Biomedical subjects

R Kishore

Publications and source records attributed to R Kishore.

At least 19 recordsLinked to original sources

PubMed2001

Stabilization of tumor necrosis factor alpha mRNA by chronic ethanol: role of A + U-rich elements and p38 mitogen-activated protein kinase signaling pathway.

Increased expression of tumor necrosis factor alpha (TNFalpha) in response to chronic ethanol has been implicated in the pathogenesis of alcoholic liver disease. However, the molecular mechanisms by which ethanol increases the levels of TNFalpha are not well characterized. Utilizing Kupffer cells isolated from rats fed an ethanol containing diet and a murine macrophage cell line, RAW264.7, exposed to ethanol in culture, we have demonstrated that exposure to chronic ethanol results in an enhanced expression of lipopolysaccharide (LPS)-induced TNFalpha. While chronic ethanol had no effect on the rate of LPS-induced TNFalpha transcription as measured by nuclear run-on experiments, TNFalpha mRNA half-life was increased by chronic ethanol. Chronic ethanol also potentiated the activation of LPS-induced p38 mitogen-activated protein (MAP) kinase in Kupffer cells, as well as in RAW264.7 cells. Specific inhibition of p38 MAP kinase activation by SB203580 in Kupffer cells or by overexpression of dominant negative p38 MAP kinase in RAW264.7 cells blocked ethanol-mediated TNFalpha mRNA stabilization. Furthermore, using chimeric reporter constructs, we have shown that A + U-rich elements in the 3'-untranslated region of TNFalpha mRNA are not sufficient to impart ethanol-mediated stabilization on TNFalpha mRNA.

3' Untranslated Regions↗

PubMed2001

Influence of hydrophobic interactions on the conformational adaptability of the beta-Ala residue.

The chemical synthesis and X-ray crystal structure analysis of a model peptide incorporating a conformationally flexible beta-Ala residue: Boc-beta-Ala-Pda, 1 (C23H46N2O3: molecular weight = 398.62) have been described. The peptide crystallized in the crystal system triclinic with space group P21: a = 5.116(3) A, b = 5.6770(10) A, c = 21.744(5) A; alpha = 87.45 degrees, beta = 86.87 degrees, gamma = 90.0 degrees; Z = 1. An attractive feature of the crystal molecular structure of 1 is the induction of a reasonably extended backbone conformation of the beta-Ala moiety, i.e. the torsion angles phi approximately -115 degrees, mu approximately 173 degrees and psi approximately 122 degrees, correspond to skew-, trans and skew+ conformation, respectively, by an unbranched hydrophobic alkyl chain, Pda, which prefers an all-anti orientation (theta1 approximately -153 degrees, theta2 approximately ellipsis theta14 approximately +/-178 degrees ). The observation is remarkable because, systematic conformational investigations of short linear beta-Ala peptides of the type Boc-beta-Ala-Xaa-OCH3 (Xaa = Aib or Acc6) have shown that the chemical and stereochemical characters of the neighboring moieties may be critical in dictating the overall folded and/or unfolded conformational features of the beta-Ala residue. The overall conformation of 1 is typical of a 'bar'. It appears convincing that, in addition to a number of hydrophobic contacts between the parallel arranged molecules, an array of conventional N-HellipsisO=C intermolecular H-bonding interactions stabilize the crystal molecular structure. Moreover, the resulting 14-membered pseudo-ring motif, generated by the amide-amide interactions between the adjacent molecules, is completely devoid of nonconventional C-HellipsisO interaction. The potentials of the conformational adaptation of the beta-Ala residue, to influence and stabilize different structural characteristics have been highlighted.

Hydrogen↗

PubMed2000

Collateral existence of folded and extended conformations of the beta-Ala moiety in a model peptide.

The single-crystal X-ray diffraction analysis of a nonchiral beta-Ala-containing model peptide, Boc-beta-Ala-Aib-OCH(3) 1 (beta-Ala, 3-aminopropionic acid; Aib, alpha-aminoisobutyric acid), establishes the coexistence of distinctly different backbone conformations in two crystallographically independent molecules, A and B, in the asymmetric unit. Interestingly, the central mu torsion angle around the -C(beta)-C(alpha)- bond of the conformationally flexible beta-Ala residue appears to be critical in dictating the overall distinct structural features, i.e., in molecule A it adopts a folded gauche conformation: mu = -71.0 degrees, whereas it favors an extended trans conformation, mu = 161.2 degrees, in molecule B. As expected, the stereochemically constrained Aib residue preferred an energetically favorable folded backbone conformation, the torsion angles being phi = 46.2 degrees and psi = 48.3 degrees for molecule A and phi = -43.6 degrees and psi = -45.5 degrees for molecule B, lying in the left-handed and right-handed helical regions of the Ramachandran map, respectively. Considering the signs as well as the magnitudes of the backbone torsional angles, molecule A typically folds into a pseudo type III' beta-turn-like structure while molecule B prefers an overall extended conformation. Entrapping the two dramatically distinct conformational characteristics in the crystalline state clearly suggests that the gauche and the trans effects of the beta-Ala moieties are indeed energetically accessible to a short linear peptide and receive strong experimental support. The analyses permitted us to emphasize that in addition to conformational constraints of the neighboring residue, the chemical nature of the side-chain acyclic substituents and the "local environments" collectively seem to influence the stabilization of the folding-unfolding behavior of the two methylene units (-CONH-CH(2)-CH(2)-CONH-) in 1.

Crystallography, X-Ray↗

PubMed2000

Ring formation drives invagination of the vulva in Caenorhabditis elegans: Ras, cell fusion, and cell migration determine structural fates.

Directed cell rearrangements occur during gastrulation, neurulation, and organ formation. Despite the identification of developmental processes in which invagination is a critical component of pattern formation, little is known regarding the underlying cellular and molecular details. Caenorhabditis elegans vulval epithelial cells undergo morphological changes that generate an invagination through the formation of seven stacked rings. Here, we study the dynamics of ring formation during multivulva morphogenesis of a let-60/ras gain-of-function mutant as a model system to explore the cellular mechanisms that drive invagination. The behavior of individual cells was analyzed in a let-60/ras mutant by three-dimensional confocal microscopy. We showed that stereotyped cell fusion events occur within the rings that form functional and nonfunctional vulvae in a let-60/ras mutant. Expression of let-60/ras gain-of-function results in abnormal cell migration, ectopic cell fusion, and structural fate transformation. Within each developing vulva the anterior and posterior halves develop autonomously. Contrary to prevailing hypotheses which proposed three cell fates (1 degrees, 2 degrees, and 3 degrees), we found that each of the seven rings is a product of a discrete structural pathway that is derived from arrays of seven distinct cell fates (A, B, C, D, E, F, and H). We have also shown how autonomous ring formation is the morphogenetic force that drives invagination of the vulva.

Animals↗

PubMed2000

Interleukin-1-mediated stabilization of mouse KC mRNA depends on sequences in both 5'- and 3'-untranslated regions.

mRNA transcribed from the mouse KC chemokine gene accumulated to significantly higher levels in multiple cell types after treatment with interleukin 1alpha (IL-1alpha) as compared with tumor necrosis factor-alpha (TNFalpha). Although TNFalpha and IL-1alpha both signaled the activation of nuclear factor kappaB and enhanced transcription of the KC gene with equal potency, only IL-1alpha treatment resulted in stabilization of KC mRNA. Nucleotide sequences that confer sensitivity for IL-1alpha-mediated mRNA stabilization were identified within the 5'- and 3'-untranslated regions (UTRs) of KC mRNA using transient transfection of chimeric plasmids containing specific portions of KC mRNA linked to the chloramphenicol acetyltransferase (CAT) gene. When plasmids containing either the 3'- or 5'-UTR of KC mRNA were used, the half-life of CAT mRNA was unaltered either in untreated or IL-1alpha-stimulated cells. In contrast, CAT mRNA transcribed from plasmids that contained both the 5'- and 3'-UTRs of the KC mRNA decayed more rapidly than control CAT mRNA, and this enhanced decay was prevented in cells treated with IL-1alpha. A cluster of four overlapping AUUUA motifs within the 3'-UTR was required, whereas the 5'-UTR region exhibited orientation dependence. These findings indicate that cooperative function of the two nucleotide sequences involves a distinct signaling pathway used by IL-1alpha but not TNFalpha.

3' Untranslated Regions↗

PubMed2000

Stabilization of a novel beta-turn-like motif by nonconventional intramolecular hydrogen-bonding interactions in a model peptide incorporating beta-alanine.

The chemical synthesis and x-ray crystal structure analysis of a model peptide incorporating a conformationally adaptable unsubstituted beta-Ala residue: Boc-beta-Ala-Acc6-OCH3 (C16H28N2O5, molecular weight = 328.41; 1) has been described. The peptide crystallized in the space group P2(1)2(1)2(1) a = 8.537 (3), b = 8.872 (10), c = 25.327 (8), alpha = beta = gamma = 90.0 degrees, Z = 4. An attractive feature of the crystal structure analysis of 1 is an accommodation of a significantly folded beta-Ala residue in a short linear peptide. The overall peptide conformation is typically folded into a beta-turn-like motif. The stabilization of the peptide backbone conformation by nonconventional C-H...O weak intramolecular hydrogen-bonding interactions, involving the ester terminal carbon atom and the ethereal oxygen of the Boc group, has been evoked. The conformational constraint that seems most apparent is the phi, psi value of the highly constrained hydrophobic Acc6 ring that may play a key role in inducing or sustaining the observed pseudo type III or III' beta-turn structure. The resulting 12-membered hydrogen bonding ring motif in 1 is distinctly different from the one found in classical beta-turn structures, stabilized by a conventional strong C=O...H-N intramolecular hydrogen bond, comprised of alpha-amino acids. The potential of the conformationally adaptable beta-Ala residue to occupy i + 1 position (left corner) of the folded beta-turn-like structure and to design and construct novel secondary structural features have been emphasized.

Hydrogen↗

PubMed2000

Entrapping unusual folding characteristics of the beta-Ala residues in a model peptide: Boc-beta-Ala-Aib-beta-Ala-NHCH3.

Crystal structure analysis of a model peptide: Boc-beta-Ala-Aib-beta-Ala-NHCH3 (beta-Ala: 3-amino propionic acid; Aib: alpha-aminoisobutyric acid) revealed distinct conformational preferences for folded [phi approximately 136 degrees, mu approximately -62 degrees, psi approximately 100 degrees] and semifolded [phi approximately 83 degrees, mu approximately -177 degrees, psi approximately -117 degrees] structures of the N-and C-terminus beta-Ala residues, respectively. The overall folded conformation is stabilized by unusual Ni...H-Ni+1 and nonconventional C-H...O intramolecular hydrogen bonding interactions.

Hydrogen Bonding↗

PubMed2000

Characterization of a novel type VII beta-turn conformation for a bio-active tetrapeptide rigin A synergy between theoretical and experimental results.

The conformational analysis of an immunomodulating tetrapeptide rigin (H-Gly-Gln-Pro-Arg-OH), shown to possess diverse immunological activity, has been investigated both theoretically and experimentally for its conformational preferences. Unrestrained molecular dynamics simulation studies in implicit dimethylsulfoxide provide strong support for the existence of a significant population of ordered reverse turn structures for the major trans isomer. Of the three different energy minimized families, generated from computer molecular modelling, only one could be complemented by most of the 1D and 2D 1H NMR parameters obtained in dimethylsulfoxide-d6. A variable temperature NMR experiment in dimethylsulfoxide-d6 revealed that the preferred conformation is not stabilized by an intramolecular hydrogen bonding interaction. An analysis of the 2D ROESY experiment provides evidence in favour of an uncommonly observed, rather ill-defined type VII beta-turn structure. A survey of the observed specific inter-and intra-residue NOE connectivities and their comparison with one of the predicted low-energy conformations, demonstrates synergy between the theoretical molecular modelling and experimentally determined NMR spectral data. The primary structure, rather than long-range interactions, appears to be critical in determining the folding behaviour of the bio-active rigin. The present structural attributes may be valuable in peptide drug design and development of the rigin analogs having more effective stimulating activity.

Hydrogen Bonding↗

PubMed2000

Infective endocarditis as a cause of fever in hemodialysis patients.

Vascular access infections are common in maintenance hemodialysis patients especially with dual lumen cuffed catheter. Persistent infections may lead to valvular seeding and the development of infective endocarditis. Though antibiotic therapy may often suffice, many patients may require surgical correction which carries a high risk of mortality. However appropriate preoperative therapy may considerably reduce the risk of surgery in maintenance hemodialysis patients.

Adult↗

PubMed1999

Cutting edge: clustered AU-rich elements are the target of IL-10-mediated mRNA destabilization in mouse macrophages.

In the present study we show that IL-10-mediated inhibition of inflammatory gene expression can be mediated by an AU-rich element (ARE) cluster present in the 3' untranslated region (3'UTR) of sensitive genes. A series of chloramphenicol acetyl transferase (CAT) reporter gene constructs were prepared in which different fragments from the IL-10-sensitive KC mRNA 3'UTR were placed downstream of the coding region of the reporter gene CAT. CAT mRNA containing the KC 3'UTR was markedly destabilized as compared with the control CAT mRNA, and the decay rate was further increased in cells stimulated with IL-10. The KC 3'UTR contains an ARE cluster and three isolated ARE motifs. The ARE cluster spanning nucleotides 378-399 appeared to be both necessary and sufficient to mediate sensitivity to IL-10 because a 116-nucleotide fragment that contains the cluster conferred sensitivity, while mutation of the sequence between positions 378 and 399 eliminated sensitivity. The destabilizing effect of IL-10 was relatively selective, as the stability of chimeric CAT mRNAs was not modulated in cells treated with IFN-gamma or IL-4.

3' Untranslated Regions↗

PubMed1999

Regulation of macrophage gene expression by pro- and anti-inflammatory cytokines.

The anti-inflammatory cytokines IL-4 and IL-10 are well recognized as important negative regulators of proinflammatory gene expression in mononuclear phagocytes. The intracellular mechanisms which mediate these responses appear to be multifactorial. IL-4 is able to markedly suppress transcriptional activation of IFNgamma-responsive genes and the promoter sequences required for both IFNgamma and IL-4 sensitivity are identical. IFNgamma-activated STAT1 and IL-4-activated STAT6 can both form complexes on the same regulatory sequence element; while STAT1 functions to promote transcription, STAT6 is inactive. STAT6 is, however, required for the suppressive activity of IL-4. In this model, IL-4 appears to suppress IFNgamma-inducible proinflammatory gene expression through the ability of STAT6 to compete with STAT1 for occupancy of promoter sites necessary for IFNgamma-induced transcriptional initiation. In a second model, IL-10 suppresses the expression of genes induced in LPS-stimulated macrophages through a pathway involving destabilization of specific mRNAs. We have demonstrated that nucleotide sequences in the 3'-untranslated region of an IL-10-sensitive gene can both destabilize a stable reporter mRNA (CAT) and confer sensitivity to IL-10-mediated destabilization. Deletion and site-specific mutagenesis have mapped this to an AU-rich sequence motif similar to that found in many cytokine and growth factor mRNAs. IL-10 is able to modulate the activity of proteins capable of binding to this sequence and one or more of these may regulate the rate of mRNA degradation. Thus mechanisms through which IL-10 and IL-4 act to dampen inflammatory responses are mechanistically distinct and involve diverse intracellular signaling pathways.

Animals↗

PubMed1999

Theoretical study of conformational flexibility of tuftsin in vacuum and in aqueous environment.

Conformational flexibility of tuftsin molecule is studied using all-atom based atom-atom potential and systematic search, simulated annealing molecular dynamics (SAMD) and molecular dynamics (MD) techniques. Latter was carried out for 650 pico seconds (ps) using AMBER 4.0 with explicit water in TIP3P model. Number of inter-atomic distances and torsional angles were monitored during SAMD and MD simulation. We found that tuftsin molecule, irrespective of any starting conformation, assumes highly folded structure with strong electrostatic interaction between Lys-2 NH3 and Arg-4 carboxylic group and weak hydrogen bond between Lys-2 CO and Arg-4 NH atoms. It had distorted but stable conformation close to inverse gamma turn.

Amino Acid Sequence↗

PubMed1998

Different requirements for signal transducer and activator of transcription 1alpha and interferon regulatory factor 1 in the regulation of low molecular mass polypeptide 2 and transporter associated with antigen processing 1 gene expression.

The components of the antigen processing machinery, low molecular mass polypeptide (LMP) 2 and transporter associated with antigen processing (TAP) 1, are encoded by closely linked genes within the major histocompatibility complex class II subregion. Although the two genes share a bi-directional promoter, LMP2 and TAP1 have differential cellular expression. TAP1 is expressed constitutively. However, LMP2 expression requires induction by interferon-gamma in most cells. The regulatory elements within the LMP2/TAP1 promoter and the transcription factors that bind these elements have been defined. However, how these transactivators regulate differential TAP1 and LMP2 gene transcription is not known. We have addressed this question by analyzing three human melanoma cell lines with distinct phenotypes of LMP2 and TAP1 expression. Whereas binding of either interferon regulatory factor 1 or Stat1 to the overlapping interferon consensus sequence-2/GAS is sufficient for regulating transcription of the TAP1 gene, binding of both factors is required for LMP2 gene transcription. This conclusion is supported by restoration of LMP2 gene transcription following transfection of wild type Stat1alpha or interferon regulatory factor 1 cDNA into cells lacking these transcription factors. The flexibility in the regulation of the TAP1 gene may reflect its role in maintaining immune surveillance. Furthermore, lack of LMP2 gene transcription in quiescent human cells suggests that LMP2 expression reflects a state of cell activation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Unilateral naris occlusion and the rat accessory olfactory bulb.

Blocking airflow through half of the nasal cavity during early life results in a 25% reduction in the size of the ipsilateral main olfactory bulb. The present study indicates that the size of the accessory bulb is relatively unaffected by the procedure.

Animals↗

PubMed1998

Development and characterization of mouse anti-human LMP2, LMP7, TAP1 and TAP2 monoclonal antibodies.

Low molecular mass polypeptides (LMP) 2 and LMP7 and transporter associated with antigen processing (TAP) subunits TAP1 and TAP2 play a crucial role in antigen processing and cell surface expression of HLA class I molecules. Since monoclonal antibodies (mAb) to these molecules will facilitate the analysis of their expression, structure and function in normal and transformed cells, in the present study we have developed these reagents. Specifically anti-LMP2 and LMP7 mAb were generated from BALB/c mice immunized with specific peptides, and anti-TAP1 and TAP2 mAb from BALB/c mice immunized with respective recombinant proteins. mAb VF101-39F7 and VF101-39G5 were shown to be specific for LMP2, mAb VF103-5D5 and VF103-8C2 for LMP7, mAb VF108-1B3 and VF108-12D6 for TAP1 and mAb VF118-1E4 and VF118-2C5 for TAP2, since they reacted specifically with the corresponding immunogens in ELISA and with the corresponding LMP and TAP subunits when tested in Western blotting with human lymphoid cell extracts. Furthermore, the mAb immunoprecipitated components with the characteristic electrophoretic mobility from lymphoid cells. Both anti-LMP and anti-TAP mAb stained keratinocytes and infiltrating lymphocytes in frozen and formalin-fixed, paraffin embedded sections of normal skin in indirect immunoperoxidase reactions. Furthermore, all the mAb except mAb VF103-5D5 stained the cytoplasm of lymphoid cells in an intracytoplasmic staining reaction. The specificity and reactivity pattern of the mAb we have characterized indicate that they will be valuable reagents to analyze the cellular expression and tissue distribution of LMP and TAP subunits.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Thermodynamic characterizations of an intramolecularly hydrogen bonded C5-structure across proteinogenic residue.

Thermodynamic investigations of a smallest possible intramolecularly hydrogen bonded C5-structure, across a Thr residue, in model peptides Boc-Xxx-Thr-NH2 (Xxx = Ile, 1 or Leu, 2), indicated unusual thermal stability of the structure in non-polar medium. An analysis of van't Hoff plots, constructed from variable temperature 1H NMR data, yielded the thermodynamic parameters of a hydrogen bonded five-membered ring. The non-significance of the spatial organizations of the preceding CdeltaH3 bearing hydrophobic proteinogenic residue on the thermal stability of the C5-structure has been observed. The results revealed that the contribution of this element of secondary structure is quantifiable and the stability appeared to be roughly comparable to other intramolecularly hydrogen bonded reverse turn structures frequently observed in polypeptides and proteins.

Amides↗

PubMed1997

Interaction of the NH2-terminal domain of fibronectin with heparin. Role of the omega-loops of the type I modules.

Determinants of the interaction of the 29-kDa NH2-terminal domain of fibronectin with heparin were explored by analysis of normal and mutant recombinant NH2-terminal fibronectin fragments produced in an insect cell Baculovirus host vector system. A genomic/cDNA clone was constructed that specified a secretable human fibronectin NH2 fragment. With the use of site-directed mutagenesis a set of 29 kDa fragments was obtained that contained glycine or glutamic acid residues in place of basic residues at various candidate sites for heparin binding in the five type I modules that make up the domain. The recombinant fragment containing the wild type sequence had a nearly normal circular dichroic spectra and a melting profile, as assayed by loss of ellipticity at 228 nm, that was indistinguishable from that of the native fragment obtained by trypsinization of plasma fibronectin. A substantial proportion of the wild type recombinant fragment bound to heparin-Sepharose, where it was eluted at the same NaCl concentration as the native fragment. The wild type fragment was capable of promoting matrix-driven translocation, a morphogenetic effect in artificial extracellular matrices that depends on the interaction of the fibronectin NH2 terminus with heparin-like molecules on the surfaces of particles. Mutant fragments in which arginines predicted to be most exposed in the folded fragment were converted to glycines retained the same affinity for heparin as the wild type fragment. In contrast, a mutant fragment in which the single basic residue (Arg99) in the minor loop ("Omega-loop") of the second type I module was converted to a glycine had an essentially normal melting profile but exhibited no binding to heparin and failed to promote matrix-driven translocation. A mutant fragment in which the single basic residue (Arg52) of the first type I module was converted to a glycine also completely lacked heparin binding activity, but one in which the single basic residue (Arg191) the fourth type I module was converted to a glycine retained the ability to bind heparin. A mutant fragment in which the single basic residue (Lys143) in the Omega-loop of the third type I module was converted to a glutamic acid lacked heparin binding activity but had a CD spectrum similar to the heparin-liganded native protein and was capable of promoting matrix-driven translocation. The results indicate that multiple residues in the Omega-loops of the fibronectin NH2-terminal domain participate in its interactions with heparin. In addition, the conformation of one of the nonbinding mutants may mimic the heparin-induced structural alteration in this fibronectin domain required for certain morphogenetic events.

Amino Acid Sequence↗

PubMed1997

Characterization of rabbit antisera elicited with human LMP2- and LMP7-specific peptides and recombinant proteins.

Anti-human LMP2 and anti-human LMP7 sera with a titer of at least 1:10,000 were developed by immunizing rabbits with LMP2- and LMP7-specific peptides corresponding to C-terminal regions of each subunit or with TrxLMP2 and TrxLMP7 recombinant proteins. IgG antibodies elicited by immunization with LMP-specific peptides or recombinant proteins displayed reactivity with their respective immunogens in ELISA. Furthermore, antibodies elicited with both types of immunogens recognize native and recombinant LMP2 and LMP7 subunits in Western blotting and are able to immunoprecipitate LMP2 and LMP7 as components of the 20S proteasome from lymphoid cell lysates. In ELISA, a subpopulation of the antibodies generated with LMP peptides and recombinant proteins corresponding to one LMP subunit is cross-reactive with the other one. This antibody subpopulation was not detectable in the affinity-purified antibody populations isolated by passing antisera over the corresponding immunogen. Neither anti-LMP2 nor anti-LMP7 sera displayed cross-reactivity with the homologous proteasome subunits Delta and MB1. In immunohistochemical reactions affinity-purified anti-LMP2 and anti-LMP7 antibodies stained cells in both frozen and formalin-fixed tissue sections of normal skin. These results indicate that the anti-LMP2 and anti-LMP7 sera elicited with peptides and recombinant proteins are both useful reagents for biochemical characterization of LMP2 and LMP7 and to analyze their expression in normal and transformed cells.

Amino Acid Sequence↗