OBJECTIVE: This study aimed to investigate the regulation of histone-like nucleoid structuring protein (H-NS) on biofilm formation and cyclic diguanylate (c-di-GMP) synthesis in Vibrio parahaemolyticus RIMD2210633. METHODS: Regulatory mechanisms were analyzed by the combined utilization of crystal violet staining, quantification of c-di-GMP, quantitative real-time polymerase chain reaction, LacZ fusion, and electrophoretic-mobility shift assay. RESULTS: The deletion of hns enhanced the biofilm formation and intracellular c-di-GMP levels in V. parahaemolyticus RIMD2210633. H-NS can bind the upstream promoter-proximal DNA regions of scrA, scrG, VP0117, VPA0198, VPA1176, VP0699, and VP2979 to repress their transcription. These genes encode a group of proteins with GGDEF and/or EAL domains associated with c-di-GMP metabolism. CONCLUSION One of the mechanisms by which H-NS represses the biofilm formation by V. parahaemolyticus RIMD2210633 may be via repression of the production of intracellular c-di-GMP.
Bacterial Proteins/metabolism* ; Biofilms ; Cyclic GMP/analogs & derivatives* ; Gene Expression Regulation, Bacterial ; Gentian Violet ; Histones/metabolism* ; Vibrio parahaemolyticus/genetics*

Escherichia coli biofilm is a complex membrane aggregation produced by the adhesion and secretion of extracellular polymeric substances by E. coli cells aggregated on specific media. Pathogenic E. coli will evade the immune system and the impact of various harmful factors in the environment after the formation of biofilm, causing sustained and even fatal damage to the host. Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger ubiquitous in bacteria and plays a crucial role in regulating biofilm formation. This paper reviewed the recent studies about the role of c-di-GMP in the movement, adhesion, and EPS production mechanism of E. coli during biofilm formation, aiming to provide a basis for inhibiting E. coli biofilm from the perspective of c-di-GMP.
Bacterial Proteins/genetics* ; Biofilms ; Cyclic GMP/analogs & derivatives* ; Escherichia coli/metabolism* ; Escherichia coli Proteins/metabolism* ; Gene Expression Regulation, Bacterial

OBJECTIVETo investigate the effect and potential mechanisms of rutaecarpine (Rut) in a rat artery balloon-injury model.

METHODSThe intimal hyperplasia model was established by rubbing the endothelia with a balloon catheter in the common carotid artery (CCA) of rats. Fifty rats were randomly divided into five groups, ie. sham, model, Rut (25, 50 and 75 mg/kg) with 10 rats of each group. The rats were treated with or without Rut (25, 50, 75 mg/kg) by intragastric administration for 14 consecutive days following injury. The morphological changes of the intima were evaluated by hematoxylin-eosin staining. The expressions of proliferating cell nuclear antigen (PCNA) and smooth muscle (SM) α-actin in the ateries were assayed by immunohistochemical staining. The mRNA expressions of c-myc, extracellular signal-regulated kinase 2 (ERK2), MAPK phosphatase-1 (MKP-1) and endothelial nitric oxide synthase (eNOS) were determined by real-time reverse transcription-polymerase chain reaction. The protein expressions of MKP-1 and phosphorylated ERK2 (p-ERK2) were examined by Western blotting. The plasma contents of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) were also determined.

RESULTSCompared with the model group, Rut treatment significantly decreased intimal thickening and ameliorated endothelial injury (P<0.05 or P<0.01). The positive expression rate of PCNA was decreased, while the expression rate of SM α-actin obviously increased in the vascular wall after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01). Furthermore, the mRNA expressions of c-myc, ERK2 and PCNA were downregulated while the expressions of eNOS and MKP-1 were upregulated (P<0.05 or P<0.01). The protein expressions of MKP-1 and the phosphorylation of ERK2 were upregulated and downregulated after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01), respectively. In addition, Rut dramatically reversed balloon injury-induced decrease of NO and cGMP in the plasma (P<0.05 or P<0.01).

CONCLUSIONRut could inhibit the balloon injury-induced carotid intimal hyperplasia in rats, possibly mediated by promotion of NO production and inhibiting ERK2 signal transduction pathways.

Actins ; metabolism ; Animals ; Carotid Arteries ; drug effects ; metabolism ; pathology ; Carotid Artery Injuries ; drug therapy ; genetics ; pathology ; Cyclic GMP ; blood ; Disease Models, Animal ; Gene Expression Regulation ; drug effects ; Hyperplasia ; Indole Alkaloids ; pharmacology ; therapeutic use ; Male ; Nitric Oxide ; blood ; Phosphorylation ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Quinazolines ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Tunica Intima ; drug effects ; pathology

RNA helicases are involved in almost every aspect of RNA, from transcription to RNA decay. DExD/H-box helicases comprise the largest SF2 helicase superfamily, which are characterized by two conserved RecA-like domains. In recent years, an increasing number of unexpected functions of these proteins have been discovered. They play important roles not only in innate immune response but also in diseases like cancers and chronic hepatitis C. In this review, we summarize the recent literatures on one member of the SF2 superfamily, the DEAD-box protein DDX41. After bacterial or viral infection, DNA or cyclic-di-GMP is released to cells. After phosphorylation of Tyr414 by BTK kinase, DDX41 will act as a sensor to recognize the invaders, followed by induction of type I interferons (IFN). After the immune response, DDX41 is degraded by the E3 ligase TRIM21, using Lys9 and Lys115 of DDX41 as the ubiquitination sites. Besides the roles in innate immunity, DDX41 is also related to diseases. An increasing number of both inherited and acquired mutations in DDX41 gene are identified from myelodysplastic syndrome and/or acute myeloid leukemia (MDS/AML) patients. The review focuses on DDX41, as well as its homolog Abstrakt in Drosophila, which is important for survival at all stages throughout the life cycle of the fly.
Agammaglobulinaemia Tyrosine Kinase ; Animals ; Bacterial Infections ; genetics ; immunology ; Cyclic GMP ; analogs & derivatives ; genetics ; immunology ; DEAD-box RNA Helicases ; genetics ; immunology ; Drosophila Proteins ; genetics ; immunology ; Drosophila melanogaster ; Humans ; Leukemia, Myeloid, Acute ; genetics ; immunology ; Mutation ; Myelodysplastic Syndromes ; genetics ; immunology ; Nuclear Proteins ; genetics ; immunology ; Protein-Tyrosine Kinases ; genetics ; immunology ; Virus Diseases ; genetics ; immunology

YfiBNR is a recently identified bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) signaling system in opportunistic pathogens. It is a key regulator of biofilm formation, which is correlated with prolonged persistence of infection and antibiotic drug resistance. In response to cell stress, YfiB in the outer membrane can sequester the periplasmic protein YfiR, releasing its inhibition of YfiN on the inner membrane and thus provoking the diguanylate cyclase activity of YfiN to induce c-di-GMP production. However, the detailed regulatory mechanism remains elusive. Here, we report the crystal structures of YfiB alone and of an active mutant YfiB(L43P) complexed with YfiR with 2:2 stoichiometry. Structural analyses revealed that in contrast to the compact conformation of the dimeric YfiB alone, YfiB(L43P) adopts a stretched conformation allowing activated YfiB to penetrate the peptidoglycan (PG) layer and access YfiR. YfiB(L43P) shows a more compact PG-binding pocket and much higher PG binding affinity than wild-type YfiB, suggesting a tight correlation between PG binding and YfiB activation. In addition, our crystallographic analyses revealed that YfiR binds Vitamin B6 (VB6) or L-Trp at a YfiB-binding site and that both VB6 and L-Trp are able to reduce YfiB(L43P)-induced biofilm formation. Based on the structural and biochemical data, we propose an updated regulatory model of the YfiBNR system.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Binding Sites ; Biofilms ; Crystallography, X-Ray ; Cyclic GMP ; analogs & derivatives ; metabolism ; Dimerization ; Molecular Dynamics Simulation ; Molecular Sequence Data ; Mutagenesis ; Protein Structure, Quaternary ; Pseudomonas aeruginosa ; metabolism ; Sequence Alignment ; Tryptophan ; chemistry ; metabolism ; Vitamin B 6 ; chemistry ; metabolism

Vascular smooth muscle cells (VSMCs) undergo phenotypic changes in response to vascular injury such as angioplasty. Protein kinase G (PKG) has an important role in the process of VSMC phenotype switching. In this study, we examined whether rosiglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma agonist, could modulate VSMC phenotype through the PKG pathway to reduce neointimal hyperplasia after angioplasty. In vitro experiments showed that rosiglitazone inhibited the phenotype change of VSMCs from a contractile to a synthetic form. The platelet-derived growth factor (PDGF)-induced reduction of PKG level was reversed by rosiglitazone treatment, resulting in increased PKG activity. This increased activity of PKG resulted in phosphorylation of vasodilator-stimulated phosphoprotein at serine 239, leading to inhibited proliferation of VSMCs. Interestingly, rosiglitazone did not change the level of nitric oxide (NO) or cyclic guanosine monophosphate (cGMP), which are upstream of PKG, suggesting that rosiglitazone influences PKG itself. Chromatin immunoprecipitation assays for the PKG promoter showed that the activation of PKG by rosiglitazone was mediated by the increased binding of Sp1 on the promoter region of PKG. In vivo experiments showed that rosiglitazone significantly inhibited neointimal formation after balloon injury. Immunohistochemistry staining for calponin and thrombospondin showed that this effect of rosiglitazone was mediated by modulating VSMC phenotype. Our findings demonstrate that rosiglitazone is a potent modulator of VSMC phenotype, which is regulated by PKG. This activation of PKG by rosiglitazone results in reduced neointimal hyperplasia after angioplasty. These results provide important mechanistic insight into the cardiovascular-protective effect of PPARgamma.
Animals ; Aorta/injuries/metabolism/*pathology ; Calcium-Binding Proteins/genetics/metabolism ; Cell Proliferation ; Cyclic GMP/metabolism ; Cyclic GMP-Dependent Protein Kinases/genetics/*metabolism ; Hyperplasia/metabolism ; Microfilament Proteins/genetics/metabolism ; Muscle, Smooth, Vascular/metabolism/pathology ; Myocytes, Smooth Muscle/drug effects/*metabolism ; Nitric Oxide/metabolism ; PPAR gamma/agonists/*metabolism ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Sp1 Transcription Factor/metabolism ; Thiazolidinediones/pharmacology ; Thrombospondins/genetics/metabolism ; Tunica Intima/metabolism/*pathology ; Vascular System Injuries/*metabolism/pathology

Initial skirmishes between the host and pathogen result in spillage of the contents of the bacterial cell. Amongst the spillage, the secondary messenger molecule, cyclic dimeric guanosine monophosphate (c di-GMP), was recently shown to be bound by stimulator of interferon genes (STING). Binding of c di-GMP by STING activates the Tank Binding Kinase (TBK1) mediated signaling cascades that galvanize the body's defenses for elimination of the pathogen. In addition to c di-GMP, STING has also been shown to function in innate immune responses against pathogen associated molecular patterns (PAMPs) originating from the DNA or RNA of pathogens. The pivotal role of STING in host defense is exemplified by the fact that STING(-/-) mice die upon infection by HSV-1. Thus, STING plays an essential role in innate immune responses against pathogens. This opens up an exciting possibility of targeting STING for development of adjuvant therapies to boost the immune defenses against invading microbes. Similarly, STING could be targeted for mitigating the inflammatory responses augmented by the innate immune system. This review summarizes and updates our current understanding of the role of STING in innate immune responses and discusses the future challenges in delineating the mechanism of STING-mediated responses.
Animals ; Cyclic GMP ; physiology ; Dimerization ; Herpes Simplex ; immunology ; pathology ; Humans ; Immunity, Innate ; Membrane Proteins ; chemistry ; genetics ; metabolism ; Protein Binding ; RNA, Viral ; metabolism ; STAT6 Transcription Factor ; metabolism ; Second Messenger Systems

Curcumin, an active ingredient of dietary spice used in curry, has been shown to exhibit anti-oxidant, anti-inflammatory and anti-proliferative properties. Using EB directed differentiation protocol of H-9 human embryonic stem (ES) cells; we evaluated the effect of curcumin (0-20 μmol/L) in enhancing such differentiation. Our results using real time PCR, western blotting and immunostaining demonstrated that curcumin significantly increased the gene expression and protein levels of cardiac specific transcription factor NKx2.5, cardiac troponin I, myosin heavy chain, and endothelial nitric oxide synthase during ES cell differentiation. Furthermore, an NO donor enhanced the curcumin-mediated induction of NKx2.5 and other cardiac specific proteins. Incubation of cells with curcumin led to a dose dependent increase in intracellular nitrite to the same extent as giving an authentic NO donor. Functional assay for second messenger(s) cyclic AMP (cAMP) and cyclic GMP (cGMP) revealed that continuous presence of curcumin in differentiated cells induced a decrease in the baseline levels of cAMP but it significantly elevated baseline contents of cGMP. Curcumin addition to a cell free assay significantly suppressed cAMP and cGMP degradation in the extracts while long term treatment of intact cells with curcumin increased the rates of cAMP and cGMP degradation suggesting that this might be due to direct suppression of some cyclic nucleotide-degrading enzyme (phosphodiesterase) by curcumin. These studies demonstrate that polyphenol curcumin may be involved in differentiation of ES cells partly due to manipulation of nitric oxide signaling.
Animals ; Antioxidants ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Curcumin ; pharmacology ; Cyclic GMP ; metabolism ; Embryoid Bodies ; drug effects ; metabolism ; physiology ; Enzyme Activators ; pharmacology ; Gene Expression ; drug effects ; Guanylate Cyclase ; genetics ; metabolism ; Homeobox Protein Nkx-2.5 ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Mice ; Myosin Heavy Chains ; genetics ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Donors ; pharmacology ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; Nitroso Compounds ; pharmacology ; Pyrazoles ; pharmacology ; Pyridines ; pharmacology ; Second Messenger Systems ; Transcription Factors ; genetics ; metabolism ; Troponin ; genetics ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg(559) through a negatively charged β-turn at its RA domain. Moreover, the three Glu residues (180-182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our study also demonstrates that substitution of Lys140 for Ala or in combination with polyglutamte mutants of Grb14 results in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu(180-182) and Lys(140), other residues in Grb14 may be involved in the electrostatic interaction with CNGA1. The RA domain is highly conserved among the members of Grb7 family of proteins, which includes Grb7, Grb10 and Grb14. Further, only Grb14 is able to modulate the channel activity, but not Grb7 and Grb10. All together, it suggests the existence of a divergence in RA domains among the members of the Grb7 family.
Animals ; Calcium ; metabolism ; Cattle ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide-Gated Cation Channels ; chemistry ; genetics ; metabolism ; GRB10 Adaptor Protein ; genetics ; metabolism ; Humans ; Kidney ; cytology ; metabolism ; Models, Molecular ; Protein Conformation ; Protein-Tyrosine Kinases ; genetics ; metabolism ; Static Electricity

Animals integrate various environmental stimuli within the nervous system to generate proper behavioral responses. However, the underlying neural circuits and molecular mechanisms are largely unknown. The insulin-like signaling pathway is known to regulate dauer formation, fat metabolism, and longevity in Caenorhabditis elegans (C. Elegans). Here, we show that this highly conserved signaling pathway also functions in the integrative response to an olfactory diacetyl and a gustatory Cu(2+) stimuli. Worms of wild-type N2 Bristol displayed a strong avoidance to the Cu(2+) barrier in the migration pathway to the attractive diacetyl. Mutants of daf-2 (insulin receptor), daf-18 (PTEN lipid phosphatase), pdk-1 (phosphoinositide-dependent kinase), akt-1/-2 (Akt/PKB kinase) and sgk-1 (serum- and glucocorticoid-inducible kinase) show severe defects in the elusion from the Cu(2+). Mutations in DAF-16, a forkhead-type transcriptional factor, suppress the integrative defects of daf-2 and akt-1/-2 mutants. We further report that neither cGMP nor TGFβ pathways, two other dauer formation regulators, likely plays a role in the integrative learning. These results suggest that the insulin-like signaling pathway constitutes an essential component for sensory integration and decision-making behavior plasticity.
Animals ; Caenorhabditis elegans ; genetics ; physiology ; Caenorhabditis elegans Proteins ; genetics ; physiology ; Chemotaxis ; genetics ; physiology ; Copper ; physiology ; Cyclic GMP ; genetics ; physiology ; Diacetyl ; metabolism ; Insulin ; metabolism ; Longevity ; Signal Transduction ; Smell ; genetics ; physiology ; Taste ; genetics ; physiology ; Transforming Growth Factor beta ; genetics ; physiology