Structure of the guinea pig neutrophil cationic peptide gene

AbstractGuinea pig neutrophils contain the antimicrobial cationic peptides GNCP-1 and GNCP-2 in the granules. In this study, the GNCP gene was isolated, and the structure was characterized. Using cDNA probes, one phage clone was isolated from a guinea pig genomic library. The gene spanned > 3 kb, and comprised three exons and two introns. Sequence analysis revealed that the gene encoded GNCP-2. Exon 1 mainly coded for the 5′ untranslated region, exon 2 coded for the prepro-peptide region of GNCP-2, and exon 3 coded for the mature peptide region of GNCP-2 and the 3′ untranslated region. Primer extension analysis indicated that the transcription initiation site was located to a thymidine residue, 93 bp upstream of the ATG initiation codon or GNCP-2 mRNA. A possible TATA box was located 24 bp upstream or the transcription start site. Interestingly, the pyrimidine-rich sequences identified in the promoter regions of the human neutrophil elastase and myeloperoxidase genes were also found in the 5′ flanking region of the GNCP-2 gene

GEP100/Arf6 Is Required for Epidermal Growth Factor-Induced ERK/Rac1 Signaling and Cell Migration in Human Hepatoma HepG2 Cells

BACKGROUND: Epidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined. METHODOLOGY/PRINCIPAL FINDINGS: We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration. CONCLUSIONS/SIGNIFICANCE: Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis

Anti-Inflammatory Action of Dexmedetomidine on Human Microglial Cells

Neuroinflammation, where inflammatory cytokines are produced in excess, contributes to the pathogenesis of delirium. Microglial cells play a central role in neuroinflammation by producing and releasing inflammatory cytokines in response to infection, tissue damage and neurodegeneration. Dexmedetomidine (DEX) is a sedative, which reduces the incidence of delirium. Thus, we hypothesized that DEX may alleviate delirium by exhibiting anti-inflammatory action on microglia. In the present study, we investigated the anti-inflammatory action of DEX on human microglial HMC3 cells. The results indicated that DEX partially suppressed the IL-6 and IL-8 production by lipopolysaccharide (LPS)-stimulated HMC3 cells as well as the phosphorylation of p38 MAPK and IκB and the translocation of NF-κB. Furthermore, DEX substantially suppressed IL-6 and IL-8 production by unstimulated HMC3 cells as wells as the phosphorylation of p38 MAPK and IκB and the translocation of NF-κB. These observations suggest that DEX exhibits anti-inflammatory action on not only LPS-stimulated but also unstimulated microglial cells via the suppression of inflammatory signaling and cytokine production

Glucosamine Downregulates the IL-1β-Induced Expression of Proinflammatory Cytokine Genes in Human Synovial MH7A Cells by <i>O</i>-GlcNAc Modification-Dependent and -Independent Mechanisms

<div><p>Osteoarthritis (OA) is one of the major joint diseases, and the synovial inflammation is involved in the pathogenesis and progression of OA. Glucosamine (GlcN) is widely used as a dietary supplement for OA, and is expected to exert the antiinflammatory action in OA. However, the detailed mechanism for the antiinflammatory action of GlcN remains poorly understood. In this study, to elucidate the molecular mechanism involved in the GlcN-medicated regulation of synovial cell activation, we comprehensively analyzed the effect of GlcN on the gene expression using a human synovial cell line MH7A by DNA microarray. The results indicated that GlcN significantly downregulates the expression of 187 genes (≤1/1.5-fold) and upregulates the expression of 194 genes (≥1.5-fold) in IL-1β-stimulated MH7A cells. Interestingly, pathway analysis indicated that among the 10 pathways into which the GlcN-regulated genes are categorized, the 4 pathways are immune-related. Furthermore, GlcN suppressed the expression of proinflammatory cytokine genes (such as IL-6, IL-8, IL-24 and TNF-α genes). In addition, GlcN-mediated <i>O</i>-GlcNAc modification was involved in the downregulation of TNF-α and IL-8 genes but not IL-6 and IL-24 genes, based on the effects of alloxan, an <i>O</i>-GlcNAc transferase inhibitor. Thus, GlcN likely exerts an antiinflammatroy action in OA by suppressing the expression of proinflammatory cytokine genes in synovial MH7A cells by <i>O</i>-GlcNAc modification-dependent and -independent mechanisms.</p></div

Pathway analysis of the GlcN-regulated genes.

<p>Pathway analysis of the GlcN-regulated genes.</p

Number of GlcN-regulated genes.

<p>Number of GlcN-regulated genes.</p

Effects of GlcN and alloxan on the levels of IL-6 and TNF-α.

<p>MH7A cells were incubated without (-) or with (+) 15 pg/ml IL-1β for 13h in the absence or presence of GlcN (5 mM) or GlcN+alloxan. IL-6 (A) and TNF-α (B) in the culture supernatants was measured by ELISA. Data are mean ± S.E. of seven to eight separate experiments. Values were compared among IL-1β-stimulated cells with or without GlcN, or GlcN+alloxan. ***<i>p</i><0.005.</p

Effect of GlcN on the expression of cytokine genes.

<p>Effect of GlcN on the expression of cytokine genes.</p

Effect of alloxan on GlcN-mediated change of gene expression.

<p>The effect of alloxan on the expression of 187 downregulated genes (≤1/1.5-fold, A) or 194 upregulated genes (≥1.5-fold, B) by GlcN (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165158#pone.0165158.s005" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165158#pone.0165158.s006" target="_blank">S3</a> Tables) was analyzed by GeneSpring Software using IL-1β-stimulated cells without (Control) or with GlcN or GlcN+alloxan. Data are average of three separate experiments, and gene expression was expressed as a ratio relative to Control.</p