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Narendra Kumar

Publications and source records attributed to Narendra Kumar.

11 recordsLinked to original sources
PubMed2006

Metal-support interactions in zeolite-supported noble metals: influence of metal crystallites on the support acidity.

The metal-support interactions on a series of catalysts of different acidities, including platinum-modified zeolites and H-MCM-41, are investigated by means of XPS, CO and pyridine adsorption, and a model reaction (ring opening of decalin). The electronic properties of Pt are influenced by the acidity of the support, and the alteration of Pt properties increases with increasing acidity of the support, as can be seen from the changes in the Pt binding energy and stretching frequency of adsorbed CO. At the same time, the presence of platinum affects the acidic properties of the supports by reducing the strength of the acid sites. This is observed directly as the changes in desorption of pyridine from the acid sites and indirectly as the suppression of cracking reactions during the ring opening of decalin on the Pt-modified catalysts. The observed results are discussed in terms of the interatomic potential model.

Journal Article↗
PubMed2005

Utilization of composted sugar industry waste (pressmud) to improve properties of sodic soil for rice cultivation.

Sulphitation pressmud (SPM) and its composts were prepared by heap, pit, NADEP and vermicomposting methods and their effects were compared with soil properties and growth, yield and nutrient uptake by rice in a sodic soil under pot conditions. Application of 15 t ha(-1) SPM and its different composts significantly increased the plant height and dry matter accumulation at different intervals, grain and straw yields and N, P and K uptake by the crop over the control. NADEP compost of SPM alone recorded the maximum and significant plant height by 8.5 to 19.3% and plant dry matter by 14.6 to 32.8% over the raw SPM at different intervals. NADEP composts of SPM alone and SPM + rice straw were also found to be superior than raw SPM by recording 34.8 and 27.8% more grain yield respectively. The SPM composts prepared by NADEP and SPM by vermicomposting methods significantly accumulated higher N and K in rice grains and straw, while NADEP compost of SPM and SPM + rice straw recorded more P in grains and straw than raw SPM. Application of SPM and its composts reduced the pH, EC and bulk density of the soil after rice harvesting, though the reductions were not significant in comparison to the control. However, these treatments increased the soil organic C by 33.33 to 69.0%, available N by 41.4 to 74.8%, available P by 47.1 to 97.8%, available K by 11.8 to 59.2% and available S by 10.3 to 90.7% over the control. NADEP composts, in general, were found to be superior than the raw SPM and other composts in residual soil nutrient content after rice crop.

Agriculture↗
PubMed2004

Molecular cloning, characterization, and overexpression of a novel [Fe]-hydrogenase isolated from a high rate of hydrogen producing Enterobacter cloacae IIT-BT 08.

Degenerate primers were designed from the conserved zone of hydA structural gene encoding for catalytic subunit of [Fe]-hydrogenase of different hydrogen producing bacteria. A 750 bp of PCR product was amplified by using the above-mentioned degenerate primers and genomic DNA of Enterobacter cloacae IIT-BT 08 as template. The amplified PCR product was cloned and sequenced. The sequence showed the presence of an ORF of 450 bp with significant similarity (40%) with C-terminal end of the conserved zone (H-cluster) of [Fe]- hydrogenase. hydA ORF was then amplified and cloned in-frame with GST in pGEX4T-1 and overexpressed in a non-hydrogen producing Escherichia coli BL-21 to produce a GST-fusion protein of a calculated molecular mass of about 42.1 kDa. Recombinant protein was purified and specifically recognized by anti-GST monoclonal antibody through Western blot. Southern hybridization confirmed the presence of this gene in E. cloacae IIT-BT 08 genome. In vitro hydrogenase assay with the overexpressed hydrogenase enzyme showed that it is catalytically active upon anaerobic adaptation. In vivo hydrogenase assay confirmed the presence of H2 gas in the gas mixture obtained from the batch culture of recombinant E. coli BL-21. A tentative molecular mechanism has been proposed about the transfer of electron from electron donor to H-cluster without the mediation of the F-cluster.

Amino Acid Sequence↗
PubMed2004

Regulation of cell motility by tyrosine phosphorylated villin.

Temporal and spatial regulation of the actin cytoskeleton is vital for cell migration. Here, we show that an epithelial cell actin-binding protein, villin, plays a crucial role in this process. Overexpression of villin in doxycyline-regulated HeLa cells enhanced cell migration. Villin-induced cell migration was modestly augmented by growth factors. In contrast, tyrosine phosphorylation of villin and villin-induced cell migration was significantly inhibited by the src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) as well as by overexpression of a dominant negative mutant of c-src. These data suggest that phosphorylation of villin by c-src is involved in the actin cytoskeleton remodeling necessary for cell migration. We have previously shown that villin is tyrosine phosphorylated at four major sites. To further investigate the role of tyrosine phosphorylated villin in cell migration, we used phosphorylation site mutants (tyrosine to phenylalanine or tyrosine to glutamic acid) in HeLa cells. We determined that tyrosine phosphorylation at residues 60, 81, and 256 of human villin played an essential role in cell migration as well as in the reorganization of the actin cytoskeleton. Collectively, these studies define how biophysical events such as cell migration are actuated by biochemical signaling pathways involving tyrosine phosphorylation of actin binding proteins, in this case villin.

Actins↗
PubMed2004

Functional dissection and molecular characterization of calcium-sensitive actin-capping and actin-depolymerizing sites in villin.

All proteins of the villin superfamily, which includes the actin-capping and -severing proteins such as gelsolin, scinderin, and severin, are calcium-regulated actin-modifying proteins. Like some of these proteins, villin has morphologically distinct effects on actin assembly depending on the free calcium concentrations. At physiological calcium (Ca2+) villin nucleates and bundles actin, whereas at higher concentrations it caps (>50 microm) and severs (>200 microM) actin filaments. Although Ca(2+)-binding sites have been described in villin, the functional characterization of these sites has not been done previously. In the present study we functionally dissect the calcium-dependent actin-capping and -depolymerizing sites in villin. Our analysis reveals that villin binds Ca2+ with a Kd of 80.5 microM, a stoichiometry of 5.97, and a Hill's coefficient of 1.2. Using the NMR structure of villin 14T and the gelsolin-actin/Ca2+ crystal structure, six putative sites that result in Ca(2+)-induced conformational changes were identified in human villin and confirmed by mutational analysis. Molecular dynamics studies support the mutational analysis and provide a model for structural difference in the A93G mutant that prevents the calcium-induced conformational changes in the S1 domain of villin. Furthermore, we determined that villin expresses at least two types of Ca(2+)-sensitive sites that determine separate functional properties; site 1 (Glu-25, Asp-44, and Glu-74) regulates actin-capping, whereas sites 1 and 2 (Asp-86, Ala-93, and Asp-61), together with the intra-domain calcium-sensitive sites in villin, regulate actin depolymerization by villin. This is the first study that employs sequential mutagenesis to biochemically and functionally characterize the calcium-sensitive sites in villin. Such mutational analysis and functional characterization of the actin-capping and -depolymerizing sites are unknown for other proteins of the villin family.

Actins↗
PubMed2004

Identification of a functional switch for actin severing by cytoskeletal proteins.

Actin severing is vital for the organization of the actin cytoskeleton during cell motility. Severing of F-actin by the homologous proteins villin and gelsolin requires unphysiologically high calcium concentrations (20-200 microM). Here we demonstrate that high calcium releases an autoinhibited conformation in villin that is maintained by two low affinity calcium binding sites (aspartic acids 467 and 715) that interact with a cluster of basic residues in the S2 domain of villin. Mutation of either of these sites as well as tyrosine phosphorylation alters the conformation of villin resulting in a protein that can sever actin in nanomolar calcium. These results suggest that tyrosine phosphorylation rather than high calcium may be the mechanism by which villin and other related proteins sever actin in vivo.

Actins↗
PubMed2003

Association of villin with phosphatidylinositol 4,5-bisphosphate regulates the actin cytoskeleton.

Villin, an epithelial cell actin-binding protein, severs actin in vitro and in vivo. Previous studies report that phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates actin severing by villin, presumably by interaction with villin. However, direct association of villin with PIP(2) has never been characterized. In this report, we presented mutational analysis to identify the PIP(2)-binding sites in villin. Villin (human) binds PIP(2) with a K(d) of 39.5 microm, a stoichiometry of 3.3, and a Hill coefficient of 1. We generated deletion mutants of villin lacking putative PIP(2)-binding sites and examined the impact of these mutations on PIP(2) binding and actin dynamics. Our analysis revealed the presence of three PIP(2)-binding sites, two in the amino-terminal core and one in the carboxyl-terminal headpiece of human villin. Synthetic peptides analogous with these sites confirmed the binding domains. Circular dichroism and quenching of intrinsic tryptophan fluorescence revealed a significant conformational change in these peptides ensuing in their association with PIP(2). By using site-directed mutagenesis (arginine 138 to alanine), we demonstrated the presence of an identical F-actin and PIP(2)-binding site in the capping and severing domain of villin. In contrast, the mutants lysine 822 and 824 to alanine demonstrated the presence of an overlapping F-actin and PIP(2)-binding site in the actin cross-linking domain of villin. Consistent with this observation, association of villin with PIP(2) inhibited the actin capping and severing functions of villin and enhanced the actin bundling function of villin. Our studies revealed that structural changes induced by association with PIP(2) could regulate the actin-modifying functions of villin. This study provided biochemical proof of the functional significance of villin association with PIP(2) and identified the molecular mechanisms involved in the regulation of actin dynamics by villin and PIP(2).

Actins↗
PubMed2002

Regulation of actin dynamics by tyrosine phosphorylation: identification of tyrosine phosphorylation sites within the actin-severing domain of villin.

We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated by phosphorylation. Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly. We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T. Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene. Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261). Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256. Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay. Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin. Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin. Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function.

Actins↗
PubMedDate not supplied

Osteochondroma of the talus in a male adolescent.

Osteochondroma of the talus is rare but must be considered as part of the differential diagnosis of any swelling in this part of the skeleton. This article describes the differential diagnosis and treatment of osteochondroma of the talus in a male adolescent. Careful surgical planning in all cases of benign-appearing lesions of small bones will prevent the recurrence of this lesion and its complications.

Adolescent↗