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D Nandi

Publications and source records attributed to D Nandi.

At least 37 records · Page 2Linked to original sources
PubMed1999

The complete primary structure of mouse 20S proteasomes.

The proteasome is a large multicatalytic proteinase that plays a role in the generation of peptides for presentation by major histocompatibility complex class I molecules. The 20S proteolytic core of mammalian proteasomes is assembled from a group of 17 protein subunits that generate a distinctive pattern of spots upon two-dimensional gel electrophoresis. The genes for most of these subunits have been cloned from humans and rats. We isolated cDNA clones for the mouse orthologues of ten of the subunits [PSMA1 (C2), PSMA2 (C3), PSMA3 (C8), PSMA4 (C9), PSMA5 (ZETA), PSMA6 (IOTA), PSMA7 (C6-I), PSMB2 (C7-I), PSMB3 (C10-II), and PSMB5 (X)] to complete the cloning of all of the mouse subunits. Using antisera raised against these subunits or their orthologues, we verified the identity of these proteins by two-dimensional NEPHGE-PAGE.

Amino Acid Sequence↗
PubMed1998

Physical and functional association of the major histocompatibility complex class I heavy chain alpha3 domain with the transporter associated with antigen processing.

CD8+ T lymphocytes recognize antigens as short, MHC class I-associated peptides derived by processing of cytoplasmic proteins. The transporter associated with antigen processing translocates peptides from the cytosol into the ER lumen, where they bind to the nascent class I molecules. To date, the precise location of the class I-TAP interaction site remains unclear. We provide evidence that this site is contained within the heavy chain alpha3 domain. Substitution of a 15 amino acid portion of the H-2Db alpha3 domain (aa 219-233) with the analogous MHC class II (H-2IAd) beta2 domain region (aa 133-147) results in loss of surface expression which can be partially restored upon incubation at 26 degrees C in the presence of excess peptide and beta2-microglobulin. Mutant H-2Db (Db219-233) associates poorly with the TAP complex, and cannot present endogenously-derived antigenic peptides requiring TAP-dependent translocation to the ER. However, this presentation defect can be overcome through use of an ER targeting sequence which bypasses TAP-dependent peptide translocation. Thus, the alpha3 domain serves as an important site of interaction (directly or indirectly) with the TAP complex and is necessary for TAP-dependent peptide loading and class I surface expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Immunoproteasome assembly: cooperative incorporation of interferon gamma (IFN-gamma)-inducible subunits.

LMP2, LMP7, and MECL are interferon gamma-inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous "immunoproteasomes" containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I-binding peptides.

Animals↗
PubMed1997

Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits.

The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.

Animals↗
PubMed1997

Cloning and characterization of mouse Lmp3 cDNA, encoding a proteasome beta subunit.

We isolated and sequenced a cDNA encoding mouse proteasome subunit LMP3 from a macrophage cDNA library. The gene encodes a 264-amino-acid protein with a calculated molecular mass of 29.11 kDa and an isoelectric point (pI) of 5.44. Comparison of the predicted protein sequence with that of the human and rat homologues, N3, revealed 11 and eight changes, respectively, in the cleaved NH2-terminal presequence of the precursor protein (pre-LMP3), and six and 10 changes, respectively, in the processed product. To corroborate the predicted molecular mass and pI, we analyzed LMP3 by immunoprecipitation with a mAb to human N3 that crossreacts with mouse LMP3. Precursor and processed forms of LMP3 were identified by 2D NEPHGE-PAGE, and their mobilities suggest the Lmp3 clone encodes the entire protein sequence.

Amino Acid Sequence↗
PubMed1997

Interferon-gamma independently activates the MHC class I antigen processing pathway and diminishes glucose responsiveness in pancreatic beta-cell lines.

The mouse pancreatic beta TC3 and beta TC6-F7 cell lines were used to characterize the effects of interferon-gamma (IFN-y) on beta-cell phenotype and function. Initially, intracellular and secreted insulin were compared in glucose-stimulated cells over time. A significant reduction in insulin content and secretion was observed on a per-cell basis in glucose-stimulated beta TC3 and beta TC6-F7 cells after 12 h of exposure to IFN-gamma. The steadystate level of pre-proinsulin mRNA expression was not affected by IFN-gamma. Thus, we postulate that IFN-gamma's inhibitory actions occur after transcription of pre-proinsulin genes. Time-course analysis of IFN-gamma-regulated mRNA expression of the two intra-MHC-encoded subunits of the proteasome (low-molecular-mass polypeptide [Lmp]-2 and Lmp-7) revealed a correlation between their induction and the inhibitory effects of IFN-gamma on glucose-stimulated insulin production. Increased expression of Lmp-2 and Lmp-7 mRNA was accompanied by a corresponding induction of LMP2 and LMP7 protein expression. Subsequently, major histocompatibility complex (MHC) class I cell-surface expression was significantly increased in IFN-gamma-treated beta TC3 and beta TC6-F7 cells. Exposure of IFN-gamma-treated beta-cells to a peptide aldehyde inhibitor of the proteasome (MG132) significantly attenuated MHC class I cell-surface expression but did not prevent the negative effects of IFN-gamma on glucose responsiveness. Enhanced expression of the MHC class I antigen processing and presentation pathway and diminished insulin production appear to be distinct pathological alterations in beta-cells exposed to the insulitic cytokine IFN-gamma.

Animals↗
PubMed1996

Identification of MECL-1 (LMP-10) as the third IFN-gamma-inducible proteasome subunit.

We show that six proteasome-associated proteins are induced by IFN-gamma, corresponding to three proteasome beta-type subunits and their precursors: the MHC-linked subunits (LMP-2 and LMP-7) and LMP-10. Concurrently, incorporation of LMP-9, LMP-17, and LMP-19 into proteasomes is reduced. LMP-10 appears to be the product of a previously cloned proteasome subunit gene, MECL-1. MECL-1 transcription is increased in the presence of IFN-gamma, whereas the transcription of two other proteasome genes, Lmp-15 and Lmp-3, is not affected. The three IFN-gamma-inducible subunits and their constitutively expressed counterparts contain most or all of the catalytic sites of the proteasome. Independent assortment of LMP-2, LMP-7, and LMP-10 into different proteasome complexes may thus generate up to 36 unique proteasome subsets. This may increase the repertoire of potentially antigenic peptides for presentation by MHC class I.

Animals↗
PubMed1996

Molecular and serological analysis of polymorphisms in the murine major histocompatibility complex-encoded proteasome subunits, LMP-2 and LMP-7.

LMP-2 and LMP-7, gamma-interferon-inducible subunits of the 20S proteasome, play an important role in antigen processing. To define the molecular basis of their polymorphism, we sequenced Lmp-2 and Lmp-7 cDNA from nine different strains of mice. Three allelic variants of both LMP-2 and LMP-7 were found, but all of the polymorphism in LMP-7 is clustered near the carboxyl terminus of the molecule. We confirmed the nucleotide sequence changes at the protein level in both the unprocessed and processed forms of the molecules by analysis of specific anti-LMP-2, anti-LMP-7 and anti-proteasome immunoprecipitates on two-dimensional PAGE gels. Interestingly, a single amino acid change at position 272 between LMP-7b,d,q and LMP-7k,s,f,x,g7, cas4 from glycine to arginine dramatically affects its migration on SDS-PAGE gels, suggesting the possibility of allele-specific posttranslational modification.

Amino Acid Sequence↗
PubMed1995

The genetics of proteasomes and antigen processing.

The T cell arm of the immune system of higher vertebrates is specific for antigenic peptides bound to cell surface major histocompatibility complex (MHC) molecules. These peptides are derived from two distinct pathways of antigen processing. The class I, or endogenous pathway, utilizes proteasomes and the ubiquitin system for protein degradation, with subsequent transport of the resulting peptides into the lumen of the endoplasmic reticulum by a specific peptide transporter, called TAP. The expression of distinct proteasome subsets is regulated by the cytokine gamma interferon (IFN-gamma). The class II, or exogenous pathway, utilizes the endosomal and lysosomal pathways for protein degradation, and a number of immune-specific accessory molecules including the class-II associated Invariant chain (Ii) and MHC-encoded HLA-DM (H2-DM in mouse) molecules.

Animals↗
PubMed1994

CD28-mediated costimulation is necessary for optimal proliferation of murine NK cells.

CD28, a cell surface molecule expressed on all murine T cells, plays a critical role in T cell activation. We show here that NK-1.1+ cells, a subpopulation of SCID splenocytes, and IL-2-activated NK cells express CD28, although at lower levels than alpha beta T cells. Optimal proliferation of highly purified asialo GM1+ NK cells from the SCID spleen was observed in response to stimulation with IL-2 and PMA, together with anti-CD28 or L-B7+ cells. Thus, in addition to IL-2, murine NK cells require CD28-mediated costimulatory signals for optimal proliferation. IL-2-activated NK cells produced enhanced levels of IFN-gamma and TNF in response to stimulation with anti-CD28 and PMA. On the other hand, we were unable to demonstrate that CD28 signaling had any effect on NK-mediated cytotoxicity. We conclude that the CD28 costimulatory pathway is functional in NK cells and plays an important role in their proliferation and cytokine production.

Animals↗
PubMed1993

Intrathymic differentiation of V gamma 3 T cells.

Whereas there is considerable information on the phenotypic and functional maturation of T cell receptor (TCR) alpha/beta thymocytes, comparatively little is known of the maturational processes that affect development of TCR-gamma/delta thymocytes. One class of gamma/delta T cells, those bearing the V gamma 3 gene product, are generated only during the early fetal stages of thymic development, and then migrate to the skin. Here we examine the intrathymic differentiation of these V gamma 3+ cells. The earliest V gamma 3 cells to appear in the thymus expressed low levels of TCR (V gamma 3low) and high levels of heat stable antigen (HSA). Over the next few days, V gamma 3+ thymocytes appeared which expressed high levels of TCR (V gamma 3high) and very low levels of HSA. The antigens CD5, CD45RB, and MEL14 were also differentially expressed on V gamma 3low versus V gamma 3high thymocytes, but the shift in expression was the opposite as compared with immature and mature TCR-alpha/beta thymocytes. Transfer experiments of sorted V gamma 3low/HSAhigh thymocytes to SCID thymic lobes showed that these cells were indeed the precursors of V gamma 3high/HSAlow thymocytes. The phenotype of the V gamma 3high thymocytes was similar to that of the postthymic V gamma 3+ cells found in the skin of adult mice. The differentiation of V gamma 3low in V gamma 3high thymocytes was also observed in fetal thymic organ culture. Addition of cyclosporin A (CsA) to these cultures had little effect on the appearance of V gamma 3low/HSAhigh cells, but blocked the appearance of V gamma 3high/HSAlow cells. These results show that, like alpha/beta T cells, V gamma 3+ thymocytes differentiate from TCRlow precursors to cells with a mature phenotype and that CsA inhibits this transition.

Animals↗
PubMed1992

Effect of pregnancy associated hypertension on immunoglobulin levels in newborns.

In a prospective study of 124 neonates born to mothers with normal pregnancy and pregnancy associated hypertension (PAH), serum IgG, IgA and IgM were estimated by single radial immunodiffusion technique. Significantly low levels of IgG were found in mothers having PAH, as compared to normal pregnancy (p less than 0.001), whereas IgA and IgM showed no difference in the two groups. There was no statistical difference in maternal and cord blood IgG in either the control or study group. IgG was significantly higher (p less than 0.001) in cord blood of babies born by vaginal route as compared to forceps (via vaginal route) or cesarean section. IgA and IgM levels did not vary with mode of delivery.

Adult↗
PubMed1991

Phenotypic analysis and gamma delta-T cell receptor repertoire of murine T cells associated with the vaginal epithelium.

T cells expressing alpha beta- and gamma delta-TCR are associated with the murine vaginal epithelium. A combination of phenotypic analysis of cell surface Ag and molecular analysis of the gamma- and delta-genes was used to demonstrate that vaginal gamma delta-T cells have several features that distinguish them from gamma delta-T cells present in other tissues. Three color flow cytofluorometric analysis demonstrated that freshly isolated vaginal gamma delta-T cells are CD4-CD8- and their expression of Thy-1 is strain dependent. Furthermore, specific differences in CD5, CD28, and p55IL-2 receptor expression were found between alpha beta- and gamma delta-T cells isolated from vaginal tissue. The vaginal gamma delta-T cells are predominantly CD45+, pgp-1+, HSA-, Ly-6C-, and MEL-14-. Both alpha beta- and gamma delta-T cells were absent in vaginal tissue from nude mice, although Thy-1-positive cells are present. It was also demonstrated that V gamma 4 and V delta 1 are the only gamma- and delta-genes that are expressed by vaginal cells. Sequence analysis of their junctions revealed that they express the V gamma 4 and V delta 1 sequences also found in fetal thymocytes. Furthermore, the V delta 1 sequence is identical in the vaginal cells and the gamma delta-T cells from the epidermal epithelium. We conclude that the vagina, like the skin, is a site where gamma delta-T cells with an invariant TCR exist.

Animals↗
PubMed1991

Gamma delta T cells in murine epithelia: origin, repertoire, and function.

The earliest TCR+ cells to appear during fetal development express products of the gamma and delta loci, and emerge as successive waves of cells bearing different V gamma gene products. These appear to emigrate and seed different epithelia. The TCR repertoire of the first two of these waves, V gamma 3 and V gamma 4, respectively, is extremely restricted. Whether the repertoire of these cells is restricted by selective processes or is shaped by developmental restrictions on rearrangements remains to be determined. These cells may function in surveillance for signals of trauma by recognizing self products induced by cellular stress.

Animals↗
PubMedDate not supplied

Effect of human chorionic gonadotrophin coadministration on the activities of ovarian Delta5-3beta-hydroxysteroid dehydrogenase, and 17beta-hydroxysteroid dehydrogenase, and ovarian and uterine histology in lithium chloride-treated albino rats.

Lithium chloride, a compound with clinical use in bipolar disorder, produces adverse effects on ovarian function in amphibian and rodent models. This study examined the effect of human chorionic gonadotrophin coadministration on ovarian steroidogenic and gametogenic activities in lithium chloride-treated rats. Relative ovarian and uterine weights, ovarian Delta(5)-3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase activities, folliculogenesis, uterine diameter, endometrial and myometrial thickness, and uterine luminal epithelial height were decreased significantly after lithium chloride treatment for 28 days at 1.6 mg/kg/day, the human therapeutic dose. These parameters were unchanged from the control level when subcutaneous (s.c.) human chorionic gonadotrophin (hCG) at 25 microg/kg/day was coadministered with the lithium chloride. The duration of the oestrous cycle was increased in lithium chloride-treated rat with longer metestrous and diestrous phases. Administration of hCG with lithium chloride prevented these estrous cycle alterations. We conclude that hCG can protect ovarian steroidogenic and gametogenic function after lithium chloride treatment.

17-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Gel purified LipL32: a prospective antigen for detection of leptospirosis.

BACKGROUND: Leptospirosis, a zoonosis, is a re-emerging disease, affecting populations across the globe. However, the current methods of diagnosis are time- consuming, cumbersome, imprecise or expensive. AIM: To develop an assay for differential and early diagnosis of Leptospirosis. METHODS AND MATERIAL: IgG based ELISA for evaluation of three antigens, namely, a gel-purified recombinant protein (rLipL32), secreted proteins and whole organism sonicates of Leptospira spp. The antigens were evaluated using, rabbit polyclonal antiserum and human sera samples. RESULTS: Studies with a rabbit polyclonal antiserum indicated the utility of these antigens in differentiating Leptospira from other common pathogenic organisms. Evaluation of these antigens with fifteen representative human serum samples indicated gel-purified rLipL32 to be a potentially useful antigen for detection of leptospirosis. The results obtained with IgG ELISA were correlated with the results of microscopic agglutination test (MAT). CONCLUSION: Gel-purified rLipL32 is a valuable antigen for early and accurate diagnosis of leptospirosis. Further evaluation of this assay in field conditions and larger sera samples will indicate its suitability in case of an epidemic.

Antibodies, Bacterial↗