Actinobacillus actinomycetemcomitans lipopolysaccharide induces interleukin-6 expression through multiple mitogen-activated protein kinase pathways in periodontal ligament fibroblasts

Actinobacillus actinomycetemcomitans plays a major role in the pathogenesis of aggressive periodontitis. Lipopolysaccharide (LPS) derived from A. actinomycetemcomitans is a key factor in inflammatory cytokine generation within periodontal tissues. In this study, we identify major mitogen-activated protein kinase (MAPK) signaling pathways induced by A. actinomycetemcomitans LPS, Escherichia coli LPS and interleukin-1 beta (IL-1 beta) in a murine periodontal ligament (mPDL) fibroblast cell line. Immunoblot analysis was used to assess the phosphorylated forms of p38, extracellular-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) MAPK following stimulation with A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta. IL-6 mRNA induction was detected via reverse transcription-polymerase chain reaction, while protein levels were quantified via enzyme-linked immunosorbent assays (ELISA). We utilized biochemical inhibitors of p38, ERK and JNK MAPK to identify the MAPK signaling pathways needed for IL-6 expression. Additional use of stable mPDL cell lines containing dominant negative mutant constructs of MAPK kinase-3 and -6 (MKK-3/6) and p38 null mutant mouse embryonic fibroblast (MEF) cells were used to substantiate the biochemical inhibitor data. Blocking p38 MAPK with SB203580 reduced the induction of IL-6 mRNA by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by > 70%, > 95% and similar to 60%, respectively. IL-6 ELISA indicated that blocking p38 MAPK reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by similar to 60%, similar to 50% and similar to 70%, respectively. All MAPK inhibitors significantly reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta whereas only p38 inhibitors consistently reduced the A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta induction of IL-6 mRNA steady-state levels. The contribution of p38 MAPK LPS-induced IL-6 expression was confirmed using MKK-3/6 dominant negative stable mPDL cell lines. Wild-type and p38 alpha(-/-) MEF cells provided additional evidence to support the role of p38 alpha MAPK in A. actinomycetemcomitans LPS-stimulated IL-6. Our results indicate that induction of IL-6 by E. coli LPS, IL-1 beta and A. actinomycetemcomitans LPS requires signaling through MKK-3-p38 alpha ERK, JNK and p38 MAPK in mPDL cells.

Genomic Strategy Identifies a Missense Mutation in WD-Repeat Domain 65 (WDR65) in an Individual With Van der Woude Syndrome

Genetic variation in the transcription factor interferon regulatory factor 6 (IRF6) causes and contributes risk for oral clefting disorders. We hypothesized that genes regulated by IRF6 are also involved in oral clefting disorders. We used five criteria to identify potential IRF6 target genes; differential gene expression in skin taken from wild-type and Irf6-deficient murine embryos, localization to the Van der Woude syndrome 2 (VWS2) locus at 1p36-1p32, overlapping expression with Irf6, presence of a conserved predicted-binding site in the promoter region, and a mutant murine phenotype that was similar to the Irf6 mutant mouse. Previously, we observed altered expression for 573 genes; 13 were located in the murine region syntenic to the VWS2 locus. Two of these genes, Wdr65 and Stratifin, met 4 of 5 criteria. Wdr65 was a novel gene that encoded a predicted protein of 1,250 amino acids with two WD domains. As potential targets for Irf6 regulation, we hypothesized that disease-causing mutations will be found in WDR65 and Stratifin in individuals with VWS or VWS-like syndromes. We identified a potentially etiologic missense mutation in WDR65 in a person with VWS who does not have an exonic mutation in IRF6. The expression and mutation data were consistent with the hypothesis that WDR65 was a novel gene involved in oral clefting. (C) 2011 Wiley-Liss, Inc.

Interação entre o gene TKN2 (KNOX-type I) e o miR156 node durante a transição de fase vegetativa para reprodutiva em tomateiro (Solanum lycopersicum)

Pós-graduação em Ciências Biológicas (Genética) - IBB

microRNA156-targeted SPL/ SBP box transcription factors regulate tomato ovary and fruit development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The Tomato (Solanum Lycopersicum cv. Micro-Tom) Natural Genetic Variation Rg1 and the DELLA Mutant Procera Control the Competence Necessary to Form Adventitious Roots and Shoots

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter's syndrome and in adaptation to a high-K diet

Type II Bartter's syndrome is a hereditary hypokalemic renal salt-wasting disorder caused by mutations in the ROMK channel (Kir1.1; Kcnj1), mediating potassium recycling in the thick ascending limb of Henle's loop (TAL) and potassium secretion in the distal tubule and cortical collecting duct (CCT). Newborns with Type II Bartter are transiently hyperkalemic, consistent with loss of ROMK channel function in potassium secretion in distal convoluted tubule and CCT. Yet, these infants rapidly develop persistent hypokalemia owing to increased renal potassium excretion mediated by unknown mechanisms. Here, we used free-flow micropuncture and stationary microperfusion of the late distal tubule to explore the mechanism of renal potassium wasting in the Romk-deficient, Type II Bartter's mouse. We show that potassium absorption in the loop of Henle is reduced in Romk-deficient mice and can account for a significant fraction of renal potassium loss. In addition, we show that iberiotoxin (IBTX)-sensitive, flow-stimulated maxi-K channels account for sustained potassium secretion in the late distal tubule, despite loss of ROMK function. IBTX-sensitive potassium secretion is also increased in high-potassium-adapted wild-type mice. Thus, renal potassium wasting in Type II Bartter is due to both reduced reabsorption in the TAL and K secretion by max-K channels in the late distal tubule. © 2006 International Society of Nephrology.

Invasin of Yersinia pseudotuberculosis is not a polyclonal activator of mouse B lymphocytes

It is known that the invasin molecule of Yersinia pseudotuberculosis stimulates human peripheral B cells in vitro. In this work we evaluated the in vivo role of invasin as polyclonal activator of B lymphocytes in the mouse experimental model, by comparing strains of Y. pseudotuberculosis expressing invasin and isogenic inv mutants. Swiss mice were infected intravenously with two strains expressing invasin (YpIII pIB1 and an isogenic virulence plasmid-cured strain, YpIII) and with two invasin mutant strains (Yp100 pIB1 and Yp100, plasmid-cured). Spleen cells were sampled on days 7, 14, 21 and 28 after infection. Immunoglobulin (Ig)-secreting spleen cells were detected by protein A plaque assay and specific antibodies were detected in sera by ELISA. The virulent strain YPIII pIB1 (wild type) did not provoke polyclonal activation of B lymphocytes in vivo. In general, fewer Ig-secreting spleen cells of all isotypes were found in the infected animals than in the control animals. Specific IgG antibodies were detected in the sera of animals infected with all strains. The peak response occurred on the 21 st day post-infection, and the Yp100 strain provoked the highest level of these antibodies. We concluded that invasin is not a polyclonal activator of murine B cells.