Site-specific mutagenesis of yeast 2-Cys peroxiredoxin improves heat or oxidative stress tolerance by enhancing its chaperone or peroxidase function

Hong, S. H.,Lee, S. S.,Chung, J. M.,Jung, H. s.,Singh, S.,Mondal, S.,Jang, H. H.,Cho, J. Y.,Bae, H. J.,Chung, B. Y. Springer Science + Business Media 2017 Protoplasma Vol.254 No.1

<P>Yeast peroxiredoxin II (yPrxII) is an antioxidant enzyme that plays a protective role against the damage caused by reactive oxygen species (ROS) in Saccharomyces cerevisiae. This enzyme consists of 196 amino acids containing 2-Cys Prx with highly conserved two active cysteine residues at positions 48 and 171. The yPrxII has dual enzymatic functions as a peroxidase and molecular chaperone. To understand the effect of additional cysteine residues on dual functions of yPrxII, S79C-yPrxII and S109C-yPrxII, the substitution of Ser with Cys residue at 79 and 109 positions, respectively, was generated. S109C-yPrxII and S79C-yPrxII showed 3.7- and 2.7-fold higher chaperone and peroxidase activity, respectively, than the wild type (WT). The improvement in enzyme activity was found to be closely associated with structural changes in proteins. S109C-yPrxII had increased beta-sheet in its secondary structure and formed high-molecular-weight (HMW) as well as low-molecular-weight (LMW) complexes, but S79C-yPrxII formed only LMW complexes. HMW complexes predominantly exhibited a chaperone function, and LMW complexes showed a peroxidase function. In addition, transgenic yeast cells over-expressing Cys-substituted yPrxII showed greater tolerance against heat and oxidative stress compared to WT-yPrxII.</P>

Effects of Egg Yolk Antibodies Produced in Response to Different Antigenic Fractions of E. coli O157:H7 on E. coli Suppression

Chae, H.S.,Singh, N.K.,Ahn, C.N.,Yoo, Y.M.,Jeong, S.G.,Ham, J.S.,Kim, D.W. Asian Australasian Association of Animal Productio 2006 Animal Bioscience Vol.19 No.11

The objective of this research was to provide the characterization and method for producing anti-E. coli O157:H7 antibodies in egg-laying hens and to determine if the antibody can restrain the proliferation of E. coli O157:H7 in-vitro. Selected antigenic fractions (whole cell, outer membrane protein and lipopolysaccharide (LPS)) from E. coli O157:H7 were injected to hens in order to produce anti-E. coli O157:H7 antibodies. The immune response and the egg yolk antibodies of laying hens against the whole cell, outer membrane protein and LPS antigens were monitored by ELISA. The level of antibodies against whole cell antigen monitored through ELISA sharply increased after the initial immunization, and it was found to be maximum on day 49 however, the level was maintained up to day 70. Antibodies (5 mg/ml) directed against the whole cell inhibited E. coli proliferation 10-13 times more than outer membrane protein or LPS. The antibody response against the whole cell antigens appeared to have higher activity in restraining the proliferation of E. coli O157:H7 than antibody against outer membrane protein or LPS. Results reflected that increasing the IgY's in the egg yolk could prevent greater economic losses due to human and animal health from pathogenic bacteria i.e. E. coli O157:H7.

A sensitive fluorescent probe for the polar solvation dynamics at protein-surfactant interfaces

Singh, P.,Choudhury, S.,Singha, S.,Jun, Y.,Chakraborty, S.,Sengupta, J.,Das, R.,Ahn, K. H.,Pal, S. Royal Society of Chemistry 2017 Physical chemistry chemical physics Vol.19 No.19

<P>Relaxation dynamics at the surface of biologically important macromolecules is important taking into account their functionality in molecular recognition. Over the years it has been shown that the solvation dynamics of a fluorescent probe at biomolecular surfaces and interfaces account for the relaxation dynamics of polar residues and associated water molecules. However, the sensitivity of the dynamics depends largely on the localization and exposure of the probe. For noncovalent fluorescent probes, localization at the region of interest in addition to surface exposure is an added challenge compared to the covalently attached probes at the biological interfaces. Here we have used a synthesized donor-acceptor type dipolar fluorophore, 6-acetyl-(2-((4-hydroxycyclohexyl)(methyl) amino) naphthalene) (ACYMAN), for the investigation of the solvation dynamics of a model protein-surfactant interface. A significant structural rearrangement of a model histone protein (H1) upon interaction with anionic surfactant sodium dodecyl sulphate (SDS) as revealed from the circular dichroism (CD) studies is nicely corroborated in the solvation dynamics of the probe at the interface. The polarization gated fluorescence anisotropy of the probe compared to that at the SDS micellar surface clearly reveals the localization of the probe at the protein-surfactant interface. We have also compared the sensitivity of ACYMAN with other solvation probes including coumarin 500 (C500) and 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl)-4H-pyran (DCM). In comparison to ACYMAN, both C500 and DCM fail to probe the interfacial solvation dynamics of a model protein-surfactant interface. While C500 is found to be delocalized from the protein-surfactant interface, DCM becomes destabilized upon the formation of the interface (protein-surfactant complex). The timescales obtained from this novel probe have also been compared with other femtosecond resolved studies and molecular dynamics simulations.</P>

Synthesis and characterization of MnO-doped titanium pyrophosphates (Ti1-x Mn x P2O7; x = 0–0.2) for intermediate-temperature proton-conducting ceramic-electrolyte fuel cells

Singh, B.,Bhardwaj, A.,Gautam, S. K.,Parkash, O.,Kumar, D.,Jadhav, H. S.,Song, S. J. Springer Science + Business Media 2017 IONICS -KIEL- Vol.23 No.7

<P>MnO-doped TiP2O7 (Ti1-x Mn (x) P2O7; x = 0-0.2) were synthesized by digesting oxide precursors with H3PO4 in a two-step synthesis method. Various compositions of Ti1-x Mn (x) P2O7 were characterized by X-ray diffraction (XRD), thermogravimetric analysis/differential scanning calorimetry (TGA/DSC), scanning electron microscopy (SEM), and electrochemical impedance spectroscopy (EIS). XRD of 1050 A degrees C sintered samples showed the formation of (TiO)P2O7 in ae15 mol% MnO-doped samples. SEM images of 1050 A degrees C sintered Ti1-x Mn (x) P2O7 samples showed that MnO acts as a sintering aid, with the higher amount of MnO leading to a better densification. It was observed that the ionic conductivities of MnO-doped sintered samples were higher than that of undoped TiP2O7 (TiP) sample. The increase in ionic conductivity of MnO-doped TiP2O7 can be attributed to MnO acting as an acceptor dopant, and increased densification of the MnO-doped samples. In unhumidified air, among various MnO-doped samples, Ti0.9Mn0.1P2O7 (TMP10) showed the highest ionic conductivity with a magnitude of 6.29 x 10(-8) S cm(-1) at 250 A degrees C and 1.33 x 10(-5) S cm(-1) at 500 A degrees C. Furthermore, the ionic conductivity of TMP10 was higher than that of Ti0.88Mn0.12P2O7 (TMP12), which could be attributed to the formation of defect pairs in TMP12. The ionic conductivity of TMP10 showed > 4 orders of magnitude increase in humidified air (pH(2)O = 0.12 atm) in 100-270 A degrees C range with a maximum of 6.55 x 10(-4) S cm(-1) at 180 A degrees C. In addition, comparison with the literature data showed that the ionic conductivity of TMP10 was higher than those observed for the high-temperature sintered TiP2O7 samples.</P>

Poly(ethylene glycol) dicarboxylate/poly(ethylene oxide) hydrogel film co-crosslinked by electron beam irradiation as an anti-adhesion barrier

Haryanto,Singh, D.,Han, S.S.,Son, J.H.,Kim, S.C. Elsevier 2015 Materials Science and Engineering C Vol.46 No.-

The cross-linked poly(ethylene glycol) dicarboxylate (PEGDC)/poly(ethylene oxide) (PEO) and poly(ethylene glycol) dimethacrylate (PEGDMA)/(PEO) hydrogels were developed for possible biomedical applications such as an anti-adhesion barrier. Various contents of PEGDC/PEO film were irradiated using an electron beam with various beam intensities in order to obtain various degrees of crosslinked hydrogels. The optimum dose (300kGy) and total crosslinker content of 10% were used to prepare crosslinked hydrogel films with three different compositions (10% PEGDC, 10% PEGDMA, 5% PEGDC-5% PEGDMA). Among them, 10% PEGDC hydrogel film exhibited the highest elongation at break (69.33+/-6.87%) with high mechanical strength. 10% PEGDC hydrogel film showed the lowest hemolysis activity (6.03+/-0.01%) and the highest tissue adherence (75.67+/-1.15cN). The result also indicated that the carboxyl groups in PEGDC affect the tissue adherence of hydrogel films via H-bonding interactions. In animal studies, 10% PEGDC anti-adhesion hydrogel film degraded within 3weeks and demonstrated better anti-adhesive effect compared to Guardix-SG®.

Production and Optimization of a Commercially Viable Alkaline Protease from a Haloalkaliphilic Bacterium

R. H. Joshi,M. S. Dodia,S. P. Singh 한국생물공학회 2008 Biotechnology and Bioprocess Engineering Vol.13 No.5

Twenty five haloalkaliphilic bacterial strains were isolated from sea water along the Coastal Gujarat (India) and screened for their ability to secret alkaline proteases. Among them, a potent strain S-20-9 (GenBank accession number EU118360), re-sembling to Halophilic Bacterium MBIC3303 on the basis of 16S rRNA gene sequencing, was selected for the optimization of enzyme production. S-20-9 produced protease optimally, under aerobic conditions during mid-stationary phase over a broad range of salt (5~25%, w/v) and pH (7~10). The optimum production was at pH 9 and 15% (w/v) NaCl. The production was suppressed by lactose, maltose, sucrose, and inorganic nitrogen sources, especially ammonium ions. Further, the pro-duction was significantly stimulated by KH₂PO₄ and suppressed by glucose. Similarly, the production was also suppressed at higher concentrations of gelatin, yeast extract, peptone, and casamino acids, indicating towards a threshold value for ni-trogen requirement. The growth and protease production were enhanced by mono-valent cation (KCl), while the divalent cations acted as inhibitors. The study holds significance as only few reports are available on the alkaline proteases from haloalkaliphilic bacteria, particularly those from moderate saline habitats.

Synthesis, magnetic and Mossbauer spectroscopic studies of Cr doped lithium ferrite nanoparticles

Srivastava, M.,Layek, S.,Singh, J.,Das, A.K.,Verma, H.C.,Ojha, A.K.,Kim, N.H.,Lee, J.H. Elsevier Sequoia 2014 JOURNAL OF ALLOYS AND COMPOUNDS Vol.591 No.-

Lithium-based ferrites are promising and potential magnetic materials for microwave applications. They possess a spinel (AB<SUB>2</SUB>O<SUB>4</SUB>) type crystal structure, where the distributions of metal cations over the tetrahedral (A) and octahedral (B) voids play a crucial role for exhibiting different physical properties. Among various parameter of synthesis, pH is an important factor that influences the cation distribution over A and B voids, overall growth of the nanoparticles and different physical properties. In the present work single phase Cr substituted lithium ferrite nanoparticles have been synthesized by the sol-gel method at different pH. The phase identification and crystallite size have been probed by X-ray diffraction studies. The crystallite size changes by 44.2-48.8nm upon varying the pH from 3.5 to 11.5. In order to investigate the cations distribution at A/B sites, Mossbauer spectroscopic measurements were done. The values of magnetic hyperfine field obtained from the Mossbauer data for the A and B sites are ~49.5T and 51T, respectively. Moreover, it is observed that the area ratio of B site to A site increases with increasing the pH. This observation further suggests that the B site is more preferable for Fe<SUP>3+</SUP> cations at higher pH than the A site. The magnetic parameter such as saturation magnetization (M<SUB>s</SUB>), remanent magnetization (M<SUB>r</SUB>), coercive field (H<SUB>C</SUB>) and squareness (S) are determined by vibrating sample magnetometer (VSM) measurements, which show a consistent increase with increasing pH. The reason for the variation in magnetic properties has been explained on the basis of increased Fe<SUP>3+</SUP> cation occupancies at the B site and size effect, which is well supported by Mossbauer spectroscopic and XRD studies.

Multi-Secondary Transformer: A Modeling Technique for Simulation - II

Patel, A.,Singh, N.P.,Gupta, L.N.,Raval, B.,Oza, K.,Thakar, A.,Parmar, D.,Dhola, H.,Dave, R.,Gupta, V.,Gajjar, S.,Patel, P.J.,Baruah, U.K. Journal of International Conference on Electrical 2014 Journal of international Conference on Electrical Vol.3 No.1

Power Transformers with more than one secondary winding are not uncommon in industrial applications. But new classes of applications where very large number of independent secondaries are used are becoming popular in controlled converters for medium and high voltage applications. Cascade H-bridge medium voltage drives and Pulse Step Modulation (PSM) based high voltage power supplies are such applications. Regulated high voltage power supplies (Fig. 1) with 35-100 kV, 5-10 MW output range with very fast dynamics (${\mu}S$ order) uses such transformers. Such power supplies are widely used in fusion research. Here series connection of isolated voltage sources with conventional switching semiconductor devices is achieved by large number of separate transformers or by single unit of multi-secondary transformer. Naturally, a transformer having numbers of secondary windings (~40) on single core is the preferred solution due to space and cost considerations. For design and simulation analysis of such a power supply, the model of a multi-secondary transformer poses special problem to any circuit analysis software as many simulation softwares provide transformer models with limited number (3-6) of secondary windings. Multi-Secondary transformer models with 3 different schemes are available. A comparison of test results from a practical Multi-secondary transformer with a simulation model using magnetic component is found to describe the behavior closer to observed test results. Earlier models assumed magnetising inductance in a linear loss less core model although in actual it is saturable core made-up of CRGO steel laminations. This article discusses a more detailed representation of flux coupled magnetic model with saturable core properties to simulate actual transformers very close to its observed parameters in test and actual usage.

Proteomic study of calpeptin-induced differentiation on calpain-interacting proteins of C2C12 myoblast.

Singh, N K,Shiwani, S,Hwang, I H Springer 2012 In vitro cellular & developmental biology Animal Vol.48 No.3

<P>Studies on skeletal muscle cell specification and development have demonstrated in the past that calpains interact with various transcriptional factors in regulating the cellular function. It has therefore, been assumed that transcriptional factors like myogenin, MyoD, Myf5, and MRF4 that are active during the myogenic differentiation might be affected and degraded by calpains. Therefore, to examine the biochemical adaptations of myoblasts during myocyte formation and muscle development comprehensively, the current study was designed to identify the effect of calpeptin (calpain inhibitors) on protein expression during differentiation of C2C12 mouse myoblast. Cells were proliferated to near 80% confluence under Dulbecco's modified eagle medium and differentiated further in 2% HS with 50?μM calpeptin. Incubated cells were collected at 0, 12, and 72?h and later the cell proteins were focused onto pH?4-7 IEF strip, followed by 12.5% SDS-PAGE. Obtained spots on the gels were compared and matched using commercial 2-DE analysis software and matched spots were identified by MALDI-ToF and/or Q-Tof systems. Conclusively, cell differentiation was observed to be active from 12 to 72?h however, calpeptin affected the differentiation process and cut down the rate of fusion by approximately 50%. Out of 41 proteins identified, 12 proteins were found to be upregulated where as 29 proteins were downregulated.</P>

Eu2+ and Mn2+ Co-doped BaMgAl10O17 Blue- and Green-Emitting Phosphor: A Luminescence and EPR Study

Singh, V.,Sivaramaiah, G.,Rao, J. L.,Singh, N.,Srivastava, A. K.,Jirimali, H. D.,Li, J.,Gao, H.,Kumaran, R. S.,Singh, P. K. Springer Science + Business Media 2016 Journal of electronic materials Vol.45 No.6

<P>Eu2+ and Mn2+ co-doped BaMgAl10O17 phosphor has been prepared by a solution combustion method. The structural, morphological and compositional analysis of the BaMgAl10O17:Eu2+ and Mn2+ powders have been studied by x-ray diffraction, scanning electron microscopy and energy dispersive x-ray spectroscopy. The electron paramagnetic resonance (EPR) spectrum exhibited resonance signals with the effective g values of g a parts per thousand 4.88 and g a parts per thousand 1.98. The resonance signal with the effective g value of g a parts per thousand 4.88 is characteristic of Eu2+ ions whereas g a parts per thousand 1.98 is due to Mn2+ ions. The number of spins participating in resonance, Gibbs free energy, magnetic susceptibility, Curie constant, effective magnetic moment, zero-field splitting parameter and hyperfine splitting constant have been evaluated. From optical and EPR correlation, it is inferred that Eu2+ and Mn2+ are present in the BaMgAl10O17 matrix.</P>