uniaxial strain, and the role of external control variables such as temperature, strain rate, chain length, and density, using molecular dynamics simulation. At temperatures higher than glass transition, stress anisotropy in the system is reduced even though the bond stretch is greater at higher temperatures. There is a significant increase in the stress level with increasing density. At higher densities, the uncoiling of the chains is suppressed and the major contribution to the deformation is by internal deformation of the chains. At faster rates of loading stress anisotropy increases. The deformation mechanism is mostly due to bond stretch and bond bending rather than overall shape and size. Stress levels increase with longer chain length. There is a critical value of the functionality of the cross-linkers beyond which the uniaxial stress developed increases caused primarily by bond stretching due to increased constraint on the motion of the monomers. Stacking of the chains in the system also plays a dominant role in the behaviour in terms of excluded volume interactions. Low density, high temperature, low values of functionality of cross-linkers, and short chain length facilitate chain uncoiling and chain slipping in cross-linked polymers.

Rare-earth carboxylates, Ln(2)(III)(mu(3)-OH)(C4H4O5)(2)(C4H2O4)]center dot 2H(2)O Ln = Ce, Pr and Nd]: synthesis, structure and properties

A new series of inorganic-organic hybrid framework compounds, Ln(2)(mu(3)-OH)(C4H4O5)(2)(C4H2O4)]center dot 2H(2)O, (Ln = Ce, Pr and Nd), have been prepared employing a hydrothermal method. Malic acid and fumaric acid form part of the structure. The malate units connect the lanthanide centers forming Ln-O-Ln two-dimensional layers, which are cross-linked by the fumarate units forming the three-dimensional structure. Extra framework water molecules form a dimer and occupy the channels. The water molecules can be reversibly adsorbed. The dehydrated structure did not show any differences in framework structure/ connectivity. The presence of lattice water provides a pathway for proton conductivity. Optical studies suggest an up-conversion behavior involving more than one photon for a neodymium compound.

Combinatorial Approach to Develop Tailored Biodegradable Poly(xylitol dicarboxylate) Polyesters

The objective of this work was to develop a versatile strategy for preparing biodegradable polymers with tunable properties for biomedical applications. A family of xylitol-based cross-linked polyesters was synthesized by melt condensation. The effect of systematic variation of chain length of the diacid, stoichiometric ratio, and postpolymerization curing time on the physicochemical properties was characterized. The degradation rate decreased as the chain length of the diacid increased. The polyesters synthesized by this approach possess a diverse spectrum of degradation (ranging from similar to 4 to 100% degradation in 7 days), mechanical strength (from 0.5 to similar to 15 MPa) and controlled release properties. The degradation was a first-order process and the rate constant of degradation decreased linearly as the hydrophobicity of the polyester increased. In controlled release studies, the order of diffusion increased with chain length and curing time. The polymers were found to be cytocompatible and are thus suitable for possible use as biodegradable polymers. This work demonstrates that this particular combinatorial approach to polymer synthesis can be used to prepare biomaterials with independently tunable properties.

Copolyesters from Soybean Oil for Use as Resorbable Biomaterials

A family of soybean oil (SO) based biodegradable cross-linked copolyesters sourced from renewable resources was developed for use as resorbable biomaterials. The polyesters were prepared by a melt condensation of epoxidized soybean oil polyol and sebacic acid with citric acid (CA) as a cross-linker. D-Mannitol (M) was added as an additional reactant to improve mechanical properties. Differential scanning calorimetry revealed that the polyester synthesized using only CA as the cross-linker was semicrystalline and elastomeric at physiological temperature. The polymers were hydrophobic in nature. The water wettability, elongation at break and the degradation rate of the polyesters decreased with increase in M content or curing time. Modeling of release kinetics of dyes showed a diffusion controlled mechanism underlies the observed sustained release from these polymers. The polyesters supported attachment and proliferation of human stem cells and were thus cytocompatible. Porous scaffolds induced osteogenic differentiation of the stern cells suggesting that these polymers are well suited for bone tissue engineering. Thus, this family of polyesters offers a low cost and green alternative as biocompatible, bioresobable polymers for potential use as resorbable biomaterials for tissue engineering and controlled release.

A novel in-situ polymer derived nano ceramic MMC by friction stir processing

Fiction stir processing (FSP) is a solid state technique used for material processing. Tool wear and the agglomeration of ceramic particles have been serious issues in FSP of metal matrix composites. In the present study, FSP has been employed to disperse the nanoscale particles of a polymer-derived silicon carbonitride (SiCN) ceramic phase into copper by an in-situ process. SiCN cross linked polymer particles were incorporated using multi-pass ESP into pure copper to form bulk particulate metal matrix composites. The polymer was then converted into ceramic through an in-situ pyrolysis process and dispersed by ESP. Multi-pass processing was carried out to remove porosity from the samples and also for the uniform dispersion of polymer derived ceramic particles. Microstructural observations were carried out using Field Emission Scanning Electron Microscopy (FE-SEM) and Transmission Electron Microscopy (TEM) of the composite. The results indicate a uniform distribution of similar to 100 nm size particles of the ceramic phase in the copper matrix after ESP. The nanocomposite exhibits a five fold increase in microhardness (260HV(100)) which is attributed to the nano scale dispersion of ceramic particles. A mechanism has been proposed for the fracturing of PDC particles during multi pass FSP. (C) 2015 Elsevier Ltd. All rights reserved

Recombinant antigen-based avidin-biotin microtiter enzyme-linked immunosorbent assay for serodiagnosis of invasive amebiasis

An immunoscreening approach was used to isolate a strongly positive cDNA clone from an Entamoeba histolytica HK-9 cDNA expression library in the phage vector lambda ZAP-II. The 1.85-kb cDNA insert was found to be truncated and encoded the cysteine-rich, immunodominant domain of the antigenic 170-kDa subunit of the amebal galactose N-acetylgalactosamine binding lectin. This domain was expressed as a glutathione S-transferase fusion protein in Escherichia coli. Inclusion bodies of the recombinant protein were solubilized with Sarkosyl, and the protein was enriched from the crude bacterial extract by thiol-affinity chromatography. The recombinant protein was used to develop a rapid, sensitive, and specific avidin-biotin microtiter enzyme-linked immunosorbent assay (ELISA) for invasive amebiasis. Sera from 38 individuals suffering from invasive amebiasis, 12 individuals with noninvasive amebiasis, 44 individuals with other infections, and 27 healthy subjects were screened by the recombinant antigen-based ELISA. The sensitivity and specificity of the assay were 90.4 and 94.3%, respectively, which correlated well with those of an ELISA developed with crude amebal antigen (r = 0.94; P < 0.0001), as well as with those of a commercially available serodiagnostic ELISA (r = 0.92; P < 0.0001). Thus, the bacterially expressed recombinant lectin can replace the crude amebal extract as an antigen in the serodiagnosis of invasive amebiasis by using avidin-biotin microtiter ELISA.

Prediction of estrus cyclicity in Asian elephants (Elephas maximus) through estimation of fecal progesterone metabolite: development of an enzyme-linked immuno-sorbent assay

Asian elephants (Dephas maximus), prominent ``flagship species'', arelisted under the category of endangered species (EN - A2c, ver. 3.1, IUCN Red List 2009) and there is a need for their conservation This requires understanding demographic and reproductive dynamics of the species. Monitoring reproductive status of any species is traditionally being carried out through invasive blood sampling and this is restrictive for large animals such as wild or semi-captive elephants due to legal. ethical, and practical reasons Hence. there is a need for a non-invasive technique to assess reproductive cyclicity profiles of elephants. which will help in the species' conservation strategies In this study. we developed an indirect competitive enzyme linked immuno-sorbent assay (ELISA) to estimate the concentration of one of the progesterone-metabolites i.e, allopregnanolone (5 alpha-P-3OH) in fecal samples of As elephants We validated the assay which had a sensitivity of 0.25 mu M at 90% binding with an EC50 value of 1 37 mu M Using female elephants. kept under semi-captive conditions in the forest camps of Mudumalar Wildlife Sanctuary, Tamil Nadu and Bandipur National Park, Karnataka, India. we measured fecal progesterone-metabolite (5 alpha-P-3OH) concentrations in six an and showed their clear correlation with those of scrum progesterone measured by a standard radio-immuno assay. Statistical analyses using a Linear Mixed Effect model showed a positive correlation (P < 0 1) between the profiles of fecal 5 alpha-P-3OH (range 0 5-10 mu g/g) and serum progesterone (range: 0 1-1 8 ng/mL) Therefore, our studies show, for the first time, that the fecal progesterone-metabolite assay could be exploited to predict estrus cyclicity and to potentially assess the reproductive status of captive and free-ranging female Asian elephants, thereby helping to plan their breeding strategy (C) 2010 Elsevier Inc.All rights reserved.

An enzyme-linked immuno focus assay for rapid detection and enumeration, and a newborn mouse model for human non-polio enteroviruses associated with acute diarrhea

We have recently reported significant association of non-polio enteroviruses (NPEVs) with acute and persistent diarrhea (18-21% of total diarrheal cases), and non-diarrheal Increased Frequency of Bowel Movements (IFoBM-ND) (about 29% of the NPEV infections) in children and that the NPEV-associated diarrhea was as significant as rotavirus diarrhea. However, their diarrhea-causing potential is yet to be demonstrated in an animal model system. Since the determination of virus titers by the traditional plaque assay takes 4-7 days, there is a need for development of a rapid method for virus titer determination to facilitate active clinical research on enterovirus-associated diarrhea. The goal of this study is to develop a cell-based rapid detection and enumeration method and to demonstrate the diarrhea-inducing potential of purified and characterized non-polio enteroviruses, which were isolated from diarrheic children. Here we describe generation of monoclonal and polyclonal antibodies against purified strains belonging to different serotypes, and development of an enzyme-linked immuno focus assay (ELIFA) for detection and enumeration of live NPEV particles in clinical and purified virus samples, and a newborn mouse model for NPEV diarrhea. Plaque-purified NPVEs, belonging to different serotypes, isolated from children with diarrhea, were grown in cell culture and purified by isopycnic CsCl density gradient centrifugation. By ELIFA, NPEVs could be detected and enumerated within 12 h post-infection. Our results demonstrated that Coxsackievirus B1 (CVB1) and CVB5 strains, isolated from diarrheic children, induced severe diarrhea in orally-inoculated 9-12 day-old mouse pups, fulfilling Koch's postulates. The methods described here would facilitate studies on NPEV-associated gastrointestinal disease. (C) 2015 Elsevier B.V. All rights reserved.

Nonlinear Enzyme Kinetics Can Lead to High Metabolic Flux Control Coefficients: Implications for the Evolution of Dominance

In a classic study, Kacser & Burns (1981, Genetics 97, 639-666) demonstrated that given certain plausible assumptions, the flux in a metabolic pathway was more or less indifferent to the activity of any of the enzymes in the pathway taken singly. It was inferred from this that the observed dominance of most wild-type alleles with respect to loss-of-function mutations did not require an adaptive, meaning selectionist, explanation. Cornish-Bowden (1987, J. theor. Biol. 125, 333-338) showed that the Kacser-Burns inference was not valid when substrate concentrations were large relative to the relevant Michaelis constants. We find that in a randomly constructed functional pathway, even when substrate levels are small, one can expect high values of control coefficients for metabolic flux in the presence of significant nonlinearities as exemplified by enzymes with Hill coefficients ranging from two to six, or by the existence of oscillatory loops. Under these conditions the flux can be quite sensitive to changes in enzyme activity as might be caused by inactivating one of the two alleles in a diploid. Therefore, the phenomenon of dominance cannot be a trivial ''default'' consequence of physiology but must be intimately linked to the manner in which metabolic networks have been moulded by natural selection.

Anthranilate Hydroxylase from Aspergillus niger: New Type of NADPH-Linked Nonheme Iron Monooxygenase

Anthranilate hydroxylase from Aspergillus niger catalyzes the oxidative deamination and dihydroxylation of anthranilic acid to 2,3-dihydroxybenzoic acid. This enzyme has been purified to homogeneity and has a molecular weight of 89,000. The enzyme is composed of two subunits of 42,000 with 2 gram-atoms of nonheme iron per mol. Fe2+-chelators like alpha,alpha'-dipyridyl and o-phenanthroline are potent inhibitors of the enzyme activity. Absorption and fluorescence spectra of the enzyme offer no evidence for the presence of other cofactors like flavin. Flavins and flavin-specific inhibitors like atebrin have no effect on the activity of the enzyme. The enzyme incorporates one atom of oxygen each from 18O2 and H218O into the product 2,3-dihydroxybenzoic acid. Based on these studies, it is concluded that anthranilate hydroxylase from A. niger is a new type of NADPH-linked nonheme iron monooxygenase.

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn2+fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.