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Biomedical subjects

Ramnik Xavier

Publications and source records attributed to Ramnik Xavier.

At least 19 recordsLinked to original sources
PubMed2006

Vav proteins control MyD88-dependent oxidative burst.

The importance of reactive oxygen intermediate (ROI) production in antimicrobial responses is demonstrated in human patients who suffer from chronic granulomatous disease (CGD) due to defective NADPH oxidase function. Exactly how bacterial products activating Toll-like receptors (TLRs) induce oxidative burst is unknown. Here, we identify the Vav family of Rho guanine nucleotide exchange factors (GEFs) as critical mediators of LPS-induced MyD88-dependent activation of Rac2, NADPH oxidase, and ROI production using mice deficient in Vav1, Vav2, and Vav3. Vav proteins are also required for p38 MAPK activation and for normal regulation of proinflammatory cytokine production, but not for other MyD88-controlled effector pathways such as those involving JNK, COX2, or iNOS and the production of reactive nitrogen intermediates (RNIs). Thus, our data indicate that Vav specifically transduces a subset of signals emanating from MyD88.

Animals↗
PubMed2006

ABCA3 inactivation in mice causes respiratory failure, loss of pulmonary surfactant, and depletion of lung phosphatidylglycerol.

The highly branched mammalian lung relies on surfactant, a mixture of phospholipids, cholesterol, and hydrophobic proteins, to reduce intraalveolar surface tension and prevent lung collapse. Human mutations in the ABCA3 transporter have been associated with childhood respiratory disease of variable severity and onset. Here, we report the generation of Abca3 null mice, which became lethargic and cyanotic and died within 1 h of birth. Tissue blots found ABCA3 expression was highest in lung but was also detectable in other tissues, including the kidney. Gross development of kidney and lung was normal in neonatal Abca3(-/-) pups, but the mice failed to inflate their lungs, leading to death from atelectatic respiratory failure. Ultrastructural analysis of the Abca3(-/-) lungs revealed an absence of surfactant from the alveolar space and a profound loss of mature lamellar bodies, the intracellular storage organelle for surfactant. Mass spectrometry measurement of >300 phospholipids in lung tissue taken from Abca3(-/-) mice showed a dramatic reduction of phosphatidylglycerol (PG) levels as well as selective reductions in phosphatidylcholine species containing short acyl chains. These results establish a requirement of ABCA3 for lamellar body formation and pulmonary surfactant secretion and suggest a unique and critical role for the transporter in the metabolism of pulmonary PG. They also demonstrate the utility of the Abca3 null mouse as a model for a devastating human disease.

ATP-Binding Cassette Transporters↗
PubMed2006

Bcl10 is phosphorylated on Ser138 by Ca2+/calmodulin-dependent protein kinase II.

Ordered assembly of scaffold proteins Carma1-Bcl10-Malt1 determines NF-kappaB activation following T cell receptor (TCR) engagement. Carma1-Bcl10 interaction and the signaling pathway are controlled by Carma1 phosphorylation, which are induced by PKCtheta and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition to Carma1 phosphorylation, previous studies have demonstrated that Bcl10 is phosphorylated in the C-terminal Ser/Thr rich region following TCR engagement. However the kinases that phosphorylate Bcl10 are incompletely understood. Here we show that CaMKII phosphorylates Bcl10 on Ser138. Furthermore, a CaMKII inhibitor, KN93, and CaMKII siRNA substantially reduce Bcl10 phosphorylation induced by phorbol myristate acetate/ionomycin. S138A mutation prolongs Bcl10-induced NF-kappaB activation, suggesting that Bcl10 phosphorylation is involved in attenuation of NF-kappaB activation. These findings suggest that CaMKII modulates NF-kappaB activation via phosphorylating Bcl10 as well as Carma1.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Centaurin beta1 down-regulates nucleotide-binding oligomerization domains 1- and 2-dependent NF-kappaB activation.

Centaurin beta1 (CENTB1), a GTPase-activating protein, is a member of the ADP-ribosylation factor family encoded by a gene located on the short arm of human chromosome 17. A yeast two-hybrid screen first suggested a direct interaction between CENTB1 and NOD2. Co-immunoprecipitation experiments confirmed direct interaction between CENTB1 and NOD2 and demonstrated similar interaction between CENTB1 and NOD1. We also demonstrate that endogenous CENTB1 interacts with endogenous NOD2 and NOD1 in SW480 and HT-29 intestinal epithelial cells. CENTB1 partially co-localized with NOD2 and NOD1 proteins in the cytoplasm of mammalian cells. CENTB1 expression in epithelial cells was highly induced by tumor necrosis factor alpha, interleukin 1beta, and the NOD1 and NOD2 ligands (gamma-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively). In addition, CENTB1 mRNA level is increased in the inflamed mucosa of patients with inflammatory bowel disease. Functionally, CENTB1 overexpression inhibited NOD1- and NOD2-dependent activation of NF-kappaB, whereas small inhibitory RNA against CENTB1 increased NF-kappaB activation following NOD1- or NOD2-mediated recognition of the bacterial components gamma-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide, respectively. In contrast, CENTB1 had no effect on NF-kappaB activation induced by Toll-like receptors. In conclusion, CENTB1 selectively down-regulates NF-kappaB activation via NODs pathways, creating a "feedback" loop and suggesting a novel role of CENTB1 in innate immune responses to bacteria and inflammatory responses.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Paeonol attenuates TNBS-induced colitis by inhibiting NF-kappaB and STAT1 transactivation.

Paeonol, a major phenolic component of Moutan Cortex, is known to have anti-inflammatory activity. However, the effect of Paeonol on colitis has not been evaluated and the molecular mechanism of its anti-inflammatory action remains unknown. The aim of this study was to determine if Paeonol enema attenuates trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. We also investigated the effects of Paeonol in colon cancer-derived CW-2 cells and T cell leukemia-derived Jurkat cells treated with tumor necrosis factor alpha (TNFalpha) and/or interferon gamma (IFNgamma), which play critical roles in TNBS-induced colitis. Paeonol enema attenuated TNBS-induced colitis judging by body weigh reduction, colon length and histological score. Myeloperoxidase activity and inducible nitric oxide synthase (iNOS) production in the colon were also reduced with Paeonol enema. In CW-2 cells, Paeonol inhibited iNOS protein and mRNA expression induced by costimulation of TNFalpha and IFNgamma. Furthermore, Paeonol reduced TNFalpha-induced NF-kappaB transactivation and IFNgamma-induced STAT1 transactivation in CW-2 cells and also in Jurkat cells. These findings suggest that Paeonol enema may be useful for the treatment of colitis.

Acetophenones↗
PubMed2006

A molecular-properties-based approach to understanding PDZ domain proteins and PDZ ligands.

PDZ domain-containing proteins and their interaction partners are mutated in numerous human diseases and function in complexes regulating epithelial polarity, ion channels, cochlear hair cell development, vesicular sorting, and neuronal synaptic communication. Among several properties of a collection of documented PDZ domain-ligand interactions, we discovered embedded in a large-scale expression data set the existence of a significant level of co-regulation between PDZ domain-encoding genes and these ligands. From this observation, we show how integration of expression data, a comparative genomics catalog of 899 mammalian genes with conserved PDZ-binding motifs, phylogenetic analysis, and literature mining can be utilized to infer PDZ complexes. Using molecular studies we map novel interaction partners for the PDZ proteins DLG1 and CARD11. These results provide insight into the diverse roles of PDZ-ligand complexes in cellular signaling and provide a computational framework for the genome-wide evaluation of PDZ complexes.

Amino Acid Motifs↗
PubMed2006

Immune cell transcriptome datasets reveal novel leukocyte subset-specific genes and genes associated with allergic processes.

BACKGROUND: The precise function of various resting and activated leukocyte subsets remains unclear. For instance, mast cells, basophils, and eosinophils play important roles in allergic inflammation but also participate in other immunologic responses. One strategy to understand leukocyte subset function is to define the expression and function of subset-restricted molecules. OBJECTIVE: To use a microarray dataset and bioinformatics strategies to identify novel leukocyte markers as well as genes associated with allergic or innate responses. METHODS: By using Affymetrix microarrays, we generated an immune transcriptome dataset composed of gene profiles from all of the major leukocyte subsets, including rare enigmatic subsets such as mast cells, basophils, and plasma cells. We also assessed whether analysis of genes expressed commonly by certain groups of leukocytes, such as allergic leukocytes, might identify genes associated with particular responses. RESULTS: Transcripts highly restricted to a single leukocyte subset were readily identified (>2000 subset-specific transcripts), many of which have not been associated previously with leukocyte functions. Transcripts expressed exclusively by allergy-related leukocytes revealed well known as well as novel molecules, many of which presumably contribute to allergic responses. Likewise, Nearest Neighbor Analysis of genes coexpressed with Toll-like receptors identified genes of potential relevance for innate immunity. CONCLUSION: Gene profiles from all of the major human leukocyte subsets provide a powerful means to identify genes associated with single leukocyte subsets, or different types of immune response. CLINICAL IMPLICATIONS: A comprehensive dataset of gene expression profiles of human leukocytes should provide new targets or biomarkers for human inflammatory diseases.

Gene Expression Profiling↗
PubMed2006

CARMA1 is critical for the development of allergic airway inflammation in a murine model of asthma.

CARMA1 has been shown to be important for Ag-stimulated activation of NF-kappaB in lymphocytes in vitro and thus could be a novel therapeutic target in inflammatory diseases such as asthma. In the present study, we demonstrate that mice with deletion in the CARMA1 gene (CARMA1(-/-)) do not develop inflammation in a murine model of asthma. Compared with wild-type controls, CARMA1(-/-) mice did not develop airway eosinophilia, had no significant T cell recruitment into the airways, and had no evidence for T cell activation in the lung or draining lymph nodes. In addition, the CARMA1(-/-) mice had significantly decreased levels of IL-4, IL-5, and IL-13, did not produce IgE, and did not develop airway hyperresponsiveness or mucus cell hypertrophy. However, adoptive transfer of wild-type Th2 cells into CARMA1(-/-) mice restored eosinophilic airway inflammation, cytokine production, airway hyperresponsiveness, and mucus production. This is the first demonstration of an in vivo role for CARMA1 in a disease process. Furthermore, the data clearly show that CARMA1 is essential for the development of allergic airway inflammation through its role in T lymphocytes, and may provide a novel means to inhibit NF-kappaB for therapy in asthma.

Adoptive Transfer↗
PubMed2006

Nore1B regulates TCR signaling via Ras and Carma1.

Nore1A was originally identified as a potential Ras effector, and Nore1B is an alternatively spliced isoform. Both share a Ras/Rap association domain (RA domain) but only Nore1A contains sequence motifs that predict SH3 domain binding and diacylglycerol/phorbol ester binding in the amino-terminal region. Here we report that Carma1 binds to Nore1A and Nore1B through the RA domain and that Carma1 interacts with active Ras in the presence of Nore1B. RNA interference against Nore1B attenuates NF-kappaB activation induced by T cell receptor (TCR) ligation, but not NF-kappaB activation induced by TNFalpha or lipoteichoic acid. In addition, Nore1B is also required for KiRas GV12-mediated ERK1 activation and Elk1 reporter activity in T cells. We also provide evidence that knockdown of Nore1B also impairs polarized redistribution of Ras at the B cell-T cell immune interface. Together, these findings suggest that endogenous Nore1B recruits active Ras to the APC-T cell interface and mediates the interaction between Ras and Carma1.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Isolation and analysis of lipid rafts in cell-cell interactions.

Lipid rafts are dynamic structures made up of proteins and lipids that float freely within the liquid-disordered bilayer of cellular membranes and have the ability to cluster to form larger, more-ordered platforms. These clustered structures have been identified in all cell types and have been shown to play critical roles in signal transduction, cellular transport, and cell-cell communication. Lipid rafts also have been implicated in facilitating bacterial/viral entry into host cells and in human disease, highlighting the significance of understanding the role lipid rafts play in physiological and pathological signaling outcomes. In this chapter, we provide protocols to isolate lipid rafts from polarized and nonpolarized cells and outline novel technologies to analyze signal transduction cascades in vivo.

Bacterial Infections↗
PubMed2005

Membrane recruitment of NOD2 in intestinal epithelial cells is essential for nuclear factor-{kappa}B activation in muramyl dipeptide recognition.

Nucleotide oligomerization domain (NOD) 2 functions as a mammalian cytosolic pathogen recognition molecule, and mutant forms have been genetically linked to Crohn's disease (CD). NOD2 associates with the caspase activation and recruitment domain of RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK)/RIP2 and activates nuclear factor (NF)-kappaB in epithelial cells and macrophages, whereas NOD2 mutant 3020insC, which is associated with CD, shows an impaired ability to activate NF-kappaB. To gain insight into the molecular mechanisms of NOD2 function, we performed a functional analysis of deletion and substitution NOD2 mutants. NOD2, but not NOD2 3020insC mutant, associated with cell surface membranes of intestinal epithelial cells. Membrane targeting and subsequent NF-kappaB activation are mediated by two leucine residues and a tryptophan-containing motif in the COOH-terminal domain of NOD2. The membrane targeting of NOD2 is required for NF-kappaB activation after the recognition of bacterial muramyl dipeptide in intestinal epithelial cells.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2005

GRIM-19 interacts with nucleotide oligomerization domain 2 and serves as downstream effector of anti-bacterial function in intestinal epithelial cells.

Nucleotide oligomerization domain 2 (NOD2) functions as a mammalian cytosolic pathogen recognition molecule, and variants have been associated with risk for Crohn disease. We recently demonstrated that NOD2 functions as an anti-bacterial factor limiting survival of intracellular invasive bacteria. To gain further insight into the mechanism of NOD2 activation and signal transduction, we performed yeast two-hybrid screening. We demonstrate that GRIM-19, a protein with homology to the NADPH dehydrogenase complex, interacts with endogenous NOD2 in HT29 cells. GRIM-19 is required for NF-kappaB activation following NOD2-mediated recognition of bacterial muramyl dipeptide. GRIM-19 also controls pathogen invasion of intestinal epithelial cells. GRIM-19 expression is decreased in inflamed mucosa of patients with inflammatory bowel diseases. GRIM-19 may be a key component in NOD2-mediated innate mucosal responses and serve to regulate intestinal epithelial cell responses to microbes.

Animals↗
PubMed2005

PDZ domains and the politics of polarity in lymphocytes.

In this issue of Immunity, Ludford-Menting at al. (2005) show that scaffold proteins previously implicated in the control of epithelial cell polarity also play an important role in determining the macroscopic organization of the engaged T cell.

Animals↗
PubMed2005

Large-scale screens for cDNAs with in vivo activity.

The pace of biological investigation has been greatly accelerated by technologies that permit comprehensive inventory of gene expression under diverse conditions. We have been developing and using approaches to understand gene function that are based on functional assessments of gene activity, using automated expression cloning followed by gene knockdown using shRNA or by gene knockout. Some recent developments in expression screens and methods to expedite the formation of targeted mutations in mice are discussed. A resource for large scale quantitative profiling of mRNA abundance is also described.

Animals↗
PubMed2004

Wave2 activates serum response element via its VCA region and functions downstream of Rac.

WAVE2 is a member of the WASP/WAVE family of protein effectors of actin reorganization and cell movement. In this report, we demonstrate that WAVE2 overexpression induces serum response element (SRE) activation through serum response factor. A WAVE2 mutant lacking the VCA region did not induce SRE activation and actin polymerization. WAVE2-induced SRE activation was blocked by exposure of cells to Latrunculin A, or overexpression of actin mutant R62D. The DeltaVCA mutant inhibited Rac V12-induced SRE activation, suggesting that WAVE2 lies downstream of Rac. Similar deletion of the VCA domain of WASP attenuated Cdc42 V12-mediated SRE activation, suggesting that WAVE2 acts in relation to Rac as WASP acts in relation to Cdc42. WAVE2 overexpression did not activate NF-kappaB.

Actins↗
PubMed2004

Discs large (Dlg1) complexes in lymphocyte activation.

T cell antigen recognition involves the formation of a structured interface between antigen-presenting and T cells that facilitates the specific transmission of activating and desensitizing stimuli. The molecular machinery that organizes the signaling molecules and controls their disposition in response to activation remains poorly understood. We show here that in T cells Discs large (Dlg1), a PDZ domain-containing protein, is recruited upon activation to cortical actin and forms complexes with early participants in T cell activation. Transient overexpression of Dlg1 attenuates basal and Vav1-induced NFAT reporter activation. Reduction of Dlg1 expression by RNA interference enhances both CD3- and superantigen-mediated NFAT activation. Attenuation of antigen receptor signaling appears to be a complex, highly orchestrated event that involves the mutual segregation of important elements of the early signaling complex.

Actins↗
PubMed2003

Homer-3 regulates activation of serum response element in T cells via its EVH1 domain.

Drosophila enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain proteins regulate signal transduction at the neuronal and immunologic synapse. Despite shared cell biologic machinery at these synapses, the regulation of client proteins that transmit synaptic activity to the nucleus is likely to be different. Homer-3, a member of the EVH1 family, is expressed in the thymus, suggesting a role for this protein in T-cell signal transduction. Upon T-cell receptor (TCR) engagement, Homer-3 was recruited to the contact area of Jurkat cells to anti-CD3 and CD28 antibody-coated beads prior to actin accumulation and was subsequently translocated into the nucleus. Overexpression of Homer-3 reduced transcriptional activation via the serum response element (SRE) in response to anti-CD3 antibody, phorbol ester, or dominant active Ha-Ras. Consistent with these results, knockdown of Homer-3 increased SRE activation. Homer-3 coprecipitated with CCAAT/enhancer binding protein beta (C/EBP beta), one of the transcription factors that binds to the SRE and has a consensus motif binding to EVH1 domain. Moreover, Homer-3 and its EVH1 domain fragment reduced transcriptional activation of C/EBP beta. These findings suggest that Homer-3 may be involved in the regulation of SRE activation in T cells via interaction between its EVH1 domain and C/EBP beta.

Amino Acid Sequence↗