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Sample records for cyclic gmp protects

  1. Cyclic GMP transporters.

    PubMed

    Sager, Georg

    2004-11-01

    The biokinetics of guanosine 3',5'-cyclic monophosphate (cGMP) is characterized by three distinct processes: synthesis by guanylate cyclases (GCs), conversion of cGMP to GMP by cyclic nucleotide phosphodiesterases (PDEs) and the excretion of unchanged cGMP by transport proteins in the cell membrane. Efflux is observed in virtually all cell types including cells which originate from brain. Studies of intact cells, in which metabolic inhibitors and probenecid reduced extrusion of cGMP and wherein cGMP was extruded against concentration gradients, indicated the existence of ATP requiring organic anion transport system(s). Functional studies of inside-out vesicles have revealed cGMP transport systems wherein translocation is coupled to hydrolysis of ATP. The extrusion of cGMP is inhibited by a number of unrelated compounds and this indicates that cGMP is substrate for multispecific transporters. Recent transfection studies suggest that members of the MRP (multidrug resistance protein) family; MRP4, MRP5 and MRP8 translocate cGMP across the cell membrane. Many of the MRPs have been detected in brain. In addition tertiary active transport by the organic anion transporter family has also been identified. At least one member (OAT1) shows relative high affinity for cGMP and is also expressed in brain. The biological significance of cGMP transporters has to be clarified. Their role in cGMP biokinetics, being responsible for one of the cellular elimination pathways, is well established. However, there is growing evidence that extracellular cGMP has effects on cell physiology and pathophysiology by an auto- or paracrine mechanism.

  2. Diversity of Cyclic Di-GMP-Binding Proteins and Mechanisms

    PubMed Central

    2015-01-01

    ABSTRACT Cyclic di-GMP (c-di-GMP) synthetases and hydrolases (GGDEF, EAL, and HD-GYP domains) can be readily identified in bacterial genome sequences by using standard bioinformatic tools. In contrast, identification of c-di-GMP receptors remains a difficult task, and the current list of experimentally characterized c-di-GMP-binding proteins is likely incomplete. Several classes of c-di-GMP-binding proteins have been structurally characterized; for some others, the binding sites have been identified; and for several potential c-di-GMP receptors, the binding sites remain to be determined. We present here a comparative structural analysis of c-di-GMP-protein complexes that aims to discern the common themes in the binding mechanisms that allow c-di-GMP receptors to bind it with (sub)micromolar affinities despite the 1,000-fold excess of GTP. The available structures show that most receptors use their Arg and Asp/Glu residues to bind c-di-GMP monomers, dimers, or tetramers with stacked guanine bases. The only exception is the EAL domains that bind c-di-GMP monomers in an extended conformation. We show that in c-di-GMP-binding signature motifs, Arg residues bind to the O-6 and N-7 atoms at the Hoogsteen edge of the guanine base, while Asp/Glu residues bind the N-1 and N-2 atoms at its Watson-Crick edge. In addition, Arg residues participate in stacking interactions with the guanine bases of c-di-GMP and the aromatic rings of Tyr and Phe residues. This may account for the presence of Arg residues in the active sites of every receptor protein that binds stacked c-di-GMP. We also discuss the implications of these structural data for the improved understanding of the c-di-GMP signaling mechanisms. PMID:26055114

  3. Cyclic GMP signaling in cardiovascular pathophysiology and therapeutics

    PubMed Central

    Tsai, Emily J.; Kass, David A.

    2009-01-01

    Cyclic guanosine 3′,5′-monophosphate (cGMP) mediates a wide spectrum of physiologic processes in multiple cell types within the cardiovascular system. Dysfunctional signaling at any step of the cascade--- cGMP synthesis, effector activation, or catabolism--- have been implicated in numerous cardiovascular diseases, ranging from hypertension to atherosclerosis to cardiac hypertrophy and heart failure. In this review, we outline each step of the cGMP signaling cascade and discuss its regulation and physiologic effects within the cardiovascular system. In addition, we illustrate how cGMP signaling becomes dysregulated in specific cardiovascular disease states. The ubiquitous role cGMP plays in cardiac physiology and pathophysiology presents great opportunities for pharmacologic modulation of the cGMP signal in the treatment of cardiovascular diseases. We detail the various therapeutic interventional strategies that have been developed or are in development, summarizing relevant preclinical and clinical studies. PMID:19306895

  4. Cyclic GMP diffusion coefficient in rod photoreceptor outer segments.

    PubMed

    Koutalos, Y; Nakatani, K; Yau, K W

    1995-01-01

    Cyclic GMP (cGMP) is the intracellular messenger that mediates phototransduction in retinal rods. As photoisomerizations of rhodopsin molecules are local events, the longitudinal diffusion of cGMP in the rod outer segment should be a contributing factor to the response of the cell to light. We have employed the truncated rod outer segment preparation from bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) to measure the cGMP diffusion coefficient. In this preparation, the distal portion of a rod outer segment was drawn into a suction pipette for measuring membrane current, and the rest of the cell was then sheared off with a glass probe, allowing bath cGMP to diffuse into the outer segment and activate the cGMP-gated channels on the surface membrane. Addition and removal of bath cGMP were fast enough to produce effectively step changes in cGMP concentration at the open end of the outer segment. When cGMP hydrolysis is inhibited by isobutylmethylxanthine (IBMX), the equation for the diffusion of cGMP inside the truncated rod outer segment has a simple analytical solution, which we have used to analyze the rise and decay kinetics of the cGMP-elicited currents. From these measurements we have obtained a cGMP diffusion coefficient of approximately 70 x 10(-8) cm2 s-1 for bullfrog rods and approximately 60 x 10(-8) cm2 s-1 for tiger salamander rods. These values are six to seven times lower than the expected value in aqueous solution. The estimated diffusion coefficient is the same at high (20-1000 microM) and low (5-10 microM) concentrations of cGMP, suggesting no significant effect from buffering over these concentration ranges.

  5. Cyclic GMP controls R. centenum cyst development

    PubMed Central

    Marden, Jeremiah N.; Dong, Qian; Roychowdhury, Sugata; Berleman, James E.; Bauer, Carl. E.

    2014-01-01

    Adenylyl cyclases are widely distributed across all kingdoms whereas guanylyl cyclases are generally thought to be restricted to eukaryotes. Here we report that the α–proteobacterium Rhodospirillum centenum secretes cGMP when developing cysts and that a guanylyl cyclase deletion strain fails to synthesize cGMP and is defective in cyst formation. The R. centenum cyclase was purified and shown to effectively synthesize cGMP from GTP in vitro, demonstrating that it is a functional guanylyl cyclase. A homolog of the Escherichia coli cAMP receptor protein (CRP) is linked to the guanylyl cyclase and when deleted is deficient in cyst development. Isothermal calorimetry (ITC) and differential scanning fluorimetry (DSF) analyses demonstrate that the recombinant CRP homolog preferentially binds to, and is stabilized by cGMP, but not cAMP. This study thus provides evidence that cGMP has a crucial role in regulating prokaryotic development. The involvement of cGMP in regulating bacterial development has broader implications as several plant-interacting bacteria contain a similar cyclase coupled by the observation that Azospirillum brasilense also synthesizes cGMP when inducing cysts. PMID:21214648

  6. Nitric oxide-cyclic GMP signaling in stem cell differentiation

    PubMed Central

    Mujoo, Kalpana; Krumenacker, Joshua S.; Murad, Ferid

    2011-01-01

    The nitric oxide-cyclic GMP (NO-cGMP) pathway mediates important physiological functions associated with various integrative body systems including the cardiovascular and nervous systems. Furthermore, NO regulates cell growth, survival, apoptosis, proliferation and differentiation at the cellular level. To understand the significance of the NO-cGMP pathway in development and differentiation, studies have been conducted both in developing embryos and stem cells. Manipulation of the NO-cGMP pathway by employing activators and inhibitors as pharmacological probes and/or genetic manipulation of NO signaling components has implicated the involvement of this pathway in regulation of stem cell differentiation. This review will focus on some of the work pertaining to the role of NO-cGMP in differentiation of stem cells into cells of various lineages particularly into myocardial cells and stem cell based therapy. PMID:22019632

  7. Cyclic AMP and cyclic GMP levels in glandular stomach of restrained rats.

    PubMed

    Zarrindast, M R; Sharghi, G; Gerayesh-Nejad, S; Djahanguiri, B

    1977-10-01

    Cyclic AMP and cyclic GMP were measured in glandular stomach of rats subjected to saline administration, cold (4 degrees C), restraint and restraint+cold after 15, 30, 60, 90 and 120 minutes. All animals subjected to restraint+cold had gastric ulceration after 2 hours. A significant but transient decrease in cAMP was observed 15 minutes after restraint+cold. A marked, sustained and significant decrease of cGMP was observed in the same group of animals. It is concluded that it seems unlikely to be a correlation between cAMP and cGMP changes of the stomach and the restraint-induced gastric ulceration.

  8. Dexamethasone-dependent modulation of cyclic GMP synthesis in podocytes.

    PubMed

    Lewko, Barbara; Waszkiewicz, Anna; Maryn, Anna; Gołos, Magdalena; Latawiec, Elżbieta; Daca, Agnieszka; Witkowski, Jacek M; Angielski, Stefan; Stępiński, Jan

    2015-11-01

    Podocytes may be direct target for glucocorticoid therapy in glomerular proteinuric disease. Permeability of podocytes largely depends on their capacity to migrate which involves the contractile apparatus in their foot processes. In this study, we examined the effect of synthetic glucocorticoid dexamethasone (DEX) on the ability of podocytes to produce cyclic guanosine monophosphate (cGMP) in the presence of vasoactive factors, atrial natriuretic peptide (ANP), nitric oxide (NO), and angiotensin II (Ang II). We investigated also the effects of cGMP and DEX on podocyte motility. Primary rat podocytes and immortalized mouse podocytes were pretreated with 1 µM DEX for 4 or 24 h. Glomerular hypertension was mimicked by subjecting the cells to mechanical stress. Total and subcellular cGMP levels were determined in podocytes incubated with 0.1 µM ANP, 1 µM S-nitroso-N-acetyl penicillamine (SNAP), and 1 µM Ang II. Cell motility was estimated by a wound-healing assay. The ANP-dependent production of cGMP increased after 4 h exposition to DEX, but was attenuated after 24 h. Adversely, a 24-h pretreatment with DEX augmented the NO-dependent cGMP synthesis. Ang II suppressed the ANP-dependent cGMP production and the effect was enhanced by DEX in mechanical stress conditions. Mechanical stress reduced total cGMP production in the presence of all stimulators, whereas extracellular to total cGMP ratio increased. 8-Br cGMP enhanced podocyte migration which was accompanied by F-actin disassembly. In the presence of DEX these effects were prevented. We conclude that DEX modulates the production of cGMP in podocytes stimulated with vasoactive factors such as Ang II, ANP, and NO, and the effect is time-dependent. cGMP increases podocyte motility, which is prevented by DEX. This mechanism may account for the antiproteinuric effect of glucocorticoids.

  9. Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.

    PubMed

    Suhasini, M; Li, H; Lohmann, S M; Boss, G R; Pilz, R B

    1998-12-01

    Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by

  10. RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP.

    PubMed

    Kellenberger, Colleen A; Wilson, Stephen C; Sales-Lee, Jade; Hammond, Ming C

    2013-04-03

    Cyclic dinucleotides are an important class of signaling molecules that regulate a wide variety of pathogenic responses in bacteria, but tools for monitoring their regulation in vivo are lacking. We have designed RNA-based fluorescent biosensors for cyclic di-GMP and cyclic AMP-GMP by fusing the Spinach aptamer to variants of a natural GEMM-I riboswitch. In live cell imaging experiments, these biosensors demonstrate fluorescence turn-on in response to cyclic dinucleotides, and they were used to confirm in vivo production of cyclic AMP-GMP by the enzyme DncV.

  11. Preparation of stable sup 125 I cyclic GMP tyrosine methyl ester suitable for 3',5' cyclic GMP radioimmunoassay by HPLC

    SciTech Connect

    Thompson, M.R.; Luttrell, M.; Giannella, R.A. )

    1990-01-01

    Determination of the concentration of cyclic guanosine monophosphate (cGMP) by radioimmunoassay (RIA) depends upon the availability of suitable radiolabeled tracers and antibody to detect the product. Reverse phase chromatographic techniques can easily separate the reaction products of chloramine-T iodination of succinyl cGMP tyrosine methyl ester. The binding characteristics of the iodination reaction products to anti-cGMP antibody have been determined. Purified succinyl cyclic nucleotide 125I-tyrosine methyl ester binds to cGMP antisera identically as commercially available tracer. The tracer is stable for greater than three months.

  12. Cyclic GMP-AMP Displays Mucosal Adjuvant Activity in Mice

    PubMed Central

    Škrnjug, Ivana

    2014-01-01

    The recently discovered mammalian enzyme cyclic GMP-AMP synthase produces cyclic GMP-AMP (cGAMP) after being activated by pathogen-derived cytosolic double stranded DNA. The product can stimulate STING-dependent interferon type I signaling. Here, we explore the efficacy of cGAMP as a mucosal adjuvant in mice. We show that cGAMP can enhance the adaptive immune response to the model antigen ovalbumin. It promotes antigen specific IgG and a balanced Th1/Th2 lymphocyte response in immunized mice. A characteristic of the cGAMP-induced immune response is the slightly reduced induction of interleukin-17 as a hallmark of Th17 activity – a distinct feature that is not observed with other cyclic di-nucleotide adjuvants. We further characterize the innate immune stimulation activity in vitro on murine bone marrow-derived dendritic cells and human dendritic cells. The observed results suggest the consideration of cGAMP as a candidate mucosal adjuvant for human vaccines. PMID:25295996

  13. A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A.

    PubMed

    Hung, Su H; Liu, Andy H; Pixley, Robin A; Francis, Penelope; Williams, LaTeeka D; Matsko, Christopher M; Barnes, Karine D; Sivendran, Sharmila; Colman, Roberta F; Colman, Robert W

    2008-06-01

    The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3',5'-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K(I)=43.4+/-7.2muM and k(cart)=0.007+/-0.0006 min(-1)). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K(d)=72 microM)>Sp-cGMPS (124), Sp-cAMPS (182)>GMP (1517), Rp-cAMPS (3762), AMP (4370 microM). NAD(+), neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.

  14. Brucella melitensis cyclic di-GMP phosphodiesterase BpdA controls expression of flagellar genes.

    PubMed

    Petersen, Erik; Chaudhuri, Pallab; Gourley, Chris; Harms, Jerome; Splitter, Gary

    2011-10-01

    Brucella melitensis encounters a variety of conditions and stimuli during its life cycle--including environmental growth, intracellular infection, and extracellular dissemination--which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP.

  15. Pharmacological modulation of secondary mediator systems--cyclic AMP and cyclic GMP--on inflammatory hyperalgesia.

    PubMed

    Cunha, F Q; Teixeira, M M; Ferreira, S H

    1999-06-01

    1. The objective of the present paper was to evaluate the relevance of neuronal balance of cyclic AMP and cyclic GMP concentration for functional regulation of nociceptor sensitivity during inflammation. 2. Injection of PGE2 (10-100 ng paw-1) evoked a dose-dependent hyperalgesic effect which was mediated via a cyclic AMP-activated protein kinase (PKA) inasmuch as hyperalgesia was blocked by the PKA inhibitor H89. 3. The PDE4 inhibitor rolipram and RP73401, but not PDE3 and PDE5 inhibitors potentiated the hyperalgesic effects of PGE2. The hyperalgesic effect of dopamine was also enhanced by rolipram. Moreover, rolipram significantly potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. This suggests that neuronal cyclic AMP mediates the prostanoid and sympathetic components of mechanical hyperalgesia. Moreover, in the neuron cyclic AMP is mainly metabolized by PDE4. 4. To examine the role of the NO/cyclic GMP pathway in modulating mechanical hyperalgesia, we tested the effects of the soluble guanylate cyclase inhibitor, ODQ. This substance counteracts the inhibitory effects of the NO donor, SNAP, on the hyperalgesia induced by PGE2. 5. The ODQ potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. In contrast, ODQ had no significant effect on the hyperalgesia induced by PGE2 and dopamine. This indicates that the hyperalgesic cytokines may activate soluble guanylate cyclase, which down-regulate the ability of these substances to cause hyperalgesia. This event appears not to be mediated by prostaglandin or dopamine. 6. In conclusion, the results presented in this paper confirm an association between (i) hyperalgesia and elevated levels of cyclic AMP as well as (ii) antinociception and elevated levels of cyclic GMP. The intracellular levels of cyclic AMP that enhance hyperalgesia are controlled by the PDE4 isoform and appear to result in activation of protein kinase A whereas the

  16. Aluminum alters cyclic AMP and cyclic GMP levels but not presynaptic cholinergic markers in rat brain in vivo.

    PubMed

    Johnson, G V; Jope, R S

    1987-02-10

    Oral administration of 0.3% aluminum (citrate or sulfate salt) for 4 weeks significantly elevated adenosine 3',5'-monophosphate (cyclic AMP) levels in rat cortex, hippocampus, striatum and cerebellum. The largest effect observed was a 60% increase in cortical cyclic AMP levels in rats administered aluminum sulfate. The effects of orally administered aluminum on guanosine 3',5'-monophosphate (cyclic GMP) levels were less widespread. Dietary aluminum citrate only elevated cyclic GMP levels in the hippocampus, while aluminum sulfate caused significant increases in the cerebellum, hippocampus and striatum. Aluminum citrate administered i.c.v. (1 mumol, 2 weeks postadministration) elevated cyclic AMP levels in the cortex, but had no effect on cyclic GMP levels. Aluminum administered either orally or i.c.v. had no effect on in vivo acetylcholine levels. However, dietary aluminum citrate significantly reduced choline levels in the cortex, hippocampus and striatum. Aluminum administered i.c.v. had no effect on choline acetyltransferase activity or on high-affinity choline transport. These results indicate that: the metabolism of cyclic AMP and of cyclic GMP are more sensitive to aluminum than are presynaptic cholinergic processes; the metabolism of cyclic AMP is more sensitive to the effects of aluminum than is the metabolism of cyclic GMP; and cortical cAMP metabolism is the most sensitive to the presence of aluminum. Possible consequences of elevated levels of cyclic nucleotides induced by aluminum in the brain are proposed.

  17. Cyclic GMP and cyclic AMP changes in response to folic acid pulses during cell development of Dictyostelium discoideum.

    PubMed

    Wurster, B; Schubiger, K; Brachet, P

    1979-06-01

    Folic acid pulses induced developmental processes in agip 71, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. Cells that had received folic acid pulses were able to form EDTA-stable cell aggregates and to complete full differentiation to fruiting bodies. In these cells no autonomous periodic activities were observed by light scattering. Folic acid pulses elicited increases in the concentrations of cyclic GMP and cyclic AMP. In undifferentiated cells, folic acid caused a rapid increase in the level of cyclic GMP without a significant change in the level of cyclic AMP. In an advanced developmental state folic acid caused an increase in cyclic AMP in addition to two successsive peaks of cyclic GMP. Experiments performed with the parent strain, Ax-2, also showed that during the development towards aggregation competence, cells acquired the ability to produce a cyclic AMP peak in response to folic acid.

  18. Stimulation of phosphatidic acid of calcium influx and cyclic GMP synthesis in neuroblastoma cells.

    PubMed

    Ohsako, S; Deguchi, T

    1981-11-10

    Phosphatidic acid added to the medium markedly elevated intracellular cyclic GMP content in cultured neuroblastoma N1E 115 cells. There was a significant elevation of cyclic GMP with 1 micrograms/ml and a maximum (70-fold) elevation with 100 micrograms/ml of phosphatidic acid. Other natural phospholipids did not increase, or increased only slightly, the cyclic GMP content in the cells. The elevation of cyclic GMP content by phosphatidic acid was absolutely dependent on extracellular calcium. Phosphatidic acid stimulated the influx of calcium into neuroblastoma cells 2- to 5-fold. The pattern of the calcium influx induced by phosphatidic acid was comparable to that of cyclic GMP elevation. The stimulation of calcium influx by phosphatidic acid was also observed in cultured heart cells, indicating that phosphatidic acid acts as a calcium ionophore or opens a specific calcium-gate in a variety of cell membranes. Treatment of neuroblastoma cells with phospholipase C increased 32Pi labeling of phosphatidic acid, stimulated the influx of calcium, and elevated the cyclic GMP content in the cells. Thus exogenous as well as endogenous phosphatidic acid stimulates the translocation of calcium across cell membranes and, as a consequence, induces the synthesis of cyclic GMP in the neuroblastoma cells.

  19. Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria*

    PubMed Central

    Valentini, Martina

    2016-01-01

    The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa. This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission. PMID:27129226

  20. Biofilms and Cyclic di-GMP (c-di-GMP) Signaling: Lessons from Pseudomonas aeruginosa and Other Bacteria.

    PubMed

    Valentini, Martina; Filloux, Alain

    2016-06-10

    The cyclic di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities, and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa This bacterium is frequently adopted as a model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation, and transmission.

  1. Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

    PubMed Central

    Galperin, Michael Y.; Gomelsky, Mark

    2013-01-01

    SUMMARY Twenty-five years have passed since the discovery of cyclic dimeric (3′→5′) GMP (cyclic di-GMP or c-di-GMP). From the relative obscurity of an allosteric activator of a bacterial cellulose synthase, c-di-GMP has emerged as one of the most common and important bacterial second messengers. Cyclic di-GMP has been shown to regulate biofilm formation, motility, virulence, the cell cycle, differentiation, and other processes. Most c-di-GMP-dependent signaling pathways control the ability of bacteria to interact with abiotic surfaces or with other bacterial and eukaryotic cells. Cyclic di-GMP plays key roles in lifestyle changes of many bacteria, including transition from the motile to the sessile state, which aids in the establishment of multicellular biofilm communities, and from the virulent state in acute infections to the less virulent but more resilient state characteristic of chronic infectious diseases. From a practical standpoint, modulating c-di-GMP signaling pathways in bacteria could represent a new way of controlling formation and dispersal of biofilms in medical and industrial settings. Cyclic di-GMP participates in interkingdom signaling. It is recognized by mammalian immune systems as a uniquely bacterial molecule and therefore is considered a promising vaccine adjuvant. The purpose of this review is not to overview the whole body of data in the burgeoning field of c-di-GMP-dependent signaling. Instead, we provide a historic perspective on the development of the field, emphasize common trends, and illustrate them with the best available examples. We also identify unresolved questions and highlight new directions in c-di-GMP research that will give us a deeper understanding of this truly universal bacterial second messenger. PMID:23471616

  2. Relaxation of intrapulmonary artery and vein by nitrogen oxide-containing vasodilators and cyclic GMP

    SciTech Connect

    Edwards, J.C.; Ignarro, L.J.; Hyman, A.L.; Kadowitz, P.J.

    1984-01-01

    The present study examines the relationship between tissue cyclic nucleotide levels and relaxation of bovine intrapulmonary arterial and venous smooth muscle in response to nitroglycerin, nitroprusside, S-nitroso-N-acetylpenicillamine and isoproterenol. Recent studies have suggested that cyclic GMP may be involved in the relaxation of vascular smooth muscle produced by nitrogen oxide-containing vasodilators and that S-nitrosothiols may act as intermediates of the latter agents. In the present study, nitroglycerin, nitroprusside and S-nitroso-N-acetylpenicillamine were more potent as relaxants of venous than arterial segments. Each of these agents elevated tissue cyclic GMP levels, but not cyclic AMP levels, before relaxation. These nitrogen oxide-containing agents were more potent as elevators of cyclic GMP levels in venous than arterial tissue and this correlated generally with their effects on vascular smooth muscle tone. Methylene blue antagonized both relaxation and increased cyclic GMP levels elicited by nitroglycerin, nitroprusside and S-nitroso-N-acetylpenicillamine. In contrast to the nitrogen oxide vasodilators, 8-bromo-cyclic GMP was equally effective in reducing induced tone in arterial or venous segments. Similarly, isoproterenol relaxed arterial and venous segments with equivalent sensitivities. Relaxation by isoproterenol was preceded by or occurred concomitantly with increased levels of cyclic AMP but not cyclic GMP and both effects were antagonized by propranolol. These findings are consistent with the hypothesis that vascular smooth muscle relaxation in response to nitrogen oxide-containing vasodilators or isoproterenol may be mediated or modulated by the intracellular accumulation of cyclic GMP or cyclic AMP, respectively.

  3. Synergistic action of cyclic GMP on catecholamine-induced chloride current in guinea-pig ventricular cells.

    PubMed Central

    Ono, K; Tareen, F M; Yoshida, A; Noma, A

    1992-01-01

    1. Effects of cyclic GMP on the catecholamine-induced chloride current (ICl) were studied using the whole-cell patch-clamp technique combined with internal perfusion in single ventricular myocytes dispersed from guinea-pig heart. 2. When ICl was activated by submaximal doses of isoprenaline (0.01-0.1 microM), adrenaline (0.5-1 microM) and histamine (0.2-0.5 microM), intracellular dialysis with cyclic GMP (10-100 microM) induced an extra increase of ICl. No further increase of ICl was induced by cyclic GMP when ICl was maximally activated. In the absence of agonists, cyclic GMP failed to induce ICl. 3. The enhancement by cyclic GMP was also observed when ICl was activated by external application of 0.2-1.0 microM-forskolin or by internal dialysis with a pipette solution containing 50-200 microM-cyclic AMP. 4. In contrast to cyclic GMP, 10-1000 microM-dibutyryl cyclic GMP and 8-bromo-cyclic GMP were ineffective in modifying ICl. 5. Milrinone (1-10 microM), a specific inhibitor of a kind of phosphodiesterase which is inhibited by cyclic GMP, also enhanced ICl activated by submaximal doses of isoprenaline. Milrinone itself did not activate ICl. 6. When ICl was enhanced by 5 microM-milrinone, an additional application of cyclic GMP failed to increase ICl. In the presence of cyclic GMP, milrinone failed to enhance ICl. 7. The above findings on ICl are analogous to the enhancement by cyclic GMP of the beta-adrenergic stimulation of the Ca2+ current reported in the same preparation, and support the hypothesis that in mammalian cardiac cells cyclic GMP potentiates elevation of cyclic AMP induced by beta-adrenergic agents, and thereby increases the amplitudes of ionic currents. PMID:1281506

  4. Optogenetic Manipulation of Cyclic Di-GMP (c-di-GMP) Levels Reveals the Role of c-di-GMP in Regulating Aerotaxis Receptor Activity in Azospirillum brasilense.

    PubMed

    O'Neal, Lindsey; Ryu, Min-Hyung; Gomelsky, Mark; Alexandre, Gladys

    2017-09-15

    Bacterial chemotaxis receptors provide the sensory inputs that inform the direction of navigation in changing environments. Recently, we described the bacterial second messenger cyclic di-GMP (c-di-GMP) as a novel regulator of a subclass of chemotaxis receptors. In Azospirillum brasilense, c-di-GMP binds to a chemotaxis receptor, Tlp1, and modulates its signaling function during aerotaxis. Here, we further characterize the role of c-di-GMP in aerotaxis using a novel dichromatic optogenetic system engineered for manipulating intracellular c-di-GMP levels in real time. This system comprises a red/near-infrared-light-regulated diguanylate cyclase and a blue-light-regulated c-di-GMP phosphodiesterase. It allows the generation of transient changes in intracellular c-di-GMP concentrations within seconds of irradiation with appropriate light, which is compatible with the time scale of chemotaxis signaling. We provide experimental evidence that binding of c-di-GMP to the Tlp1 receptor activates its signaling function during aerotaxis, which supports the role of transient changes in c-di-GMP levels as a means of adjusting the response of A. brasilense to oxygen gradients. We also show that intracellular c-di-GMP levels in A. brasilense change with carbon metabolism. Our data support a model whereby c-di-GMP functions to imprint chemotaxis receptors with a record of recent metabolic experience, to adjust their contribution to the signaling output, thus allowing the cells to continually fine-tune chemotaxis sensory perception to their metabolic state.IMPORTANCE Motile bacteria use chemotaxis to change swimming direction in response to changes in environmental conditions. Chemotaxis receptors sense environmental signals and relay sensory information to the chemotaxis machinery, which ultimately controls the swimming pattern of cells. In bacteria studied to date, differential methylation has been known as a mechanism to control the activity of chemotaxis receptors and modulates

  5. NO, nitrotyrosine, and cyclic GMP in signal transduction

    NASA Technical Reports Server (NTRS)

    Hanafy, K. A.; Krumenacker, J. S.; Murad, F.

    2001-01-01

    Over the past 25 years, the role of nitric oxide (NO) in biology has evolved from being recognized as an environmental pollutant to an endogenously produced substance involved in cell communication and signal transduction. NO is produced by a family of enzymes called nitric oxide synthases (NOSs), which can be stimulated by a variety of factors that mediate responses to various stimuli. NO can initiate its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC), or through several other chemical reactions. Activation of sGC results in the production of 3',5'-cyclic guanosine monophosphate (cGMP), an intracellular second messenger signaling molecule, which can subsequently mediate such diverse physiological events such as vasodilatation and immunomodulation. Chemically reactive NO can affect physiological changes through modifications to cellular proteins, one of which is tyrosine nitration. The demonstration that NO is involved in so many biological pathways indicates the importance of this endogenously produced substance, and suggests that there is much more to be discovered about its role in biology in years to come.

  6. NO, nitrotyrosine, and cyclic GMP in signal transduction

    NASA Technical Reports Server (NTRS)

    Hanafy, K. A.; Krumenacker, J. S.; Murad, F.

    2001-01-01

    Over the past 25 years, the role of nitric oxide (NO) in biology has evolved from being recognized as an environmental pollutant to an endogenously produced substance involved in cell communication and signal transduction. NO is produced by a family of enzymes called nitric oxide synthases (NOSs), which can be stimulated by a variety of factors that mediate responses to various stimuli. NO can initiate its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC), or through several other chemical reactions. Activation of sGC results in the production of 3',5'-cyclic guanosine monophosphate (cGMP), an intracellular second messenger signaling molecule, which can subsequently mediate such diverse physiological events such as vasodilatation and immunomodulation. Chemically reactive NO can affect physiological changes through modifications to cellular proteins, one of which is tyrosine nitration. The demonstration that NO is involved in so many biological pathways indicates the importance of this endogenously produced substance, and suggests that there is much more to be discovered about its role in biology in years to come.

  7. Cyclic-di-GMP signalling meets extracellular polysaccharide synthesis in Bacillus subtilis.

    PubMed

    Kampf, Jan; Stülke, Jörg

    2017-06-01

    In order to resist harmful environmental conditions, many bacteria form multicellular aggregates called biofilms. In these biofilms, they protect themselves in a self-produced matrix consisting of extracellular polysaccharides, proteins and DNA. In many bacteria, biofilm formation is stimulated in the presence of the second messenger cyclic di-GMP. In this issue of Environmental Microbiology Reports, Bedrunka and Graumann have studied matrix production by the proteins encoded in the Bacillus subtilis ydaJKLMN operon. For the first time, they were able to provide a link between c-di-GMP signalling and matrix production in this bacterium. The work demonstrates that the c-di-GMP receptor protein YdaK forms a membrane-bound complex with the YdaM and YdaN proteins, and that this interaction with YdaK is required for polysaccharide production by YdaL, YdaM and YdaN. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Ca sup 2+ current is regulated by cyclic GMP-dependent protein kinase in mammalian cardiac myocytes

    SciTech Connect

    Mery, P-F.; Fischmeister, R. ); Lohmann, S.M.; Walter, U. )

    1991-02-15

    Regulation of cardiac contraction by neurotransmitters and hormones is often correlated with regulation of the L-type Ca{sup 2+}-channel current (I{sub Ca}) through the opposite actions for two second messengers, cyclic AMP and cyclic GMP. While cyclic AMP stimulation of I{sub Ca} is mediated by the activation of cyclic AMP-dependent protein kinase, inhibition of I{sub Ca} by cyclic GMP in frog heart is largely mediated by activation of cyclic AMP phosphodiesterase. The present patch-clamp study reveals that, in rat ventricular cells, cyclic GMP can also regulate I{sub Ca} via activation of endogenous cyclic GMP-dependent protein kinase (cGMP-PK). Indeed, the effect of cyclic GMP on I{sub Ca} was mimicked by intracellular perfusion with the proteolytic active fragment of purified cGMP-PK. Moreover, cGMP-PK immunoreactivity was detected in pure rat ventricular myocytes by using a specific polyclonal antibody. These results demonstrate a dual mechanism for the inhibitory action of cyclic GMP in heart, as well as a physiological role for cGMP-PK in the control of mammalian heart function.

  9. Molecular mechanisms of gravity-dependent signaling in human melanocytic cells involve cyclic GMP

    NASA Astrophysics Data System (ADS)

    Ivanova, Krassimira; Lambers, Britta; Block, Ingrid; Bromeis, Birgit; Das, Pranab K.; Gerzer, Rupert

    2005-08-01

    Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) is an important messenger in melanocyte signaling we have compared the regulation of cGMP levels in human melanocytes and melanoma cells with different metastatic potential under hypergravity conditions. We were able to demonstrate that long-term exposure to hypergravity stimulates cGMP efflux in cultured human melanocytes and non- metastatic melanoma cells, whereas highly metastatic melanoma cells appear to be insensitive to hypergravity, most probably, due to an up-regulated cGMP efflux at 1g. Here we report that these effects are associated with the expression of the multidrug resistance proteins 4 and 5 known to act as selective export pumps for amphiphilic anions like cGMP. Thus, an altered gravity vector may induce cGMP-dependent signaling events in melanocytic cells that could be important for malignant transformation.

  10. Radiation Effects on Cyclic AMP, Cyclic GMP, and Amino Acid Levels in the CSF of the Primate

    DTIC Science & Technology

    1980-11-07

    rradiation . An analysis of brain areas obtained by biopsy of irradiated animals showed significant decreases in only the cerebellar cyclic AMP and cyclic...GMP. No appreciablk ¢±anges were found in the CSF amino acid composition. Acc. !-,’r ror UV , . c" l. __ ..:j’od I7 .... ’U r~rd0 /or cpy I NCI

  11. A new role for a classical gene: white transports cyclic GMP.

    PubMed

    Evans, Jennifer M; Day, Jonathan P; Cabrero, Pablo; Dow, Julian A T; Davies, Shireen-Anne

    2008-03-01

    Guanosine 3'-5' cyclic monophosphate (cGMP) and adenosine 3'-5' cyclic monophosphate (cAMP) are important regulators of cell and tissue function. However, cGMP and cAMP transport have received relatively limited attention, especially in model organisms where such studies can be conducted in vivo. The Drosophila Malpighian (renal) tubule transports cGMP and cAMP and utilises these as signalling molecules. We show here via substrate competition and drug inhibition studies that cAMP transport - but not cGMP transport - requires the presence of di- or tri-carboxylates; and that transport of both cyclic nucleotides occurs via ATP binding cassette sub-family G2 (ABCG2), but not via ABC sub-family C (ABCC), transporters. In Drosophila, the white (w) gene is known for the classic eye colour mutation. However, gene expression data show that of all adult tissues, w is most highly expressed in Malpighian tubules. Furthermore, as White is a member of the ABCG2 transporter class, it is a potential candidate for a tubule cGMP transporter. Assay of cGMP transport in w(-) (mutant) tubules shows that w is required for cGMP transport but not cAMP transport. Targeted over-expression of w in w(-) tubule principal cells significantly increases cGMP transport compared with that in w(-) controls. Conversely, treatment of wild-type tubules with cGMP increases w mRNA expression levels, implying that cGMP is a physiologically relevant substrate for White. Immunocytochemical localisation reveals that White is expressed in intracellular vesicles in tubule principal cells, suggesting that White participates in vesicular transepithelial transport of cGMP.

  12. Occurrence of Cyclic di-GMP-Modulating Output Domains in Cyanobacteria: an Illuminating Perspective

    PubMed Central

    Agostoni, Marco; Koestler, Benjamin J.; Waters, Christopher M.; Williams, Barry L.; Montgomery, Beronda L.

    2013-01-01

    ABSTRACT Microorganisms use a variety of metabolites to respond to external stimuli, including second messengers that amplify primary signals and elicit biochemical changes in a cell. Levels of the second messenger cyclic dimeric GMP (c-di-GMP) are regulated by a variety of environmental stimuli and play a critical role in regulating cellular processes such as biofilm formation and cellular motility. Cyclic di-GMP signaling systems have been largely characterized in pathogenic bacteria; however, proteins that can impact the synthesis or degradation of c-di-GMP are prominent in cyanobacterial species and yet remain largely underexplored. In cyanobacteria, many putative c-di-GMP synthesis or degradation domains are found in genes that also harbor light-responsive signal input domains, suggesting that light is an important signal for altering c-di-GMP homeostasis. Indeed, c-di-GMP-associated domains are often the second most common output domain in photoreceptors—outnumbered only by a histidine kinase output domain. Cyanobacteria differ from other bacteria regarding the number and types of photoreceptor domains associated with c-di-GMP domains. Due to the widespread distribution of c-di-GMP domains in cyanobacteria, we investigated the evolutionary origin of a subset of genes. Phylogenetic analyses showed that c-di-GMP signaling systems were present early in cyanobacteria and c-di-GMP genes were both vertically and horizontally inherited during their evolution. Finally, we compared intracellular levels of c-di-GMP in two cyanobacterial species under different light qualities, confirming that light is an important factor for regulating this second messenger in vivo. PMID:23943760

  13. Bile Acids and Bicarbonate Inversely Regulate Intracellular Cyclic di-GMP in Vibrio cholerae

    PubMed Central

    Koestler, Benjamin J.

    2014-01-01

    Vibrio cholerae is a Gram-negative bacterium that persists in aquatic reservoirs and causes the diarrheal disease cholera upon entry into a human host. V. cholerae employs the second messenger molecule 3′,5′-cyclic diguanylic acid (c-di-GMP) to transition between these two distinct lifestyles. c-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and hydrolyzed by phosphodiesterase (PDE) enzymes. Bacteria typically encode many different DGCs and PDEs within their genomes. Presumably, each enzyme senses and responds to cognate environmental cues by alteration of enzymatic activity. c-di-GMP represses the expression of virulence factors in V. cholerae, and it is predicted that the intracellular concentration of c-di-GMP is low during infection. Contrary to this model, we found that bile acids, a prevalent constituent of the human proximal small intestine, increase intracellular c-di-GMP in V. cholerae. We identified four c-di-GMP turnover enzymes that contribute to increased intracellular c-di-GMP in the presence of bile acids, and deletion of these enzymes eliminates the bile induction of c-di-GMP and biofilm formation. Furthermore, this bile-mediated increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted by intestinal epithelial cells. Our results lead us to propose that V. cholerae senses distinct microenvironments within the small intestine using bile and bicarbonate as chemical cues and responds by modulating the intracellular concentration of c-di-GMP. PMID:24799624

  14. Cyclic GMP is involved in auxin signalling during Arabidopsis root growth and development

    PubMed Central

    Nan, Wenbin; Wang, Xiaomin; Bi, Yurong

    2014-01-01

    The second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) plays an important role in plant development and responses to stress. Recent studies indicated that cGMP is a secondary signal generated in response to auxin stimulation. cGMP also mediates auxin-induced adventitious root formation in mung bean and gravitropic bending in soybean. Nonetheless, the mechanism of the participation of cGMP in auxin signalling to affect these growth and developmental processes is largely unknown. In this report we provide evidence that indole-3-acetic acid (IAA) induces cGMP accumulation in Arabidopsis roots through modulation of the guanylate cyclase activity. Application of 8-bromo-cGMP (a cell-permeable cGMP derivative) increases auxin-dependent lateral root formation, root hair development, primary root growth, and gene expression. In contrast, inhibitors of endogenous cGMP synthesis block these processes induced by auxin. Data also showed that 8-bromo-cGMP enhances auxin-induced degradation of Aux/IAA protein modulated by the SCFTIR1 ubiquitin-proteasome pathway. Furthermore, it was found that 8-bromo-cGMP is unable to directly influence the auxin-dependent TIR1-Aux/IAA interaction as evidenced by pull-down and yeast two-hybrid assays. In addition, we provide evidence for cGMP-mediated modulation of auxin signalling through cGMP-dependent protein kinase (PKG). Our results suggest that cGMP acts as a mediator to participate in auxin signalling and may govern this process by PKG activity via its influence on auxin-regulated gene expression and auxin/IAA degradation. PMID:24591051

  15. Cyclic GMP is involved in auxin signalling during Arabidopsis root growth and development.

    PubMed

    Nan, Wenbin; Wang, Xiaomin; Yang, Lei; Hu, Yanfeng; Wei, Yuantao; Liang, Xiaolei; Mao, Lina; Bi, Yurong

    2014-04-01

    The second messenger cyclic guanosine 3',5'-monophosphate (cGMP) plays an important role in plant development and responses to stress. Recent studies indicated that cGMP is a secondary signal generated in response to auxin stimulation. cGMP also mediates auxin-induced adventitious root formation in mung bean and gravitropic bending in soybean. Nonetheless, the mechanism of the participation of cGMP in auxin signalling to affect these growth and developmental processes is largely unknown. In this report we provide evidence that indole-3-acetic acid (IAA) induces cGMP accumulation in Arabidopsis roots through modulation of the guanylate cyclase activity. Application of 8-bromo-cGMP (a cell-permeable cGMP derivative) increases auxin-dependent lateral root formation, root hair development, primary root growth, and gene expression. In contrast, inhibitors of endogenous cGMP synthesis block these processes induced by auxin. Data also showed that 8-bromo-cGMP enhances auxin-induced degradation of Aux/IAA protein modulated by the SCF(TIR1) ubiquitin-proteasome pathway. Furthermore, it was found that 8-bromo-cGMP is unable to directly influence the auxin-dependent TIR1-Aux/IAA interaction as evidenced by pull-down and yeast two-hybrid assays. In addition, we provide evidence for cGMP-mediated modulation of auxin signalling through cGMP-dependent protein kinase (PKG). Our results suggest that cGMP acts as a mediator to participate in auxin signalling and may govern this process by PKG activity via its influence on auxin-regulated gene expression and auxin/IAA degradation.

  16. Functional Characterization of Core Components of the Bacillus subtilis Cyclic-Di-GMP Signaling Pathway

    PubMed Central

    Gao, Xiaohui; Mukherjee, Sampriti; Matthews, Paige M.; Hammad, Loubna A.; Kearns, Daniel B.

    2013-01-01

    Bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is an intracellular second messenger that regulates adaptation processes, including biofilm formation, motility, and virulence in Gram-negative bacteria. In this study, we have characterized the core components of a c-di-GMP signaling pathway in the model Gram-positive bacterium Bacillus subtilis. Specifically, we have directly identified and characterized three active diguanylate cyclases, DgcP, DgcK, and DgcW (formerly YtrP, YhcK, and YkoW, respectively), one active c-di-GMP phosphodiesterase, PdeH (formerly YuxH), and a cyclic-diguanylate (c-di-GMP) receptor, DgrA (formerly YpfA). Furthermore, elevation of c-di-GMP levels in B. subtilis led to inhibition of swarming motility, whereas biofilm formation was unaffected. Our work establishes paradigms for Gram-positive c-di-GMP signaling, and we have shown that the concise signaling system identified in B. subtilis serves as a powerful heterologous host for the study of c-di-GMP enzymes from bacteria predicted to possess larger, more-complex signaling systems. PMID:23893111

  17. Mutant analysis suggests that cyclic GMP mediates the cyclic AMP-induced Ca2+ uptake in Dictyostelium.

    PubMed

    Menz, S; Bumann, J; Jaworski, E; Malchow, D

    1991-05-01

    Previous work has shown that streamer F (stmF) mutants of Dictyostelium discoideum exhibit prolonged chemotactic elongation in aggregation fields. The mutants carry an altered structural gene for cyclic GMP phosphodiesterase resulting in low activities of this enzyme. Chemotactic stimulation by cyclic AMP causes a rapid transient increase in the cyclic GMP concentration followed by association of myosin heavy chains with the cytoskeleton. Both events persist several times longer in stmF mutants than in the parental strain, indicating that the change in association of myosin with the cytoskeleton is transmitted directly or indirectly by cyclic GMP. We measured the cyclic AMP-induced Ca2+ uptake with a Ca(2+)-sensitive electrode and found that Ca2+ uptake was prolonged in stmF mutants but not in the parental strain. The G alpha 2 mutant strain HC33 (fgdA), devoid of InsP3 release and receptor/guanylate cyclase coupling, lacked Ca2+ uptake. However, the latter response and cyclic GMP formation were normal in the signal-relay mutant strain agip 53 where cyclic AMP-stimulated cyclic AMP synthesis is absent. LiCl, which inhibits InsP3 formation in Dictyostelium, blocked Ca2+ uptake in a dose-dependent manner. The data indicate that the receptor-mediated Ca2+ uptake depends on the InsP3 pathway and is regulated by cyclic GMP. The rate of Ca2+ uptake was correlated in time with the association of myosin with the cytoskeleton, suggesting that Ca2+ uptake is involved in the motility response of the cells.

  18. Application of Synthetic Peptide Arrays To Uncover Cyclic Di-GMP Binding Motifs

    PubMed Central

    Düvel, Juliane; Bense, Sarina; Möller, Stefan; Bertinetti, Daniela; Schwede, Frank; Morr, Michael; Eckweiler, Denitsa; Genieser, Hans-Gottfried; Jänsch, Lothar; Herberg, Friedrich W.; Frank, Ronald

    2015-01-01

    ABSTRACT High levels of the universal bacterial second messenger cyclic di-GMP (c-di-GMP) promote the establishment of surface-attached growth in many bacteria. Not only can c-di-GMP bind to nucleic acids and directly control gene expression, but it also binds to a diverse array of proteins of specialized functions and orchestrates their activity. Since its development in the early 1990s, the synthetic peptide array technique has become a powerful tool for high-throughput approaches and was successfully applied to investigate the binding specificity of protein-ligand interactions. In this study, we used peptide arrays to uncover the c-di-GMP binding site of a Pseudomonas aeruginosa protein (PA3740) that was isolated in a chemical proteomics approach. PA3740 was shown to bind c-di-GMP with a high affinity, and peptide arrays uncovered LKKALKKQTNLR to be a putative c-di-GMP binding motif. Most interestingly, different from the previously identified c-di-GMP binding motif of the PilZ domain (RXXXR) or the I site of diguanylate cyclases (RXXD), two leucine residues and a glutamine residue and not the charged amino acids provided the key residues of the binding sequence. Those three amino acids are highly conserved across PA3740 homologs, and their singular exchange to alanine reduced c-di-GMP binding within the full-length protein. IMPORTANCE In many bacterial pathogens the universal bacterial second messenger c-di-GMP governs the switch from the planktonic, motile mode of growth to the sessile, biofilm mode of growth. Bacteria adapt their intracellular c-di-GMP levels to a variety of environmental challenges. Several classes of c-di-GMP binding proteins have been structurally characterized, and diverse c-di-GMP binding domains have been identified. Nevertheless, for several c-di-GMP receptors, the binding motif remains to be determined. Here we show that the use of a synthetic peptide array allowed the identification of a c-di-GMP binding motif of a putative c-di-GMP

  19. The ins and outs of cyclic di-GMP signaling in Vibrio cholerae.

    PubMed

    Conner, Jenna G; Zamorano-Sánchez, David; Park, Jin Hwan; Sondermann, Holger; Yildiz, Fitnat H

    2017-04-01

    The second messenger nucleotide cyclic dimeric guanosine monophosphate (c-di-GMP) governs many cellular processes in the facultative human pathogen Vibrio cholerae. This organism copes with changing environmental conditions in aquatic environments and during transitions to and from human hosts. Modulation of c-di-GMP allows V. cholerae to shift between motile and sessile stages of life, thus allowing adaptation to stressors and environmental conditions during its transmission cycle. The V. cholerae genome encodes a large set of proteins predicted to degrade and produce c-di-GMP. A subset of these enzymes has been demonstrated to control cellular processes - particularly motility, biofilm formation, and virulence - through transcriptional, post-transcriptional, and translational mechanisms. Recent studies have identified and characterized enzymes that modulate or sense c-di-GMP levels and have led towards mechanistic understanding of c-di-GMP regulatory circuits in V. cholerae. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Secreted Cyclic Di-GMP Induces Stalk Cell Differentiation in the Eukaryote Dictyostelium discoideum.

    PubMed

    Chen, Zhi-hui; Schaap, Pauline

    2016-01-01

    Cyclic di-GMP (c-di-GMP) is currently recognized as the most widely used intracellular signal molecule in prokaryotes, but roles in eukaryotes were only recently discovered. In the social amoeba Dictyostelium discoideum, c-di-GMP, produced by a prokaryote-type diguanylate cyclase, induces the differentiation of stalk cells, thereby enabling the formation of spore-bearing fruiting bodies. In this review, we summarize the currently known mechanisms that control the major life cycle transitions of Dictyostelium and focus particularly on the role of c-di-GMP in stalk formation. Stalk cell differentiation has characteristics of autophagic cell death, a process that also occurs in higher eukaryotes. We discuss the respective roles of c-di-GMP and of another signal molecule, differentiation-inducing factor 1, in autophagic cell death in vitro and in stalk formation in vivo.

  1. Relaxation of guinea-pig trachea by sodium nitroprusside: cyclic GMP and nitric oxide not involved.

    PubMed Central

    Sadeghi-Hashjin, G.; Folkerts, G.; Henricks, P. A.; van de Loo, P. G.; Dik, I. E.; Nijkamp, F. P.

    1996-01-01

    1. Sodium nitroprusside (SNP) completely relaxed the guinea-pig isolated, perfused trachea in a concentration-dependent manner. Although SNP was less potent by about 2 orders of magnitude, its maximal effect was 25% higher compared to isoprenaline. 2. SNP (3.2 microM) increased cyclic GMP levels by 300% and relaxed guinea-pig isolated, perfused trachea by 54%. The SNP-induced relaxations of the preparations were not affected by the guanylate cyclase inhibitor, methylene blue. Moreover, zaprinast, a cyclic GMP-specific phosphodiesterase inhibitor which was supposed to enhance SNP-induced relaxations, decreased the maximal relaxation by 22% (P < 0.001). 3. In contrast, 8Br-cyclic GMP (10 microM) increased the cyclic GMP levels by 1100% without inducing a marked relaxation. 4. SNP (10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; a direct donor of nitric oxide; 10 microM), relaxed the tissues by 75% and 25%, respectively, without any nitric oxide (NO) release by SNP (< 1 pmol 100 microliters-1), but a substantial NO release by SNAP (560 pmol 100 microliters-1). 5. It is concluded that the SNP-induced tracheal relaxations are probably not mediated by cyclic GMP and NO. PMID:8762066

  2. Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles.

    PubMed

    Shuhaibar, Leia C; Egbert, Jeremy R; Norris, Rachael P; Lampe, Paul D; Nikolaev, Viacheslav O; Thunemann, Martin; Wen, Lai; Feil, Robert; Jaffe, Laurinda A

    2015-04-28

    Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2-4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes.

  3. Intercellular signaling via cyclic GMP diffusion through gap junctions restarts meiosis in mouse ovarian follicles

    PubMed Central

    Shuhaibar, Leia C.; Egbert, Jeremy R.; Norris, Rachael P.; Lampe, Paul D.; Nikolaev, Viacheslav O.; Thunemann, Martin; Wen, Lai; Feil, Robert; Jaffe, Laurinda A.

    2015-01-01

    Meiosis in mammalian oocytes is paused until luteinizing hormone (LH) activates receptors in the mural granulosa cells of the ovarian follicle. Prior work has established the central role of cyclic GMP (cGMP) from the granulosa cells in maintaining meiotic arrest, but it is not clear how binding of LH to receptors that are located up to 10 cell layers away from the oocyte lowers oocyte cGMP and restarts meiosis. Here, by visualizing intercellular trafficking of cGMP in real-time in live follicles from mice expressing a FRET sensor, we show that diffusion of cGMP through gap junctions is responsible not only for maintaining meiotic arrest, but also for rapid transmission of the signal that reinitiates meiosis from the follicle surface to the oocyte. Before LH exposure, the cGMP concentration throughout the follicle is at a uniformly high level of ∼2–4 μM. Then, within 1 min of LH application, cGMP begins to decrease in the peripheral granulosa cells. As a consequence, cGMP from the oocyte diffuses into the sink provided by the large granulosa cell volume, such that by 20 min the cGMP concentration in the follicle is uniformly low, ∼100 nM. The decrease in cGMP in the oocyte relieves the inhibition of the meiotic cell cycle. This direct demonstration that a physiological signal initiated by a stimulus in one region of an intact tissue can travel across many layers of cells via cyclic nucleotide diffusion through gap junctions could provide a general mechanism for diverse cellular processes. PMID:25775542

  4. Clinical relevance of cyclic GMP modulators: A translational success story of network pharmacology.

    PubMed

    Oettrich, J M; Dao, V T; Frijhoff, J; Kleikers, Pwm; Casas, A I; Hobbs, A J; Schmidt, H H H W

    2016-04-01

    Therapies that modulate cyclic guanosine-3'-5'-monophosphate (cGMP) have emerged as one of the most successful areas in recent drug discovery and clinical pharmacology. Historically, their focus has been on cardiovascular disease phenotypes; however, cGMP's relevance is likely to go beyond this rather limited organ-based set of indications. Moreover, the multitude of targets and their apparent interchangeability is a proof-of-concept of network pharmacology.

  5. A new adjuvant delivery system 'cyclic di-GMP/YSK05 liposome' for cancer immunotherapy.

    PubMed

    Miyabe, Hiroko; Hyodo, Mamoru; Nakamura, Takashi; Sato, Yusuke; Hayakawa, Yoshihiro; Harashima, Hideyoshi

    2014-06-28

    Cyclic dinucleotides are of importance in the field of microbiology and immunology. They function as second messengers and are thought to participate in the signal transduction of cytosolic DNA immune responses. One such dinucleotide, cyclic di-GMP (c-di-GMP), stimulates the immune system. It is thought that c-di-GMP is recognized by ATP dependent RNA helicase (DDX41) in the cytosol, forms a complex with the Stimulator of interferon genes protein (STING), triggers a signal via the tank binding kinase 1-interferon regulatory factor 3 (TBK1-IRF3) pathway and induces the production of type I interferons. Therefore c-di-GMP can be thought of as a new class of adjuvant. However, because c-di-GMP contains two phosphate groups, this prevents its use as an adjuvant because it cannot pass through the cell membrane, even though the target molecule of c-di-GMP is located in the cytoplasm. Our group has been developing a series of liposomal drug delivery systems and recently investigated YSK05 which is a synthetic, pH sensitive lipid that has a high fusogenicity. We utilized this lipid as a carrier to transport c-di-GMP into the cytosol to then use c-di-GMP as an adjuvant. Based on screening experiments, YSK05/POPE/cholesterol=40/25/35 was found to induce IFN-β in Raw264.7 cells. The induction of IFN-β from c-di-GMP liposomes was inhibited by adding BX795, a TBK1 inhibitor, indicating that the production of IFN-β caused the activation of the STING-TBK1 pathway. C-di-GMP liposomes also showed significantly higher levels of expression of CD80, CD86 and MHC class I. The c-di-GMP/YSK05 liposome facilitated antigen specific cytotoxic T cell activity and the inhibition of tumor growth in a mouse model. These findings indicate that c-di-GMP/YSK05 liposomes could be used, not only to transfer c-di-GMP to the cytosol and induce an innate immune system but also as a platform for investigating the mechanism of immune sensing with cyclic dinucleotides in vitro and in vivo.

  6. Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

    DOE PAGES

    Campbell, James C.; Kim, Jeong Joo; Li, Kevin Y.; ...

    2016-01-14

    Membrane-bound cGMP-dependent protein kinase (PKG) II is an important regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKGII binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415more » of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.« less

  7. Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

    SciTech Connect

    Campbell, James C.; Kim, Jeong Joo; Li, Kevin Y.; Huang, Gilbert Y.; Reger, Albert S.; Matsuda, Shinya; Sankaran, Banumathi; Link, Todd M.; Yuasa, Keizo; Ladbury, John E.; Casteel, Darren E.; Kim, Choel

    2016-01-14

    Membrane-bound cGMP-dependent protein kinase (PKG) II is an important regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKGII binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.

  8. Polyphosphate, cyclic AMP, guanosine tetraphosphate, and c-di-GMP reduce in vitro Lon activity

    PubMed Central

    Osbourne, Devon O; Soo, Valerie WC; Konieczny, Igor; Wood, Thomas K

    2014-01-01

    Lon protease is conserved from bacteria to humans and regulates cellular processes by degrading different classes of proteins including antitoxins, transcriptional activators, unfolded proteins, and free ribosomal proteins. Since we found that Lon has several putative cyclic diguanylate (c-di-GMP) binding sites and since Lon binds polyphosphate (polyP) and lipid polysaccharide, we hypothesized that Lon has an affinity for phosphate-based molecules that might regulate its activity. Hence we tested the effect of polyP, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), guanosine tetraphosphate (ppGpp), c-di-GMP, and GMP on the ability of Lon to degrade α-casein. Inhibition of in vitro Lon activity occurred for polyP, cAMP, ppGpp, and c-di-GMP. We also demonstrated by HPLC that Lon is able to bind c-di-GMP. Therefore, four cell signals were found to regulate the activity of Lon protease. PMID:24874800

  9. Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II*

    PubMed Central

    Campbell, James C.; Kim, Jeong Joo; Li, Kevin Y.; Huang, Gilbert Y.; Reger, Albert S.; Matsuda, Shinya; Sankaran, Banumathi; Link, Todd M.; Yuasa, Keizo; Ladbury, John E.; Casteel, Darren E.; Kim, Choel

    2016-01-01

    Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II. PMID:26769964

  10. The cyclic-di-GMP signaling pathway in the Lyme disease spirochete, Borrelia burgdorferi

    PubMed Central

    Novak, Elizabeth A.; Sultan, Syed Z.; Motaleb, Md. A.

    2014-01-01

    In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP) signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases) and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any) c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease. PMID:24822172

  11. Cyclic-di-GMP levels affect Pseudomonas aeruginosa fitness in the presence of imipenem.

    PubMed

    Nicastro, Gianlucca G; Kaihami, Gilberto H; Pereira, Thays O; Meireles, Diogo A; Groleau, Marie-Christine; Déziel, Eric; Baldini, Regina L

    2014-05-01

    A large number of genes coding for enzymes predicted to synthesize and degrade 3'-5'-cyclic diguanylic acid (c-di-GMP) is found in most bacterial genomes and this dinucleotide emerged as an intracellular signal-controlling bacterial behaviour. An association between high levels of c-di-GMP and antibiotic resistance may be expected because c-di-GMP regulates biofilm formation and this mode of growth leads to enhanced antibiotic resistance. However, a clear understanding of this correlation has not been established. We found that increased levels of c-di-GMP in Pseudomonas aeruginosa improve fitness in the presence of imipenem, even when grown as planktonic cells. P. aeruginosa post-transcriptionally regulates the amounts of five porins in response to c-di-GMP, including OprD, responsible for imipenem uptake. Cells with low c-di-GMP levels are consequently more sensitive to this antibiotic. Main efflux pumps or β-lactamase genes did not show altered mRNA levels in P. aeruginosa strains with modified different c-di-GMP concentrations. Together, our findings show that c-di-GMP levels modulate fitness of planktonic cultures in the presence of imipenem. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. A new nonhydrolyzable reactive cGMP analogue, (Rp)-Guanosine-3′, 5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

    PubMed Central

    Hung, Su H.; Liu, Andy H.; Pixley, Robin A.; Francis, Penelope; Williams, LaTeeka D.; Matsko, Christopher M.; Barnes, Karine D.; Sivendran, Sharmila; Colman, Roberta F.; Colman, Robert W.

    2008-01-01

    The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′, 5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (KI = 43.4 ± 7.2 μM and kcart = 0.007 ± 0.0006 min−1). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (Kd = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). NAD+, neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes. PMID:18394675

  13. Enhanced Vascular Effects of Cyclic GMP in Septic Rat Aorta

    DTIC Science & Technology

    1988-01-01

    9). Modulation of KCI-induced contraction by methylene Measurements of aortic contraction. The thoracic aorta blue and 8-bromo-cGMP. Four rings with...was added to the organ 1.3 CaCl 2, 1.2 MgSO4. 7H 20, 1.2 KH2PO4, 25.0 NaHCO3, baths of both rings contracted with KCI after maximum and 11.7 glucose ...pH 7.4) while bubbled with 95% 02- contraction was attained, and any additional increments 5% CO2. in tension were measured . A dose of 8-bromo-cGMP

  14. The nitric oxide/cyclic GMP pathway: a potential major regulator of cochlear physiology.

    PubMed

    Fessenden, J D; Schacht, J

    1998-04-01

    The nitric oxide (NO)/cyclic guanosine monophosphate (GMP) pathway is now recognized as a major regulatory system in cell physiology and tissue homeostasis. This pathway may control processes as diverse as muscle relaxation, gut peristalsis, neurotransmission and hormonal secretion. It is also involved in the development and function of sensory systems such as vision and olfaction. This review will detail the NO/cyclic GMP pathway, evaluate studies in the auditory system and discuss its potential participation in cochlear blood flow, supporting cell physiology and excitotoxicity.

  15. Brucella melitensis Cyclic di-GMP Phosphodiesterase BpdA Controls Expression of Flagellar Genes▿†

    PubMed Central

    Petersen, Erik; Chaudhuri, Pallab; Gourley, Chris; Harms, Jerome; Splitter, Gary

    2011-01-01

    Brucella melitensis encounters a variety of conditions and stimuli during its life cycle—including environmental growth, intracellular infection, and extracellular dissemination—which necessitates flexibility of bacterial signaling to promote virulence. Cyclic-di-GMP is a bacterial secondary signaling molecule that plays an important role in adaptation to changing environments and altering virulence in a number of bacteria. To investigate the role of cyclic-di-GMP in B. melitensis, all 11 predicted cyclic-di-GMP-metabolizing proteins were separately deleted and the effect on virulence was determined. Three of these cyclic-di-GMP-metabolizing proteins were found to alter virulence. Deletion of the bpdA and bpdB genes resulted in attenuation of virulence of the bacterium, while deletion of the cgsB gene produced a hypervirulent strain. In a Vibrio reporter system to monitor apparent alteration in levels of cyclic-di-GMP, both BpdA and BpdB displayed a phenotype consistent with cyclic-di-GMP-specific phosphodiesterases, while CgsB displayed a cyclic-di-GMP synthase phenotype. Further analysis found that deletion of bpdA resulted in a dramatic decrease in flagellar promoter activities, and a flagellar mutant showed similar phenotypes to the bpdA and bpdB mutant strains in mouse models of infection. These data indicate a potential role for regulation of flagella in Brucella melitensis via cyclic-di-GMP. PMID:21856843

  16. Cyclic Di-GMP modulates the disease progression of Erwinia amylovora.

    PubMed

    Edmunds, Adam C; Castiblanco, Luisa F; Sundin, George W; Waters, Christopher M

    2013-05-01

    The second messenger cyclic di-GMP (c-di-GMP) is a nearly ubiquitous intracellular signal molecule known to regulate various cellular processes, including biofilm formation, motility, and virulence. The intracellular concentration of c-di-GMP is inversely governed by diguanylate cyclase (DGC) enzymes and phosphodiesterase (PDE) enzymes, which synthesize and degrade c-di-GMP, respectively. The role of c-di-GMP in the plant pathogen and causal agent of fire blight disease Erwinia amylovora has not been studied previously. Here we demonstrate that three of the five predicted DGC genes in E. amylovora (edc genes, for Erwinia diguanylate cyclase), edcA, edcC, and edcE, are active diguanylate cyclases. We show that c-di-GMP positively regulates the secretion of the main exopolysaccharide in E. amylovora, amylovoran, leading to increased biofilm formation, and negatively regulates flagellar swimming motility. Although amylovoran secretion and biofilm formation are important for the colonization of plant xylem tissues and the development of systemic infections, deletion of the two biofilm-promoting DGCs increased tissue necrosis in an immature-pear infection assay and an apple shoot infection model, suggesting that c-di-GMP negatively regulates virulence. In addition, c-di-GMP inhibited the expression of hrpA, a gene encoding the major structural component of the type III secretion pilus. Our results are the first to describe a role for c-di-GMP in E. amylovora and suggest that downregulation of motility and type III secretion by c-di-GMP during infection plays a key role in the coordination of pathogenesis.

  17. Coordinated Cyclic-Di-GMP Repression of Salmonella Motility through YcgR and Cellulose

    PubMed Central

    Zorraquino, Violeta; García, Begoña; Latasa, Cristina; Echeverz, Maite; Toledo-Arana, Alejandro; Valle, Jaione

    2013-01-01

    Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition. PMID:23161026

  18. Bile acids and bicarbonate inversely regulate intracellular cyclic di-GMP in Vibrio cholerae.

    PubMed

    Koestler, Benjamin J; Waters, Christopher M

    2014-07-01

    Vibrio cholerae is a Gram-negative bacterium that persists in aquatic reservoirs and causes the diarrheal disease cholera upon entry into a human host. V. cholerae employs the second messenger molecule 3',5'-cyclic diguanylic acid (c-di-GMP) to transition between these two distinct lifestyles. c-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and hydrolyzed by phosphodiesterase (PDE) enzymes. Bacteria typically encode many different DGCs and PDEs within their genomes. Presumably, each enzyme senses and responds to cognate environmental cues by alteration of enzymatic activity. c-di-GMP represses the expression of virulence factors in V. cholerae, and it is predicted that the intracellular concentration of c-di-GMP is low during infection. Contrary to this model, we found that bile acids, a prevalent constituent of the human proximal small intestine, increase intracellular c-di-GMP in V. cholerae. We identified four c-di-GMP turnover enzymes that contribute to increased intracellular c-di-GMP in the presence of bile acids, and deletion of these enzymes eliminates the bile induction of c-di-GMP and biofilm formation. Furthermore, this bile-mediated increase in c-di-GMP is quenched by bicarbonate, the intestinal pH buffer secreted by intestinal epithelial cells. Our results lead us to propose that V. cholerae senses distinct microenvironments within the small intestine using bile and bicarbonate as chemical cues and responds by modulating the intracellular concentration of c-di-GMP. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  19. An Extended Cyclic Di-GMP Network in the Predatory Bacterium Bdellovibrio bacteriovorus

    PubMed Central

    Rotem, Or; Nesper, Jutta; Borovok, Ilya; Gorovits, Rena; Kolot, Mikhail; Pasternak, Zohar; Shin, Irina; Glatter, Timo; Pietrokovski, Shmuel; Jenal, Urs

    2015-01-01

    ABSTRACT Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and

  20. Cyclic Di-GMP Regulates Type IV Pilus-Dependent Motility in Myxococcus xanthus

    PubMed Central

    Skotnicka, Dorota; Petters, Tobias; Heering, Jan; Hoppert, Michael; Kaever, Volkhard

    2015-01-01

    ABSTRACT The nucleotide-based second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) is involved in regulating a plethora of processes in bacteria that are typically associated with lifestyle changes. Myxococcus xanthus undergoes major lifestyle changes in response to nutrient availability, with the formation of spreading colonies in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. Here, we investigated the function of c-di-GMP in M. xanthus and show that this bacterium synthesizes c-di-GMP during growth. Manipulation of the c-di-GMP level by expression of either an active, heterologous diguanylate cyclase or an active, heterologous phosphodiesterase correlated with defects in type IV pilus (T4P)-dependent motility, whereas gliding motility was unaffected. An increased level of c-di-GMP correlated with reduced transcription of the pilA gene (which encodes the major pilin of T4P), reduced the assembly of T4P, and altered cell agglutination, whereas a decreased c-di-GMP level correlated with altered cell agglutination. The systematic inactivation of the 24 genes in M. xanthus encoding proteins containing GGDEF, EAL, or HD-GYP domains, which are associated with c-di-GMP synthesis, degradation, or binding, identified three genes encoding proteins important for T4P-dependent motility, whereas all mutants had normal gliding motility. Purified DmxA had diguanylate cyclase activity, whereas the hybrid histidine protein kinases TmoK and SgmT, each of which contains a GGDEF domain, did not have diguanylate cyclase activity. These results demonstrate that c-di-GMP is important for T4P-dependent motility in M. xanthus. IMPORTANCE We provide the first direct evidence that M. xanthus synthesizes c-di-GMP and demonstrate that c-di-GMP is important for T4P-dependent motility, whereas we did not obtain evidence that c-di-GMP regulates gliding motility. The data presented uncovered a novel mechanism for regulation of T4P

  1. Control of Ca2+ in rod outer segment disks by light and cyclic GMP.

    PubMed

    George, J S; Hagins, W A

    1983-05-26

    Photons absorbed in vertebrate rods and cones probably cause electrochemical changes at the photoreceptor plasma membrane by changing the cytoplasmic concentration of a diffusible transmitter substance, reducing the Na+ current flowing into the outer segment of the cell in the dark, to produce the observed membrane hyperpolarization that is the initial excitatory response. Cyclic GMP has been proposed as the transmitter because a light-activated cyclic GMP phosphodiesterase (PDE) has been found in rod disk membranes and because intracellularly injected cyclic GMP reduces rod membrane potentials. Free Ca2+ has also been proposed because increasing external [Ca2+] quickly and reversibly reduces the dark current and divalent cationophores increase the Ca2+ sensitivity. Ca2+ efflux from rod outer segments (ROS) of intact retinas occurs simultaneously with light responses. Vesicles prepared from ROS disk membranes become more permeable on illumination, releasing trapped ions or molecules, but intact outer segment disks have not previously been found to store sufficient Ca2+ in darkness and to release enough in light to meet the theoretical requirements for control of the dark current by varying cytoplasmic Ca2+ (refs 14-18). We now report experiments that show the required Ca2+ storage and release from rod disk membranes suspended in media containing high-energy phosphate esters and electrolytes approximating the cytoplasmic composition of live rod cells. Cyclic GMP stimulates Ca2+ uptake by ROS disks in such media.

  2. Atrial natriuretic peptide receptor heterogeneity and effects on cyclic GMP accumulation

    SciTech Connect

    Leitman, D.C.

    1988-01-01

    The effects of atrial natriuretic peptide (ANP), oxytocin (OT) and vasopressin (AVP) on guanylate cyclase activity and cyclic GMP accumulation were examined, since these hormones appear to be intimately associated with blood pressure and intravascular volume homeostasis. ANP was found to increase cyclic GMP accumulation in ten cell culture systems, which were derived from blood vessels, adrenal cortex, kidney, lung, testes and mammary gland. ANP receptors were characterized in intact cultured cells using {sup 125}I-ANP{sub 8-33}. Specific {sup 125}I-ANP binding was saturable and of high affinity. Scratchard analysis of the binding data for all cell types exhibited a straight line, indicating that these cells possessed a single class of binding sites. Despite the presence of linear Scatchard plots, these studies demonstrated that cultured cells possess two functionally and physically distinct ANP-binding sites. Most of the ANP-binding sites in cultured cells have a molecular size of 66,000 daltons under reducing conditions. The identification of cultured cell types in which hormones (ANP and oxytocin) regulate guanylate cyclase activity and increase cyclic GMP synthesis will provide valuable systems to determine the mechanisms of hormone-receptor coupling to guanylate cyclase and the cellular processes regulated by cyclic GMP.

  3. Modulation by cyclic GMP of the odour sensitivity of vertebrate olfactory receptor cells

    NASA Technical Reports Server (NTRS)

    Leinders-Zufall, T.; Shepherd, G. M.; Zufall, F.

    1996-01-01

    Recent evidence has indicated a significant role for the cGMP second messenger system in vertebrate olfactory transduction but no clear functions have been identified for cGMP so far. Here, we have examined the effects of 8-Br-cGMP and carbon monoxide (CO) on odour responses of salamander olfactory receptor neurons using perforated patch recordings. We report that 8-Br-cGMP strongly down-regulates the odour sensitivity of the cells, with a K1/2 of 460 nM. This adaptation-like effect can be mimicked by CO, an activator of soluble guanylyl cyclase, with a K1/2 of 1 microM. Sensitivity modulation is achieved through a regulatory chain of events in which cGMP stimulates a persistent background current due to the activation of cyclic nucleotide-gated channels. This in turn leads to sustained Ca2+ entry providing a negative feedback signal. One consequence of the Ca2+ entry is a shift to the right of the stimulus-response curve and a reduction in saturating odour currents. Together, these two effects can reduce the sensory generator current by up to twenty-fold. Thus, cGMP functions to control the gain of the G-protein coupled cAMP pathway. Another consequence of the action of cGMP is a marked prolongation of the odour response kinetics. The effects of CO/cGMP are long-lasting and can continue for minutes. Hence, we propose that cGMP helps to prevent saturation of the cell's response by adjusting the operational range of the cAMP cascade and contributes to olfactory adaptation by decreasing the sensitivity of olfactory receptor cells to repeated odour stimuli.

  4. Modulation by cyclic GMP of the odour sensitivity of vertebrate olfactory receptor cells

    NASA Technical Reports Server (NTRS)

    Leinders-Zufall, T.; Shepherd, G. M.; Zufall, F.

    1996-01-01

    Recent evidence has indicated a significant role for the cGMP second messenger system in vertebrate olfactory transduction but no clear functions have been identified for cGMP so far. Here, we have examined the effects of 8-Br-cGMP and carbon monoxide (CO) on odour responses of salamander olfactory receptor neurons using perforated patch recordings. We report that 8-Br-cGMP strongly down-regulates the odour sensitivity of the cells, with a K1/2 of 460 nM. This adaptation-like effect can be mimicked by CO, an activator of soluble guanylyl cyclase, with a K1/2 of 1 microM. Sensitivity modulation is achieved through a regulatory chain of events in which cGMP stimulates a persistent background current due to the activation of cyclic nucleotide-gated channels. This in turn leads to sustained Ca2+ entry providing a negative feedback signal. One consequence of the Ca2+ entry is a shift to the right of the stimulus-response curve and a reduction in saturating odour currents. Together, these two effects can reduce the sensory generator current by up to twenty-fold. Thus, cGMP functions to control the gain of the G-protein coupled cAMP pathway. Another consequence of the action of cGMP is a marked prolongation of the odour response kinetics. The effects of CO/cGMP are long-lasting and can continue for minutes. Hence, we propose that cGMP helps to prevent saturation of the cell's response by adjusting the operational range of the cAMP cascade and contributes to olfactory adaptation by decreasing the sensitivity of olfactory receptor cells to repeated odour stimuli.

  5. Estimating the rate constant of cyclic GMP hydrolysis by activated phosphodiesterase in photoreceptors

    NASA Astrophysics Data System (ADS)

    Reingruber, Jürgen; Holcman, David

    2008-10-01

    The early steps of light response occur in the outer segment of rod and cone photoreceptor. They involve the hydrolysis of cGMP, a soluble cyclic nucleotide, that gates ionic channels located in the outer segment membrane. We shall study here the rate by which cGMP is hydrolyzed by activated phosphodiesterase (PDE). This process has been characterized experimentally by two different rate constants βd and βsub: βd accounts for the effect of all spontaneously active PDE in the outer segment, and βsub characterizes cGMP hydrolysis induced by a single light-activated PDE. So far, no attempt has been made to derive the experimental values of βd and βsub from a theoretical model, which is the goal of this work. Using a model of diffusion in the confined rod geometry, we derive analytical expressions for βd and βsub by calculating the flux of cGMP molecules to an activated PDE site. We obtain the dependency of these rate constants as a function of the outer segment geometry, the PDE activation and deactivation rates and the aqueous cGMP diffusion constant. Our formulas show good agreement with experimental measurements. Finally, we use our derivation to model the time course of the cGMP concentration in a transversally well-stirred outer segment.

  6. Bacterial Signal Transduction by Cyclic Di-GMP and Other Nucleotide Second Messengers

    PubMed Central

    Gründling, Angelika; Jenal, Urs; Ryan, Robert; Yildiz, Fitnat

    2015-01-01

    The first International Symposium on c-Di-GMP Signaling in Bacteria (22 to 25 March 2015, Harnack-Haus, Berlin, Germany) brought together 131 molecular microbiologists from 17 countries to discuss recent progress in our knowledge of bacterial nucleotide second messenger signaling. While the focus was on signal input, synthesis, degradation, and the striking diversity of the modes of action of the current second messenger paradigm, i.e., cyclic di-GMP (c-di-GMP), “classics” like cAMP and (p)ppGpp were also presented, in novel facets, and more recent “newcomers,” such as c-di-AMP and c-AMP-GMP, made an impressive appearance. A number of clear trends emerged during the 30 talks, on the 71 posters, and in the lively discussions, including (i) c-di-GMP control of the activities of various ATPases and phosphorylation cascades, (ii) extensive cross talk between c-di-GMP and other nucleotide second messenger signaling pathways, and (iii) a stunning number of novel effectors for nucleotide second messengers that surprisingly include some long-known master regulators of developmental pathways. Overall, the conference made it amply clear that second messenger signaling is currently one of the most dynamic fields within molecular microbiology, with major impacts in research fields ranging from human health to microbial ecology. PMID:26055111

  7. Cyclic-di-GMP signalling regulates motility and biofilm formation in Bordetella bronchiseptica

    PubMed Central

    Sisti, Federico; Ha, Dae-Gon; O'Toole, George A.; Hozbor, Daniela

    2013-01-01

    The signalling molecule bis-(3′–5′)-cyclic-dimeric guanosine monophosphate (c-di-GMP) is a central regulator of diverse cellular functions, including motility, biofilm formation, cell cycle progression and virulence, in bacteria. Multiple diguanylate cyclase and phosphodiesterase-domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) modulate the levels of the second messenger c-di-GMP to transmit signals and obtain such specific cellular responses. In the genus Bordetella this c-di-GMP network is poorly studied. In this work, we evaluated the expression of two phenotypes in Bordetella bronchiseptica regulated by c-di-GMP, biofilm formation and motility, under the influence of ectopic expression of Pseudomonas aeruginosa proteins with EAL or GGDEF domains that regulates the c-di-GMP level. In agreement with previous reports for other bacteria, we observed that B. bronchiseptica is able to form biofilm and reduce its motility only when GGDEF domain protein is expressed. Moreover we identify a GGDEF domain protein (BB3576) with diguanylate cyclase activity that participates in motility and biofilm regulation in B. bronchiseptica. These results demonstrate for the first time, to our knowledge, the presence of c-di-GMP regulatory signalling in B. bronchiseptica. PMID:23475948

  8. The Nitric Oxide/Cyclic GMP Pathway in Organ Transplantation: Critical Role in Successful Lung Preservation

    NASA Astrophysics Data System (ADS)

    Pinsky, David J.; Naka, Yoshifumi; Chowdhury, Nepal C.; Liao, Hui; Oz, Mehmet C.; Michler, Robert E.; Kubaszewski, Eugeniusz; Malinski, Tadeusz; Stern, David M.

    1994-12-01

    Reestablishment of vascular homeostasis following ex vivo preservation is a critical determinant of successful organ transplantation. Because the nitric oxide (NO) pathway modulates pulmonary vascular tone and leukocyte/endothelial interactions, we hypothesized that reactive oxygen intermediates would lead to decreased NO (and hence cGMP) levels following pulmonary reperfusion, leading to increased pulmonary vascular resistance and leukostasis. Using an orthotopic rat model of lung transplantation, a porphyrinic microsensor was used to make direct in vivo measurements of pulmonary NO. NO levels measured at the surface of the transplanted lung plummeted immediately upon reperfusion, with levels moderately increased by topical application of superoxide dismutase. Because cGMP levels declined in preserved lungs after reperfusion, this led us to buttress the NO pathway by adding a membrane-permeant cGMP analog to the preservation solution. Compared with grafts stored in its absence, grafts stored with supplemental 8-Br-cGMP and evaluated 30 min after reperfusion demonstrated lower pulmonary vascular resistances with increased graft blood flow, improved arterial oxygenation, decreased neutrophil infiltration, and improved recipient survival. These beneficial effects were dose dependent, mimicked by the type V phosphodiesterase inhibitor 2-o-propoxyphenyl-8-azapurin-6-one, and inhibited by a cGMP-dependent protein kinase antagonist, the R isomer of 8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate. Augmenting the NO pathway at the level of cGMP improves graft function and recipient survival following lung transplantation.

  9. A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii.

    PubMed

    Avelar, Gabriela Mól; Glaser, Talita; Leonard, Guy; Richards, Thomas A; Ulrich, Henning; Gomes, Suely L

    2015-09-01

    Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K(+) channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K(+) selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K(+) channel and the rhodopsin-guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K(+) channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Expression and Genetic Activation of Cyclic Di-GMP-Specific Phosphodiesterases in Escherichia coli

    PubMed Central

    Reinders, Alberto; Hee, Chee-Seng; Ozaki, Shogo; Mazur, Adam; Boehm, Alex; Schirmer, Tilman

    2015-01-01

    ABSTRACT Intracellular levels of the bacterial second messenger cyclic di-GMP (c-di-GMP) are controlled by antagonistic activities of diguanylate cyclases and phosphodiesterases. The phosphodiesterase PdeH was identified as a key regulator of motility in Escherichia coli, while deletions of any of the other 12 genes encoding potential phosphodiesterases did not interfere with motility. To analyze the roles of E. coli phosphodiesterases, we demonstrated that most of these proteins are expressed under laboratory conditions. We next isolated suppressor mutations in six phosphodiesterase genes, which reinstate motility in the absence of PdeH by reducing cellular levels of c-di-GMP. Expression of all mutant alleles also led to a reduction of biofilm formation. Thus, all of these proteins are bona fide phosphodiesterases that are capable of interfering with different c-di-GMP-responsive output systems by affecting the global c-di-GMP pool. This argues that E. coli possesses several phosphodiesterases that are inactive under laboratory conditions because they lack appropriate input signals. Finally, one of these phosphodiesterases, PdeL, was studied in more detail. We demonstrated that this protein acts as a transcription factor to control its own expression. Motile suppressor alleles led to a strong increase of PdeL activity and elevated pdeL transcription, suggesting that enzymatic activity and transcriptional control are coupled. In agreement with this, we showed that overall cellular levels of c-di-GMP control pdeL transcription and that this control depends on PdeL itself. We thus propose that PdeL acts both as an enzyme and as a c-di-GMP sensor to couple transcriptional activity to the c-di-GMP status of the cell. IMPORTANCE Most bacteria possess multiple diguanylate cyclases and phosphodiesterases. Genetic studies have proposed that these enzymes show signaling specificity by contributing to distinct cellular processes without much cross talk. Thus, spatial

  11. [Biosynthesis of cyclic GMP in plant cells - new insight into guanylate cyclases].

    PubMed

    Świeżawska, Brygida; Marciniak, Katarzyna; Szmidt-Jaworska, Adriana

    2015-01-01

    Cyclic 3',5'-guanosine monophosphate (cGMP) is involved in many physiological processes in plants. Concentration of this second messenger in plant cell is determined by guanylyl cyclases (GCs) responsible for cGMP synthesis and phosphodiesterases (PDEs) involved in cGMP inactivation. First discovered plant GCs were localized in cytosol, but few years ago a new family of plasma membrane proteins with guanylyl cyclase activity was identified in Arabidopsis thaliana. These proteins belong to the family of a leucine-rich repeat receptor-like kinases (LRR-RLK) with extracellular leucine-rich repeat domain, a transmembrane-spanning domain, and an intracellular kinase domain. A novel class of guanylyl cyclases contain the GC catalytic center encapsulated within the intracellular kinase domain. These molecules are different to animal GCs in that the GC catalytic center is nested within the kinase domain. In presented paper we summarized the most recent data concerning plant guanylyl cyclases.

  12. Fifty ways to inhibit motility via cyclic di-GMP: the emerging Pseudomonas aeruginosa swarming story.

    PubMed

    McCarter, Linda L; Gomelsky, Mark

    2015-02-01

    There are numerous ways by which cyclic dimeric GMP (c-di-GMP) inhibits motility. Kuchma et al. (S. L. Kuchma, N. J. Delalez, L. M. Filkins, E. A. Snavely, J. P. Armitage, and G. A. O'Toole, J. Bacteriol. 197:420-430, 2015, http://dx.doi.org/10.1128/JB.02130-14) offer a new, previously unseen way of swarming motility inhibition in Pseudomonas aeruginosa PA14. This bacterium possesses a single flagellum with one rotor and two sets of stators, only one of which can provide torque for swarming. The researchers discovered that elevated levels of c-di-GMP inhibit swarming by skewing stator selection in favor of the nonfunctional, "bad" stators. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. Role of Na+-K+ ATPase in cyclic GMP-mediated relaxation of canine pulmonary artery smooth muscle cells

    PubMed Central

    Tamaoki, J; Tagaya, E; Nishimura, K; Isono, K; Nagai, A

    1997-01-01

    Sodium-potassium adenosine triphosphatase (Na+-K+ ATPase) plays a role in the regulation of vascular tone, but contribution of this enzyme to nitrovasodilator-induced pulmonary vasodilatation remains uncertain. We thus studied the interaction between guanosine 3′:5′-cyclic monophosphate (cyclic GMP) and Na+-K+ ATPase in smooth muscle cells isolated from canine pulmonary artery. To assess the contractile properties, changes in smooth muscle cell length were determined microscopically. Application of potassium chloride (KCl) shortened the cell length, an effect which was reduced by sodium nitroprusside and 8-bromo-cyclic GMP in a concentration-dependent manner. Pretreatment of cells with the cyclic GMP-dependent kinase inhibitor KT 5823 (2 μM) abolished the effects of sodium nitroprusside and 8-bromo-cyclic GMP. Ouabain (0.3 μM) did not alter the KCl-induced muscle shortening, but inhibited the relaxant responses to sodium nitroprusside and 8-bromo-cyclic GMP. Incubation of smooth muscle cells with sodium nitroprusside concentration-dependently increased intracellular cyclic GMP levels and ouabain-sensitive 86Rb uptake, and these values were significantly correlated. In the presence of KT 5823, sodium nitroprusside increased cyclic GMP levels but did not alter ouabain-sensitive 86Rb uptake. These results suggest that there is a link between accumulation of intracellular cyclic GMP and activation of sarcolemmal Na+-K+ ATPase in pulmonary artery smooth muscle cells and that this link may be involved in the sodium nitroprusside-induced pulmonary vasodilatation. PMID:9298536

  14. Disintegration of aerobic granules: role of second messenger cyclic di-GMP.

    PubMed

    Wan, Chunli; Zhang, Peng; Lee, Duu-Jong; Yang, Xue; Liu, Xiang; Sun, Supu; Pan, Xiangliang

    2013-10-01

    Loss of structural stability of aerobic granular process is the challenge for its field applications to treat wastewaters. The second messenger, cyclic diguanylate (c-di-GMP), is widely used by bacteria to regulate the synthesis of exopolysaccharide. This study for the first time confirmed the correlation between concentration of intracellular c-di-GMP and the granular stability under sequencing batch reactor (MBR) mode. In the presence of manganese ions (Mn(2+)), the concentrations of intracellular c-di-GMP and of extracellular polysaccharides and proteins in granules were declined. Clone library study revealed that the polysaccharide producers. Acinetobacter sp., Thauera sp., Bdellovibrio sp. and Paracoccus sp. were lost after Mn(2+) addition. The findings reported herein confirmed that the c-di-GMP is a key chemical factor epistatic to quorum sensing to determine granular stability. Stimulation of synthesis of intracellular c-di-GMP presents a potential way to enhance long-term stability of aerobic granules. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. The effect of cyclic AMP and cyclic GMP phosphodiesterase inhibitors on the superoxide burst of guinea-pig peritoneal macrophages.

    PubMed Central

    Turner, N. C.; Wood, L. J.; Burns, F. M.; Gueremy, T.; Souness, J. E.

    1993-01-01

    1. The cyclic nucleotide phosphodiesterase (PDE) activity of guinea-pig peritoneal macrophages was partially characterized and the effects of selective and non-selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP PDE) and guanosine 3':5'-cyclic monophosphate (cyclic GMP PDE) phosphodiesterases on superoxide generation were investigated using peritoneal macrophages from horse-serum pretreated guinea-pigs. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and the PDE I/V selective inhibitor, zaprinast, inhibited spontaneous superoxide generation with IC50s of 30.7 +/- 11.3 microM and 145 +/- 17 microM respectively (n = 6 and 5). The concentration-response curves for the PDE IV selective inhibitors rolipram and Ro20-1724 were biphasic; mean maximum inhibitions were 56.9 +/- 5.9% and 66.8 +/- 10.5% respectively at 300 microM, but in 2 out of 6 (rolipram) and 2 out of 5 (Ro20-1724) experiments inhibition was < 50%. The PDE III inhibitor SK&F 94120 was without effect. Spontaneous superoxide generation was reduced 57 +/- 10% by 1 microM prostaglandin E2 (PGE2) and 62.6 +/- 3.76% by 1 microM salbutamol. 3. The increase in superoxide generation elicited by FMLP (10(-9)-10(-5)M) was unaffected by any of the PDE inhibitors studied. Inhibition of FMLP-stimulated superoxide generation by PGE2 was enhanced in the presence of 10 microM IBMX. 4. Macrophages were found to contain a predominantly membrane bound cyclic AMP PDE (90% of total activity) which was unaffected by cyclic GMP or calcium/calmodulin. The cyclic AMP PDE activity in the cytosolic fraction was enhanced in the presence of calcium/calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8387385

  16. Nocturnal decreases in nitric oxide and cyclic GMP contents in the chick brain and their prevention by light.

    PubMed

    Guerrero, J M; Pablos, M I; Ortiz, G G; Agapito, M T; Reiter, R J

    1996-10-01

    The diurnal variations in the contents of nitric oxide (NO) and cyclic GMP were studied in the chick brain. NO and cyclic GMP contents in the chick brain were lower at night than during the day and were inversely correlated with high night-time tissue melatonin levels. Furthermore, when animals were kept in light at night, tissue melatonin levels remained at low diurnal values, whereas NO and cyclic GMP contents remained high. Since we have previously shown that physiological concentrations of melatonin inhibit nitric oxide synthase (NOS) activity in different brain areas, the nocturnal decrease in brain NO and cyclic GMP contents may be, in part, a consequence of the nocturnal inhibitory effect of melatonin on NOS activity.

  17. Immunohistochemical Localization of Cyclic GMP-Dependent Protein Kinase in Mammalian Brain

    NASA Astrophysics Data System (ADS)

    Lohmann, Suzanne M.; Walter, Ulrich; Miller, Penelope E.; Greengard, Paul; de Camilli, Pietro

    1981-01-01

    The distribution of cyclic GMP-dependent protein kinase in rat brain has been studied by an immunological approach involving radioimmunoassay and fluorescence immunohistochemistry. Data obtained by radioimmunoassay indicate that cyclic GMP-dependent protein kinase is 20- to 40-fold more concentrated in cerebellum than in other brain regions. Immunohistochemical experiments demonstrate that the high concentration of immunoreactivity of the protein kinase in cerebellum is attributable to Purkinje cells. Immunoreactivity in these cells is homogeneously distributed throughout the cell (perikarya, dendrites, and axons) with the exception of the nucleus. No other neurons either in the cerebellum or in other brain regions were stained by antiserum to the protein kinase. Immunoreactivity, however, was found throughout the brain on smooth muscle cells of blood vessels.

  18. Desensitization of AMPA receptors and AMPA-NMDA receptor interaction: an in vivo cyclic GMP microdialysis study in rat cerebellum.

    PubMed Central

    Fedele, E.; Raiteri, M.

    1996-01-01

    1. Desensitization is an important characteristic of glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type. 2. Stimulation of N-methyl-D-aspartate (NMDA) or AMPA receptors in cerebellum results in increased production of cyclic GMP. We have investigated AMPA receptor desensitization in vivo by monitoring extracellular cyclic GMP during intracerebellar microdialysis in conscious unrestrained adult rats. 3. Local infusion of AMPA (10 to 100 microM) caused dose-related elevations of cyclic GMP levels. The effect of AMPA was prevented by the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NOARG). 4. In the absence of AMPA, DNQX lowered the basal levels of cyclic GMP whereas the NMDA receptor channel antagonist dizocilpine (MK-801) was ineffective. 5. Cyclothiazide, a blocker of AMPA receptor desensitization, potentiated the cyclic GMP response to exogenous AMPA. Moreover, cyclothiazide (100-300 microM) produced on its own dose-dependent elevations of extracellular cyclic GMP. The cyclothiazide-induced response was prevented not only by DNQX but also by MK-801. 6. While the cyclic GMP response elicited by AMPA was totally insensitive to MK-801, the response produced by AMPA (10 microM) plus cyclothiazide (30 microM) was strongly attenuated by the NMDA receptor antagonist (30 microM). 7. The results suggest that (a) AMPA receptors linked to the NO-cyclic GMP pathway in the cerebellum can undergo desensitization in vivo during exposure to exogenous AMPA; cyclothiazide inhibits such desensitization; (b) AMPA receptors (but not NMDA receptors) are 'tonically' activated and kept in a partly desensitized state by endogenous glutamate; (c) if cyclothiazide is present, activation of AMPA receptors may permit endogenous activation of NMDA receptors. PMID:8882607

  19. A minimalist biosensor: Quantitation of cyclic di-GMP using the conformational change of a riboswitch aptamer

    PubMed Central

    Kellenberger, Colleen A; Sales-Lee, Jade; Pan, Yuchen; Gassaway, Madalee M; Herr, Amy E; Hammond, Ming C

    2015-01-01

    Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA. PMID:26114964

  20. Characterization of noradrenaline-stimulated cyclic GMP formation in brain astrocytes in culture.

    PubMed Central

    Agulló, L; García, A

    1992-01-01

    Cyclic GMP accumulation induced by noradrenaline in astrocyte-enriched primary cultures from rat cerebrum involves synthesis of NO, as evidenced by the competitive inhibition exerted by the NO synthase inhibitor NG-monomethyl-L-arginine (IC50 = 3 microM). Furthermore, the noradrenaline effect was potently inhibited by haemoglobin (IC50 = 25 nM) and potentiated by superoxide dismutase, indicating that NO synthesis and cyclic GMP formation may occur in different subsets of astrocytes. Investigation of the receptors implicated by using selective adrenoceptor agonists and antagonists indicates that about 75% of the NO-dependent noradrenaline response is mediated by alpha 1-adrenoceptors and the rest by beta-adrenoceptors, with no evidence for potentiating effects between the two receptor types. This noradrenaline effect appears to require Ca2+ entry, since it is strongly dependent on extracellular Ca2+ but is not affected by conditions that will abolish intracellular Ca2+ mobilization (incubation with neomycin or pretreatment with carbachol). Inhibition by pretreatment with pertussis toxin is in agreement with involvement of the alpha 1A-adrenoceptor subtype in this Ca(2+)-dependent effect. However, implication of an unknown alpha 1-adrenoceptor subtype cannot be disregarded, because a similar inhibition is exerted by the presumably selective alpha 1B- and alpha 1C-adrenoceptor blocking agent chloroethylclonidine. Treatment of the cultures with the protein kinase C activator phorbol 12-myristate 13-acetate inhibits to a great extent the noradrenaline-induced cyclic GMP formation. PMID:1334410

  1. Role of nitric oxide/cyclic GMP in myocardial adenosine A1 receptor-inotropic response.

    PubMed

    Sterin-Borda, Leonor; Gómez, Ricardo M; Borda, Enri

    2002-01-01

    In this study we have determined the different signalling pathways involved in adenosine A(1)-receptor (A(1)-receptor)-dependent inhibition of contractility in rat isolated atria. N-cyclopentyladenosine (CPA) stimulation of A(1)-receptor exerts: negative inotropic response, inositol phosphates accumulation, stimulation of nitric oxide synthase (NOS), increased production of nitric oxide (NO) and cyclic GMP. Inhibitors of phospholipase C (PLC), protein kinase C (PKC), calcium/calmodulin, NOS and guanylate cyclase shifted the dose-response curve of CPA on contractility to the right. Those inhibitors also attenuated the A(1)-receptor-dependent increase in cyclic GMP and activation of NOS. These results suggest that CPA activation of A(1)-receptors exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving calcium/calmodulin and PKC, leading to activation of NOS and soluble guanylate cyclase.

  2. Role of nitric oxide/cyclic GMP in myocardial adenosine A1 receptor-inotropic response

    PubMed Central

    Sterin-Borda, Leonor; Gómez, Ricardo M; Borda, Enri

    2002-01-01

    In this study we have determined the different signalling pathways involved in adenosine A1-receptor (A1-receptor)-dependent inhibition of contractility in rat isolated atria. N-cyclopentyladenosine (CPA) stimulation of A1-receptor exerts: negative inotropic response, inositol phosphates accumulation, stimulation of nitric oxide synthase (NOS), increased production of nitric oxide (NO) and cyclic GMP. Inhibitors of phospholipase C (PLC), protein kinase C (PKC), calcium/calmodulin, NOS and guanylate cyclase shifted the dose-response curve of CPA on contractility to the right. Those inhibitors also attenuated the A1-receptor-dependent increase in cyclic GMP and activation of NOS. These results suggest that CPA activation of A1-receptors exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving calcium/calmodulin and PKC, leading to activation of NOS and soluble guanylate cyclase. PMID:11815380

  3. Role of nitric oxide and cyclic GMP signaling in melanocyte response to hypergravity

    NASA Astrophysics Data System (ADS)

    Ivanova, Krassimira; Lambers, Britta; Tsiockas, Wasiliki; Block, Ingrid; Gerzer, Rupert

    Nitric oxide (NO) has a prominent role in many (patho)physiological processes in the skin including erythema, inflammation, and cancerogenesis. The soluble guanylyl cyclase (sGC), a key transducer in NO signaling, catalyzes the formation of the second messenger guanosine 3´,5´-cyclic monophosphate (cyclic cGMP or cGMP). For human melanocytes, which are responsible for skin pigmentation by synthesizing the pigment melanin, it has been reported that the NO/sGC/cGMP pathway is involved in UVB-induced melanogenesis. Melanin acts as a scavenger for free radicals that may arise during metabolic stress. It may also act as a photosensitizer that generates active oxygen species upon UV irradiation, which may initiate hypopigmentary disorders (e.g., vitiligo) as well as UV-induced oncogene cell transformation. In addition, melanoma, a deadly skin cancer, which arises from transformed melanocytes, is characterized by a resistance to chemotherapy. In our studies we have shown that NO can induce perturbation of melanocyte-extracellular matrix component interactions, which may contribute to loss of melanocytes or melanoma metastasis. Such NO effects appear to be modulated partly via cGMP. Moreover, we found that different guanylyl cyclase isoforms are responsible for cGMP synthesis in melanocytic cells. Normal human melanocytes and nonmetastatic melanoma cells predominantly express sGC, which appears to be associated with melanogenesis, whereas absence of NO-sensitive GC, but up-regulated activities of the natriuretic peptide-sensitive membrane guanylyl cyclase isoforms were found in highly metastatic phenotypes. Due to the growing interest in the regulation of signaling activities in normal and transformed cells under altered gravity conditions, we have further investigated whether the NO/cGMP signaling is involved in melanocyte response to gravitational stress. We found that normal human melanocytes and non-metastatic melanoma cell lines, but not highly metastatic cells

  4. CRP-Cyclic AMP Regulates the Expression of Type 3 Fimbriae via Cyclic di-GMP in Klebsiella pneumoniae

    PubMed Central

    Lin, Ching-Ting; Lin, Tien-Huang; Wu, Chien-Chen; Wan, Lei; Huang, Chun-Fa; Peng, Hwei-Ling

    2016-01-01

    Klebsiella pneumoniae is the predominant pathogen isolated from liver abscesses of diabetic patients in Asian countries. However, the effects of elevated blood glucose levels on the virulence of this pathogen remain largely unknown. Type 3 fimbriae, encoded by the mrkABCDF genes, are important virulence factors in K. pneumoniae pathogenesis. In this study, the effects of exogenous glucose and the intracellular cyclic AMP (cAMP) signaling pathway on type 3 fimbriae expression regulation were investigated. The production of MrkA, the major subunit of type 3 fimbriae, was increased in glucose-rich medium, whereas cAMP supplementation reversed the effect. MrkA production was markedly increased by cyaA or crp deletion, but slightly decreased by cpdA deletion. In addition, the mRNA levels of mrkABCDF genes and the activity of PmrkA were increased in Δcrp strain, as well as the mRNA levels of mrkHIJ genes that encode cyclic di-GMP (c-di-GMP)-related regulatory proteins that influence type 3 fimbriae expression. Moreover, the activities of PmrkHI and PmrkJ were decreased in ΔlacZΔcrp strain. These results indicate that CRP-cAMP down-regulates mrkABCDF and mrkHIJ at the transcriptional level. Further deletion of mrkH or mrkI in Δcrp strain diminished the production of MrkA, indicating that MrkH and MrkI are required for the CRP regulation of type 3 fimbriae expression. Furthermore, the high activity of PmrkHI in the ΔlacZΔcrp strain was diminished in ΔlacZΔcrpΔmrkHI, but increased in the ΔlacZΔcrpΔmrkJ strain. Deletion of crp increased the intracellular c-di-GMP concentration and reduced the phosphodiesterase activity. Moreover, we found that the mRNA levels of multiple genes related to c-di-GMP metabolism were altered in Δcrp strain. These indicate that CRP regulates type 3 fimbriae expression indirectly via the c-di-GMP signaling pathway. In conclusion, we found evidence of a coordinated regulation of type 3 fimbriae expression by the CRP-cAMP and c-di-GMP

  5. Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs

    NASA Technical Reports Server (NTRS)

    Kandasamy, S. B.; Williaes, B. A.

    1983-01-01

    It is shown that the intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP) induced hyperthermia in guinea pigs which was not mediated through prostaglandins or norepinephrine since a prostaglandin synthesis inhibitor and an alpha-adrenergic receptor blocking agent did not antagonize the hyperthermia. However, the hyperthermic response to Db-cAMP was attenuated by the central administration of a beta-adrenergic receptor antagonist, which indicates that cAMP may be involved, through beta-adrenergic receptors, in the central regulation of heat production and conservation. The central administration of Db-cGMP produced hypothermia which was not mediated via histamine H1 or H2 receptors and serotonin. The antagonism of hypothermia induced by Db-cGMP and acetylcholine + physostigmine by central administration of a cholinergic muscarine receptor antagonist and not by a cholinergic nicotinic receptor antagonist suggests that cholinoceptive neurons and endogenous cGMP may regulate heat loss through cholinergic muscarine receptors. It is concluded that these results indicate a regulatory role in thermoregulation provided by a balance between opposing actions of cAMP and cGMP in guinea pigs.

  6. Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs

    NASA Technical Reports Server (NTRS)

    Kandasamy, S. B.; Williaes, B. A.

    1983-01-01

    It is shown that the intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP) induced hyperthermia in guinea pigs which was not mediated through prostaglandins or norepinephrine since a prostaglandin synthesis inhibitor and an alpha-adrenergic receptor blocking agent did not antagonize the hyperthermia. However, the hyperthermic response to Db-cAMP was attenuated by the central administration of a beta-adrenergic receptor antagonist, which indicates that cAMP may be involved, through beta-adrenergic receptors, in the central regulation of heat production and conservation. The central administration of Db-cGMP produced hypothermia which was not mediated via histamine H1 or H2 receptors and serotonin. The antagonism of hypothermia induced by Db-cGMP and acetylcholine + physostigmine by central administration of a cholinergic muscarine receptor antagonist and not by a cholinergic nicotinic receptor antagonist suggests that cholinoceptive neurons and endogenous cGMP may regulate heat loss through cholinergic muscarine receptors. It is concluded that these results indicate a regulatory role in thermoregulation provided by a balance between opposing actions of cAMP and cGMP in guinea pigs.

  7. Cyclic GMP Balance Is Critical for Malaria Parasite Transmission from the Mosquito to the Mammalian Host

    PubMed Central

    Lakshmanan, Viswanathan; Fishbaugher, Matthew E.; Morrison, Bob; Baldwin, Michael; Macarulay, Michael; Vaughan, Ashley M.; Mikolajczak, Sebastian A.

    2015-01-01

    ABSTRACT Transmission of malaria occurs during Anopheles mosquito vector blood meals, when Plasmodium sporozoites that have invaded the mosquito salivary glands are delivered to the mammalian host. Sporozoites display a unique form of motility that is essential for their movement across cellular host barriers and invasion of hepatocytes. While the molecular machinery powering motility and invasion is increasingly well defined, the signaling events that control these essential parasite activities have not been clearly delineated. Here, we identify a phosphodiesterase (PDEγ) in Plasmodium, a regulator of signaling through cyclic nucleotide second messengers. Reverse transcriptase PCR (RT-PCR) analysis and epitope tagging of endogenous PDEγ detected its expression in blood stages and sporozoites of Plasmodium yoelii. Deletion of PDEγ (pdeγ−) rendered sporozoites nonmotile, and they failed to invade the mosquito salivary glands. Consequently, PDEγ deletion completely blocked parasite transmission by mosquito bite. Strikingly, pdeγ− sporozoites showed dramatically elevated levels of cyclic GMP (cGMP), indicating that a perturbation in cyclic nucleotide balance is involved in the observed phenotypic defects. Transcriptome sequencing (RNA-Seq) analysis of pdeγ− sporozoites revealed reduced transcript abundance of genes that encode key components of the motility and invasion apparatus. Our data reveal a crucial role for PDEγ in maintaining the cyclic nucleotide balance in the malaria parasite sporozoite stage, which in turn is essential for parasite transmission from mosquito to mammal. PMID:25784701

  8. Regulation of biofilm formation and cellular buoyancy through modulating intracellular cyclic di-GMP levels in engineered cyanobacteria.

    PubMed

    Agostoni, Marco; Waters, Christopher M; Montgomery, Beronda L

    2016-02-01

    The second messenger cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP) has been implicated in the transition between motile and sessile lifestyles in bacteria. In this study, we demonstrate that biofilm formation, cellular aggregation or flocculation, and cellular buoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 (Synechocystis) and Fremyella diplosiphon. Synechocystis is a unicellular cyanobacterium and displays lower levels of c-di-GMP; F. diplosiphon is filamentous and displays higher intracellular c-di-GMP levels. We transformed Synechocystis and F. diplosiphon with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae or Escherichia coli, respectively. These engineered strains allowed us to modulate intracellular c-di-GMP levels. Biofilm formation and cellular deposition were induced in the DGC-expressing Synechocystis strain which exhibited high intracellular levels of c-di-GMP; whereas strains expressing PDE in Synechocystis and F. diplosiphon to drive low intracellular levels of c-di-GMP exhibited enhanced cellular buoyancy. In addition, the PDE-expressing F. diplosiphon strain showed elevated chlorophyll levels. These results imply roles for coordinating c-di-GMP homeostasis in regulating native cyanobacterial phenotypes. Engineering exogenous DGC or PDE proteins to regulate intracellular c-di-GMP levels represents an effective tool for uncovering cryptic phenotypes or modulating phenotypes in cyanobacteria for practical applications in biotechnology applicable in photobioreactors and in green biotechnologies, such as energy-efficient harvesting of cellular biomass or the treatment of metal-containing wastewaters.

  9. Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate.

    PubMed

    Cooley, Richard B; Smith, T Jarrod; Leung, Wilfred; Tierney, Valerie; Borlee, Bradley R; O'Toole, George A; Sondermann, Holger

    2015-06-22

    We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Cyclic Di-GMP-Regulated Periplasmic Proteolysis of a Pseudomonas aeruginosa Type Vb Secretion System Substrate

    PubMed Central

    Cooley, Richard B.; Smith, T. Jarrod; Leung, Wilfred; Tierney, Valerie; Borlee, Bradley R.; O'Toole, George A.

    2015-01-01

    ABSTRACT We previously identified a second-messenger-regulated signaling system in the environmental bacterium Pseudomonas fluorescens which controls biofilm formation in response to levels of environmental inorganic phosphate. This system contains the transmembrane cyclic di-GMP (c-di-GMP) receptor LapD and the periplasmic protease LapG. LapD regulates LapG and controls the ability of this protease to process a large cell surface adhesin protein, LapA. While LapDG orthologs can be identified in diverse bacteria, predictions of LapG substrates are sparse. Notably, the opportunistic pathogen Pseudomonas aeruginosa harbors LapDG orthologs, but neither the substrate of LapG nor any associated secretion machinery has been identified to date. Here, we identified P. aeruginosa CdrA, a protein known to mediate cell-cell aggregation and biofilm maturation, as a substrate of LapG. We also demonstrated LapDG to be a minimal system sufficient to control CdrA localization in response to changes in the intracellular concentration of c-di-GMP. Our work establishes this biofilm signaling node as a regulator of a type Vb secretion system substrate in a clinically important pathogen. IMPORTANCE Here, the biological relevance of a conserved yet orphan signaling system in the opportunistic pathogen Pseudomonas aeruginosa is revealed. In particular, we identified the adhesin CdrA, the cargo of a two-partner secretion system, as a substrate of a periplasmic protease whose activity is controlled by intracellular c-di-GMP levels and a corresponding transmembrane receptor via an inside-out signaling mechanism. The data indicate a posttranslational control mechanism of CdrA via c-di-GMP, in addition to its established transcriptional regulation via the same second messenger. PMID:26100041

  11. Cyclic di-GMP allosterically inhibits the CRP-like protein (Clp) of Xanthomonas axonopodis pv. citri.

    PubMed

    Leduc, Jason L; Roberts, Gary P

    2009-11-01

    The protein Clp from Xanthomonas axonopodis pv. citri regulates pathogenesis and is a member of the CRP (cyclic AMP receptor protein) superfamily. We show that unlike the DNA-binding activity of other members of this family, the DNA-binding activity of Clp is allosterically inhibited by its effector and that cyclic di-GMP serves as that effector at physiological concentrations.

  12. Identification of a noncatalytic cGMP-binding domain conserved in both the cGMP-stimulated and photoreceptor cyclic nucleotide phosphodiesterases.

    PubMed Central

    Charbonneau, H; Prusti, R K; LeTrong, H; Sonnenburg, W K; Mullaney, P J; Walsh, K A; Beavo, J A

    1990-01-01

    Partial amino acid sequence has been determined for the cone, alpha' subunit of the bovine photoreceptor cyclic nucleotide phosphodiesterase (PDE) and deduced from nucleotide sequences of a partial cDNA clone. These sequences identify the alpha' subunit as the product of a gene that is distinct from those encoding the alpha or beta subunits of the membrane-associated rod photoreceptor PDE. Comparisons between the recently determined cGMP-stimulated-PDE sequence and those of the alpha and alpha' photoreceptor PDE subunits reveal an unexpected sequence similarity. In addition to the catalytic domain conserved in eukaryotic PDEs, all three PDEs possess a second conserved segment of approximately 340 residues that contains two internally homologous repeats. Limited proteolysis and direct photolabeling studies indicate that the noncatalytic, cGMP-binding site(s) in the cGMP-stimulated PDE is located within this conserved domain, suggesting that it also may serve this function in the photoreceptor PDEs. Moreover, other PDEs that do not bind cGMP at noncatalytic sites do not contain this conserved domain. The function of the conserved segment in the photoreceptor PDEs is not known, but the homology to allosteric sites of the cGMP-stimulated PDE suggests a role in cGMP binding and modulation of enzyme activity. Images PMID:2153290

  13. Mangiferin prevents guinea pig tracheal contraction via activation of the nitric oxide-cyclic GMP pathway.

    PubMed

    Vieira, Aline B; Coelho, Luciana P; Insuela, Daniella B R; Carvalho, Vinicius F; dos Santos, Marcelo H; Silva, Patricia Mr; Martins, Marco A

    2013-01-01

    Previous studies have described the antispasmodic effect of mangiferin, a natural glucoside xanthone (2-C-β-Dgluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) that is present in mango trees and other plants, but its mechanism of action remains unknown. The aim of this study was to examine the potential contribution of the nitric oxide-cyclic GMP pathway to the antispasmodic effect of mangiferin on isolated tracheal rings preparations. The functional effect of mangiferin on allergic and non-allergic contraction of guinea pig tracheal rings was assessed in conventional organ baths. Cultured tracheal rings were exposed to mangiferin or vehicle, and nitric oxide synthase (NOS) 3 and cyclic GMP (cGMP) levels were quantified using western blotting and enzyme immunoassays, respectively. Mangiferin (0.1-10 µM) inhibited tracheal contractions induced by distinct stimuli, such as allergen, histamine, 5-hydroxytryptamine or carbachol, in a concentration-dependent manner. Mangiferin also caused marked relaxation of tracheal rings that were precontracted by carbachol, suggesting that it has both anti-contraction and relaxant properties that are prevented by removing the epithelium. The effect of mangiferin was inhibited by the nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) (100 µM), and the soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM), but not the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ22536) (100 µM). The antispasmodic effect of mangiferin was also sensitive to K⁺ channel blockers, such as tetraethylammonium (TEA), glibenclamide and apamin. Furthermore, mangiferin inhibited Ca²⁺-induced contractions in K⁺ (60 mM)-depolarised tracheal rings preparations. In addition, mangiferin increased NOS3 protein levels and cGMP intracellular levels in cultured tracheal rings. Finally, mangiferin-induced increase in cGMP levels was abrogated by co-incubation with either ODQ or L

  14. Mangiferin Prevents Guinea Pig Tracheal Contraction via Activation of the Nitric Oxide-Cyclic GMP Pathway

    PubMed Central

    Vieira, Aline B.; Coelho, Luciana P.; Insuela, Daniella B. R.; Carvalho, Vinicius F.; dos Santos, Marcelo H.; Silva, Patricia MR.; Martins, Marco A.

    2013-01-01

    Previous studies have described the antispasmodic effect of mangiferin, a natural glucoside xanthone (2-C-β-Dgluco-pyranosyl-1,3,6,7-tetrahydroxyxanthone) that is present in mango trees and other plants, but its mechanism of action remains unknown. The aim of this study was to examine the potential contribution of the nitric oxide-cyclic GMP pathway to the antispasmodic effect of mangiferin on isolated tracheal rings preparations. The functional effect of mangiferin on allergic and non-allergic contraction of guinea pig tracheal rings was assessed in conventional organ baths. Cultured tracheal rings were exposed to mangiferin or vehicle, and nitric oxide synthase (NOS) 3 and cyclic GMP (cGMP) levels were quantified using western blotting and enzyme immunoassays, respectively. Mangiferin (0.1–10 µM) inhibited tracheal contractions induced by distinct stimuli, such as allergen, histamine, 5-hydroxytryptamine or carbachol, in a concentration-dependent manner. Mangiferin also caused marked relaxation of tracheal rings that were precontracted by carbachol, suggesting that it has both anti-contraction and relaxant properties that are prevented by removing the epithelium. The effect of mangiferin was inhibited by the nitric oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME) (100 µM), and the soluble guanylate cyclase inhibitor, 1H-[1], [2], [4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 µM), but not the adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (SQ22536) (100 µM). The antispasmodic effect of mangiferin was also sensitive to K+ channel blockers, such as tetraethylammonium (TEA), glibenclamide and apamin. Furthermore, mangiferin inhibited Ca2+-induced contractions in K+ (60 mM)-depolarised tracheal rings preparations. In addition, mangiferin increased NOS3 protein levels and cGMP intracellular levels in cultured tracheal rings. Finally, mangiferin-induced increase in cGMP levels was abrogated by co-incubation with either ODQ or L

  15. Liposomes loaded with a STING pathway ligand, cyclic di-GMP, enhance cancer immunotherapy against metastatic melanoma.

    PubMed

    Nakamura, Takashi; Miyabe, Hiroko; Hyodo, Mamoru; Sato, Yusuke; Hayakawa, Yoshihiro; Harashima, Hideyoshi

    2015-10-28

    Malignant melanomas escape immunosurveillance via the loss/down-regulation of MHC-I expression. Natural killer (NK) cells have the potential to function as essential effector cells for eliminating melanomas. Cyclic di-GMP (c-di-GMP), a ligand of the stimulator of interferon genes (STING) signal pathway, can be thought of as a new class of adjuvant against cancer. However, it is yet to be tested, because technologies for delivering c-di-GMP to the cytosol are required. Herein, we report that c-di-GMP efficiently activates NK cells and induces antitumor effects against malignant melanomas when loaded in YSK05 lipid containing liposomes, by assisting in the efficient delivery of c-di-GMP to the cytosol. The intravenous administration of c-di-GMP encapsulated within YSK05-liposomes (c-di-GMP/YSK05-Lip) into mice efficiently induced the production of type I interferon (IFN) as well as the activation of NK cells, resulting in a significant antitumor effect in a lung metastasis mouse model using B16-F10. This antitumor effect was dominated by NK cells. The infiltration of NK cells was observed in the lungs with B16-F10 melanomas. These findings indicate that the c-di-GMP/YSK05-Lip induces MHC-I non-restricted antitumor immunity mediated by NK cells. Consequently, c-di-GMP/YSK05-Lip represents a potentially new adjuvant system for use in immunotherapy against malignant melanomas.

  16. The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

    PubMed

    Khan, Mike; Harms, Jerome S; Marim, Fernanda M; Armon, Leah; Hall, Cherisse L; Liu, Yi-Ping; Banai, Menachem; Oliveira, Sergio C; Splitter, Gary A; Smith, Judith A

    2016-12-01

    Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  17. Cyclic di-GMP is Essential for the Survival of the Lyme Disease Spirochete in Ticks

    PubMed Central

    He, Ming; Ouyang, Zhiming; Troxell, Bryan; Xu, Haijun; Moh, Akira; Piesman, Joseph; Norgard, Michael V.; Gomelsky, Mark; Yang, X. Frank

    2011-01-01

    Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that modulates many biological processes. Although its role in bacterial pathogenesis during mammalian infection has been documented, the role of c-di-GMP in a pathogen's life cycle within a vector host is less understood. The enzootic cycle of the Lyme disease pathogen Borrelia burgdorferi involves both a mammalian host and an Ixodes tick vector. The B. burgdorferi genome encodes a single copy of the diguanylate cyclase gene (rrp1), which is responsible for c-di-GMP synthesis. To determine the role of c-di-GMP in the life cycle of B. burgdorferi, an Rrp1-deficient B. burgdorferi strain was generated. The rrp1 mutant remains infectious in the mammalian host but cannot survive in the tick vector. Microarray analyses revealed that expression of a four-gene operon involved in glycerol transport and metabolism, bb0240-bb0243, was significantly downregulated by abrogation of Rrp1. In vitro, the rrp1 mutant is impaired in growth in the media containing glycerol as the carbon source (BSK-glycerol). To determine the contribution of the glycerol metabolic pathway to the rrp1 mutant phenotype, a glp mutant, in which the entire bb0240-bb0243 operon is not expressed, was generated. Similar to the rrp1 mutant, the glp mutant has a growth defect in BSK-glycerol medium. In vivo, the glp mutant is also infectious in mice but has reduced survival in ticks. Constitutive expression of the bb0240-bb0243 operon in the rrp1 mutant fully rescues the growth defect in BSK-glycerol medium and partially restores survival of the rrp1 mutant in ticks. Thus, c-di-GMP appears to govern a catabolic switch in B. burgdorferi and plays a vital role in the tick part of the spirochetal enzootic cycle. This work provides the first evidence that c-di-GMP is essential for a pathogen's survival in its vector host. PMID:21738477

  18. Role of cyclic di-GMP in Xylella fastidiosa biofilm formation, plant virulence, and insect transmission.

    PubMed

    Chatterjee, Subhadeep; Killiny, Nabil; Almeida, Rodrigo P P; Lindow, Steven E

    2010-10-01

    Xylella fastidiosa must coordinately regulate a variety of traits contributing to biofilm formation, host plant and vector colonization, and transmission between plants. Traits such as production of extracellular polysaccharides (EPS), adhesins, extracellular enzymes, and pili are expressed in a cell-density-dependent fashion mediated by a cell-to-cell signaling system involving a fatty acid diffusible signaling factor (DSF). The expression of gene PD0279 (which has a GGDEF domain) is downregulated in the presence of DSF and may be involved in intracellular signaling by modulating the levels of cyclic di-GMP. PD0279, designated cyclic di-GMP synthase A (cgsA), is required for biofilm formation, plant virulence, and vector transmission. cgsA mutants exhibited a hyperadhesive phenotype in vitro and overexpressed gumJ, hxfA, hxfB, xadA, and fimA, which promote attachment of cells to surfaces and, hence, biofilm formation. The mutants were greatly reduced in virulence to grape albeit still transmissible by insect vectors, although at a reduced level compared with transmission rates of the wild-type strain, despite the fact that similar numbers of cells of the cgsA mutant were acquired by the insects from infected plants. High levels of EPS were measured in cgsA mutants compared with wild-type strains, and scanning electron microscopy analysis also revealed a thicker amorphous layer surrounding the mutants. Overexpression of cgsA in a cgsA-complemented mutant conferred the opposite phenotypes in vitro. These results suggest that decreases of cyclic di-GMP result from the accumulation of DSF as cell density increases, leading to a phenotypic transition from a planktonic state capable of colonizing host plants to an adhesive state that is insect transmissible.

  19. Taurine prevents ammonia-induced accumulation of cyclic GMP in rat striatum by interaction with GABAA and glycine receptors.

    PubMed

    Hilgier, Wojciech; Oja, Simo S; Saransaari, Pirjo; Albrecht, Jan

    2005-05-10

    Previously, we had shown that ammonium chloride (ammonia)-induced accumulation of cyclic GMP in the microdialysates of rat striatum is blocked by taurine. In this study, coinfusion with taurine of a GABAA receptor antagonist bicuculline or a glycine receptor antagonist strychnine (100 microM each), separately, restored ammonia-induced release of cGMP to the extracellular fluid to approximately 29% and 18% of the level measured in the absence of taurine, respectively. Simultaneous coinfusion of both antagonists or of 100 muM picrotoxin, which is an antagonist of both GABAA and Gly receptors, offsets most of the taurine block. Ammonia-induced accumulation of cyclic GMP was attenuated by approximately 12% upon coinfusion of a GABAA receptor agonist muscimol (100 microM). The results suggest that stimulation of both GABAA and glycine receptors is involved in the mechanism by which taurine limits the activation of the NMDA/NO/cGMP pathway by ammonia in the striatum.

  20. Identification of a cyclic-di-GMP-modulating response regulator that impacts biofilm formation in a model sulfate reducing bacterium

    PubMed Central

    Rajeev, Lara; Luning, Eric G.; Altenburg, Sara; Zane, Grant M.; Baidoo, Edward E. K.; Catena, Michela; Keasling, Jay D.; Wall, Judy D.; Fields, Matthew W.; Mukhopadhyay, Aindrila

    2014-01-01

    We surveyed the eight putative cyclic-di-GMP-modulating response regulators (RRs) in Desulfovibrio vulgaris Hildenborough that are predicted to function via two-component signaling. Using purified proteins, we examined cyclic-di-GMP (c-di-GMP) production or turnover in vitro of all eight proteins. The two RRs containing only GGDEF domains (DVU2067, DVU0636) demonstrated c-di-GMP production activity in vitro. Of the remaining proteins, three RRs with HD-GYP domains (DVU0722, DVUA0086, and DVU2933) were confirmed to be Mn2+-dependent phosphodiesterases (PDEs) in vitro and converted c-di-GMP to its linear form, pGpG. DVU0408, containing both c-di-GMP production (GGDEF) and degradation domains (EAL), showed c-di-GMP turnover activity in vitro also with production of pGpG. No c-di-GMP related activity could be assigned to the RR DVU0330, containing a metal-dependent phosphohydrolase HD-OD domain, or to the HD-GYP domain RR, DVU1181. Studies included examining the impact of overexpressed cyclic-di-GMP-modulating RRs in the heterologous host E. coli and led to the identification of one RR, DVU0636, with increased cellulose production. Evaluation of a transposon mutant in DVU0636 indicated that the strain was impaired in biofilm formation and demonstrated an altered carbohydrate:protein ratio relative to the D. vulgaris wild type biofilms. However, grown in liquid lactate/sulfate medium, the DVU0636 transposon mutant showed no growth impairment relative to the wild-type strain. Among the eight candidates, only the transposon disruption mutant in the DVU2067 RR presented a growth defect in liquid culture. Our results indicate that, of the two diguanylate cyclases (DGCs) that function as part of two-component signaling, DVU0636 plays an important role in biofilm formation while the function of DVU2067 has pertinence in planktonic growth. PMID:25120537

  1. Cyclic GMP-AMP Containing Mixed Phosphodiester Linkages Is An Endogenous High Affinity Ligand for STING

    PubMed Central

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2′-OH of GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and 5′-phosphate of GMP. This molecule, termed 2′3′-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2′3′-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation. PMID:23747010

  2. Rod outer segment structure influences the apparent kinetic parameters of cyclic GMP phosphodiesterase

    PubMed Central

    1994-01-01

    Cyclic GMP hydrolysis by the phosphodiesterase (PDE) of retinal rod outer segments (ROS) is a key amplification step in phototransduction. Definitive estimates of the turnover number, kcat, and of the Km are crucial to quantifying the amplification contributed by the PDE. Published estimates for these kinetic parameters vary widely; moreover, light-dependent changes in the Km of PDE have been reported. The experiments and analyses reported here account for most observed variations in apparent Km, and they lead to definitive estimates of the intrinsic kinetic parameters in amphibian rods. We first obtained a new and highly accurate estimate of the ratio of holo-PDE to rhodopsin in the amphibian ROS, 1:270. We then estimated the apparent kinetic parameters of light-activated PDE of suspensions of disrupted frog ROS whose structural integrity was systematically varied. In the most severely disrupted ROS preparation, we found Km = 95 microM and kcat = 4,400 cGMP.s-1. In suspensions of disc-stack fragments of greater integrity, the apparent Km increased to approximately 600 microM, though kcat remained unchanged. In contrast, the Km for cAMP was not shifted in the disc stack preparations. A theoretical analysis shows that the elevated apparent Km of suspensions of disc stacks can be explained as a consequence of diffusion with hydrolysis in the disc stack, which causes active PDEs nearer the center of the stack to be exposed to a lower concentration of cyclic GMP than PDEs at the disc stack rim. The analysis predicts our observation that the apparent Km for cGMP is elevated with no accompanying decrease in kcat. The analysis also predicts the lack of a Km shift for cAMP and the previously reported light dependence of the apparent Km for cGMP. We conclude that the intrinsic kinetic parameters of the PDE do not vary with light or structural integrity, and are those of the most severely disrupted disc stacks. PMID:7931138

  3. Systematic Identification of Cyclic-di-GMP Binding Proteins in Vibrio cholerae Reveals a Novel Class of Cyclic-di-GMP-Binding ATPases Associated with Type II Secretion Systems

    PubMed Central

    Shang, Xiaoran; Orr, Mona W.; Goodson, Jonathan R.; Galperin, Michael Y.; Yildiz, Fitnat H.; Lee, Vincent T.

    2015-01-01

    Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates a variety of complex processes through a diverse set of c-di-GMP receptor proteins. We have utilized a systematic approach to identify c-di-GMP receptors from the pathogen Vibrio cholerae using the Differential Radial Capillary Action of Ligand Assay (DRaCALA). The DRaCALA screen identified a majority of known c-di-GMP binding proteins in V. cholerae and revealed a novel c-di-GMP binding protein, MshE (VC0405), an ATPase associated with the mannose sensitive hemagglutinin (MSHA) type IV pilus. The known c-di-GMP binding proteins identified by DRaCALA include diguanylate cyclases, phosphodiesterases, PilZ domain proteins and transcription factors VpsT and VpsR, indicating that the DRaCALA-based screen of open reading frame libraries is a feasible approach to uncover novel receptors of small molecule ligands. Since MshE lacks the canonical c-di-GMP-binding motifs, a truncation analysis was utilized to locate the c-di-GMP binding activity to the N-terminal T2SSE_N domain. Alignment of MshE homologs revealed candidate conserved residues responsible for c-di-GMP binding. Site-directed mutagenesis of these candidate residues revealed that the Arg9 residue is required for c-di-GMP binding. The ability of c-di-GMP binding to MshE to regulate MSHA dependent processes was evaluated. The R9A allele, in contrast to the wild type MshE, was unable to complement the ΔmshE mutant for the production of extracellular MshA to the cell surface, reduction in flagella swimming motility, attachment to surfaces and formation of biofilms. Testing homologs of MshE for binding to c-di-GMP identified the type II secretion ATPase of Pseudomonas aeruginosa (PA14_29490) as a c-di-GMP receptor, indicating that type II secretion and type IV pili are both regulated by c-di-GMP. PMID:26506097

  4. Cyclic Di-GMP Signaling Contributes to Pseudomonas aeruginosa-Mediated Catheter-Associated Urinary Tract Infection

    PubMed Central

    Cole, Stephanie J.

    2015-01-01

    ABSTRACT Bis-(3′-5′) cyclic dimeric GMP (c-di-GMP) controls the lifestyle transition between the sessile and motile states in many Gram-negative bacteria, including the opportunistic human pathogen Pseudomonas aeruginosa. Under laboratory conditions, high concentrations of c-di-GMP decrease motility and promote biofilm formation, while low concentrations of c-di-GMP promote motility and decease biofilm formation. Here we sought to determine the contribution of c-di-GMP signaling to biofilm formation during P. aeruginosa-mediated catheter-associated urinary tract infection (CAUTI). Using a murine CAUTI model, a decrease in CFU was detected in the bladders and kidneys of mice infected with strains overexpressing the phosphodiesterases (PDEs) encoded by PA3947 and PA2133 compared to those infected with wild-type P. aeruginosa. Conversely, overexpression of the diguanylate cyclases (DGCs) encoded by PA3702 and PA1107 increased the number of bacteria in bladder and significantly increased dissemination of bacteria to the kidneys compared to wild-type infection. To determine which of the DGCs and PDEs contribute to c-di-GMP signaling during infection, a panel of PA14 in-frame deletion mutants lacking DGCs and PDEs were tested in the CAUTI model. Results from these infections revealed five mutants, three containing GGDEF domains (ΔPA14_26970, ΔPA14_72420, and ΔsiaD) and two containing dual GGDEF-EAL domains (ΔmorA and ΔPA14_07500), with decreased colonization of the bladder and dissemination to the kidneys. These results indicate that c-di-GMP signaling influences P. aeruginosa-mediated biofilms during CAUTI. IMPORTANCE Biofilm-based infections are an important cause of nosocomial infections, since they resist the immune response and traditional antibiotic treatment. Cyclic di-GMP (c-di-GMP) is a second messenger that promotes biofilm formation in many Gram-negative pathogens, including Pseudomonas aeruginosa. Here we determined the contribution of c-di-GMP

  5. cyclic GMP Mediated Inhibition of Spontaneous Germinal Vesicle Breakdown Both with and without Cumulus in Mouse Oocyte.

    PubMed

    Hwang, Heekyung; Cheon, Yong-Pil

    2016-12-01

    Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest.

  6. cyclic GMP Mediated Inhibition of Spontaneous Germinal Vesicle Breakdown Both with and without Cumulus in Mouse Oocyte

    PubMed Central

    Hwang, Heekyung; Cheon, Yong-Pil

    2016-01-01

    ABSTRACT Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest. PMID:28144640

  7. Blockage and permeation of divalent cations through the cyclic GMP-activated channel from tiger salamander retinal rods.

    PubMed Central

    Colamartino, G; Menini, A; Torre, V

    1991-01-01

    1. Blockage and permeation of divalent cations through channels activated by guanosine 3',5'-cyclic monophosphate (cyclic GMP) were studied in membrane patches excised from retinal rods of the tiger salamander Ambystoma tigrinum by rapidly changing the ionic medium bathing the intracellular side of the excised membrane. 2. The Na+ current, observed when 110 mM-NaCl was present on both sides of the membrane patch, was reduced by the addition of 1 mM of the chloride salts of Ca2+, Mg2+, Sr2+, Ba2+ or Mn2+ to the bathing medium. The sequence of blocking potency at +60 mV was Mg2+ greater than Mn2+ approximately Ba2+ greater than Ca2+ greater than Sr2+, while at -60 mV it was Ba2+ greater than Ca2+ greater than Sr2+ greater than Mn2+ approximately Mg2+. For all divalent cations the blocking effect depended, in a complex way, on the membrane potential. 3. The blocking effect of Ca2+ and Mg2+ increased when the concentration of cyclic GMP was reduced from 100 to 5 microM. At -60 mV 1 mM-Ca2+ blocked about 34% of the Na+ current in the presence of 100 microM-cyclic GMP, while in the presence of 5 microM-cyclic GMP, 1 mM-Ca2+ blocked about 56% of the Na+ current. 4. When, in the presence of 100 microM-cyclic GMP, 110 mM-NaCl at the intracellular side was replaced by equiosmolar amounts of chloride salts of divalent cations (73.3 mM) a small outward current carried by divalent cations could be observed at large positive membrane potentials. At +60 mV the ratio between the current carried by Na+, Sr2+, Ca2+, Ba2+, Mg2+ and Mn2+ was 83.3:1.4:1:0.58:0.33:0.25. 5. In agreement with previous observations the dependence of the Na+ current on the concentration of cyclic GMP shows a clear co-operativity among cyclic GMP molecules.4+ cyclic GMP-gated channel in excised patches is similar to but not identical to the selectivity sequence of divalent cations through the channel in intact rods. PMID:1725182

  8. Cyclic-GMP-AMP Is An Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA

    PubMed Central

    Chen, Xiang; Du, Fenghe; Shi, Heping; Chen, Chuo; Chen, Zhijian J.

    2013-01-01

    Cytosolic DNA induces type-I interferons and other cytokines that are important for antimicrobial defense but can also result in autoimmunity. This DNA signaling pathway requires the adaptor protein STING and the transcription factor IRF3, but the mechanism of DNA sensing is unclear. Here we showed that mammalian cytosolic extracts synthesized cyclic-GMP-AMP (cGAMP) in vitro from ATP and GTP in the presence of DNA but not RNA. DNA transfection or DNA virus infection of mammalian cells also triggered cGAMP production. cGAMP bound to STING, leading to the activation of IRF3 and induction of interferon-β. Thus, cGAMP represents the first cyclic di-nucleotide in metazoa and it functions as an endogenous second messenger that triggers interferon production in response to cytosolic DNA. PMID:23258412

  9. Structure of the GAF domain, a ubiquitous signaling motif and a new class of cyclic GMP receptor

    PubMed Central

    Ho, Yew-Seng J.; Burden, Lisa M.; Hurley, James H.

    2000-01-01

    GAF domains are ubiquitous motifs present in cyclic GMP (cGMP)-regulated cyclic nucleotide phosphodiesterases, certain adenylyl cyclases, the bacterial transcription factor FhlA, and hundreds of other signaling and sensory proteins from all three kingdoms of life. The crystal structure of the Saccharomyces cerevisiae YKG9 protein was determined at 1.9 Å resolution. The structure revealed a fold that resembles the PAS domain, another ubiquitous signaling and sensory transducer. YKG9 does not bind cGMP, but the isolated first GAF domain of phosphodiesterase 5 binds with Kd = 650 nM. The cGMP binding site of the phosphodiesterase GAF domain was identified by homology modeling and site-directed mutagenesis, and consists of conserved Arg, Asn, Lys and Asp residues. The structural and binding studies taken together show that the cGMP binding GAF domains form a new class of cyclic nucleotide receptors distinct from the regulatory domains of cyclic nucleotide-regulated protein kinases and ion channels. PMID:11032796

  10. Cyclic-di-GMP regulates lipopolysaccharide modification and contributes to Pseudomonas aeruginosa immune evasion.

    PubMed

    McCarthy, Ronan R; Mazon-Moya, Maria J; Moscoso, Joana A; Hao, Youai; Lam, Joseph S; Bordi, Christophe; Mostowy, Serge; Filloux, Alain

    2017-03-06

    Pseudomonas aeruginosa is a Gram-negative bacterial pathogen associated with acute and chronic infections. The universal cyclic-di-GMP second messenger is instrumental in the switch from a motile lifestyle to resilient biofilm as in the cystic fibrosis lung. The SadC diguanylate cyclase is associated with this patho-adaptive transition. Here, we identify an unrecognized SadC partner, WarA, which we show is a methyltransferase in complex with a putative kinase, WarB. We established that WarA binds to cyclic-di-GMP, which potentiates its methyltransferase activity. Together, WarA and WarB have structural similarities with the bifunctional Escherichia coli lipopolysaccharide (LPS) O antigen regulator WbdD. Strikingly, WarA influences P. aeruginosa O antigen modal distribution and interacts with the LPS biogenesis machinery. LPS is known to modulate the immune response in the host, and by using a zebrafish infection model, we implicate WarA in the ability of P. aeruginosa to evade detection by the host.

  11. The bacterial second messenger c-di-GMP: probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs.

    PubMed

    Shanahan, Carly A; Strobel, Scott A

    2012-12-14

    The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs.

  12. The bacterial second messenger c-di-GMP: Probing interactions with protein and RNA binding partners using cyclic dinucleotide analogs

    PubMed Central

    Shanahan, Carly A.; Strobel, Scott A.

    2013-01-01

    The ability of bacteria to adapt to a changing environment is essential for their survival. One mechanism used to facilitate behavioral adaptations is the second messenger signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP). c-di-GMP is widespread throughout the bacterial domain and plays a vital role in regulating the transition between the motile planktonic lifestyle and the sessile biofilm forming state. This second messenger also controls the virulence response of pathogenic organisms and is thought to be connected to quorum sensing, the process by which bacteria communicate with each other. The intracellular concentration of c-di-GMP is tightly regulated by the opposing enzymatic activities of diguanlyate cyclases and phosphodiesterases, which synthesize and degrade the second messenger, respectively. The change in the intracellular concentration of c-di-GMP is directly sensed by downstream targets of the second messenger, both protein and RNA, which induce the appropriate phenotypic response. This review will summarize our current state of knowledge of c-di-GMP signaling in bacteria with a focus on protein and RNA binding partners of the second messenger. Efforts towards the synthesis of c-di-GMP and its analogs are discussed as well as studies aimed at targeting these macromolecular effectors with chemically synthesized cyclic dinucleotide analogs. PMID:23108253

  13. Cyclic Di-GMP Regulates Multiple Cellular Functions in the Symbiotic Alphaproteobacterium Sinorhizobium meliloti

    PubMed Central

    Schäper, Simon; Krol, Elizaveta; Skotnicka, Dorota; Kaever, Volkhard; Hilker, Rolf; Søgaard-Andersen, Lotte

    2015-01-01

    ABSTRACT Sinorhizobium meliloti undergoes major lifestyle changes between planktonic states, biofilm formation, and symbiosis with leguminous plant hosts. In many bacteria, the second messenger 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) promotes a sessile lifestyle by regulating a plethora of processes involved in biofilm formation, including motility and biosynthesis of exopolysaccharides (EPS). Here, we systematically investigated the role of cdG in S. meliloti Rm2011 encoding 22 proteins putatively associated with cdG synthesis, degradation, or binding. Single mutations in 21 of these genes did not cause evident changes in biofilm formation, motility, or EPS biosynthesis. In contrast, manipulation of cdG levels by overproducing endogenous or heterologous diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) affected these processes and accumulation of N-Acyl-homoserine lactones in the culture supernatant. Specifically, individual overexpression of the S. meliloti genes pleD, SMb20523, SMb20447, SMc01464, and SMc03178 encoding putative DGCs and of SMb21517 encoding a single-domain PDE protein had an impact and resulted in increased levels of cdG. Compared to the wild type, an S. meliloti strain that did not produce detectable levels of cdG (cdG0) was more sensitive to acid stress. However, it was symbiotically potent, unaffected in motility, and only slightly reduced in biofilm formation. The SMc01790-SMc01796 locus, homologous to the Agrobacterium tumefaciens uppABCDEF cluster governing biosynthesis of a unipolarly localized polysaccharide, was found to be required for cdG-stimulated biofilm formation, while the single-domain PilZ protein McrA was identified as a cdG receptor protein involved in regulation of motility. IMPORTANCE We present the first systematic genome-wide investigation of the role of 3′,5′-cyclic di-GMP (c-di-GMP, or cdG) in regulation of motility, biosynthesis of exopolysaccharides, biofilm formation, quorum sensing, and symbiosis in a

  14. Intestinal GPS: bile and bicarbonate control cyclic di-GMP to provide Vibrio cholerae spatial cues within the small intestine.

    PubMed

    Koestler, Benjamin J; Waters, Christopher M

    2014-01-01

    The second messenger cyclic di-GMP (c-di-GMP) regulates numerous phenotypes in response to environmental stimuli to enable bacteria to transition between different lifestyles. Here we discuss our recent findings that the human pathogen Vibrio cholerae recognizes 2 host-specific signals, bile and bicarbonate, to regulate intracellular c-di-GMP. We have demonstrated that bile acids increase intracellular c-di-GMP to promote biofilm formation. We have also shown that this bile-mediated increase of intracellular c-di-GMP is negated by bicarbonate, and that this interaction is dependent on pH, suggesting that V. cholerae uses these 2 environmental cues to sense and adapt to its relative location in the small intestine. Increased intracellular c-di-GMP by bile is attributed to increased c-di-GMP synthesis by 3 diguanylate cyclases (DGCs) and decreased expression of one phosphodiesterase (PDE) in the presence of bile. The molecular mechanisms by which bile controls the activity of the 3 DGCs and the regulators of bile-mediated transcriptional repression of the PDE are not yet known. Moreover, the impact of varying concentrations of bile and bicarbonate at different locations within the small intestine and the response of V. cholerae to these cues remains unclear. The native microbiome and pharmaceuticals, such as omeprazole, can impact bile and pH within the small intestine, suggesting these are potential unappreciated factors that may alter V. cholerae pathogenesis.

  15. Cation interactions within the cyclic GMP-activated channel of retinal rods from the tiger salamander.

    PubMed Central

    Zimmerman, A L; Baylor, D A

    1992-01-01

    1. The ionic dependence of current through the 3',5'-cyclic guanosine monophosphate (cyclic GMP)-activated channels of salamander rods was studied in excised inside-out membrane patches from isolated outer segments. Voltage-clamp experiments on transducing rods were performed so that the channels in intact cells could be compared with those in excised patches. 2. The reversal potential of the cyclic GMP-induced patch current was close to the Na+ equilibrium potential when the concentration of NaCl on the cytoplasmic surface of a patch was varied at constant external NaCl concentration. Fitting the Goldman-Hodgkin-Katz equation indicated that the apparent ratio of permeabilities for Na+ and Cl- was at least 50. This confirms a previous report that the channel's Na+ permeability is much larger than its Cl- permeability. 3. Na+ currents through the channel did not obey the independence principle. The outward patch current at large positive potential began to saturate with increasing concentrations of internal Na+, as if permeation required Na+ to bind to a site with an apparent dissociation constant around 180 mM. 4. In symmetrical NaCl solutions containing very low concentrations of divalent cations the current-voltage relation measured from excised patches 50 microseconds after switching the voltage showed mild outward rectification. By 1 ms the rectification was more pronounced. The rectification at 50 microseconds is attributed to voltage dependence of Na+ permeation. The additional rectification at later times is attributed to voltage dependence of the channel's probability of being open, depolarization favouring the open state. 5. In symmetrical Mg2+ solutions the cyclic GMP-induced patch currents were smaller and the outward rectification was more pronounced. 6. Addition of Mg2+ or Ca2+ to an internal Na+ solution blocked the cyclic GMP-induced Na+ current through the channels, as if by occupying a single binding site with an affinity in the 0.1-2 mM range

  16. Inhibition of PDE5A1 guanosine cyclic monophosphate (cGMP) hydrolysing activity by sildenafil analogues that inhibit cellular cGMP efflux.

    PubMed

    Subbotina, Anna; Ravna, Aina W; Lysaa, Roy A; Abagyan, Ruben; Bugno, Ryszard; Sager, Georg

    2017-06-01

    To determine the ability of 11 sildenafil analogues to discriminate between cyclic nucleotide phosphodiesterases (cnPDEs) and to characterise their inhibitory potencies (Ki values) of PDE5A1-dependent guanosine cyclic monophosphate (cGMP) hydrolysis. Sildenafil analogues were identified by virtual ligand screening (VLS) and screened for their ability to inhibit adenosine cyclic monophosphate (cAMP) hydrolysis by PDE1A1, PDE1B1, PDE2A1, PDE3A, PDE10A1 and PDE10A2, and cGMP hydrolysis by PDE5A, PDE6C, PDE9A2 for a low (1 nm) and high concentration (10 μm). Complete IC50 plots for all analogues were performed for PDE5A-dependent cGMP hydrolysis. Docking studies and scoring were made using the ICM molecular modelling software. The analogues in a low concentration showed no or low inhibition of PDE1A1, PDE1B1, PDE2A1, PDE3A, PDE10A1 and PDE10A2. In contrast, PDE5A and PDE6C were markedly inhibited to a similar extent by the analogues in a low concentration, whereas PDE9A2 was much less inhibited. The analogues showed a relative narrow range of Ki values for PDE5A inhibition (1.2-14 nm). The sildenafil molecule was docked in the structure of PDE5A1 co-crystallised with sildenafil. All the analogues had similar binding poses as sildenafil. Sildenafil analogues that inhibit cellular cGMP efflux are potent inhibitors of PDE5A and PDE6C. © 2017 The Authors. Journal of Pharmacy and Pharmacology published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society.

  17. Mechanistic insight into the conserved allosteric regulation of periplasmic proteolysis by the signaling molecule cyclic-di-GMP

    PubMed Central

    Chatterjee, Debashree; Cooley, Richard B; Boyd, Chelsea D; Mehl, Ryan A; O'Toole, George A; Sondermann, Holger

    2014-01-01

    Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation, a prevalent adaptation strategy in response to changing environments. In Pseudomonas fluorescens, this process is regulated by the Lap system and the second messenger cyclic-di-GMP. High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG, preventing it from cleaving a cell surface-bound adhesin, thereby promoting cell adhesion. In this study, we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapD's HAMP domain. LapD appears to act as a coincidence detector, whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase. Given the conservation of key elements of this receptor system in many bacterial species, the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling. DOI: http://dx.doi.org/10.7554/eLife.03650.001 PMID:25182848

  18. Effects of saline loading during head down tilt on ANP and cyclic GMP levels and on urinary fluid excretion

    NASA Astrophysics Data System (ADS)

    Drummer, C.; Lang, R. E.; Baisch, F.; Blomqvist, G.; Heer, M.; Gerzer, R.

    In the present study the renal and humoral effects of acute saline infusions were investigated in six healthy male volunteers before, during and after a ten day period of -6° head-down-tilt (HDT). During the whole 23-day study period the subjects received a standardized diet including 40 ml water and 125 mg NaCl per kg body weight per day. After the infusion of 0.9% saline (22 ml/kg within 20 minutes) plasma atrial natriuretic peptide (ANP) levels were only slightly increased (not significant) at the end of the infusion, while plasma cyclic GMP levels were significantly increased by about 40% (p<0.05) one hour later. No difference was observed in the plasma ANP and cyclic GMP changes between the pre-HDT, the HDT and the post-HDT infusion experiment. Urine flow, sodium excretion and urinary cyclic GMP excretion were significantly increased (p<0.05 and below) by 100 to 300% during the second and third hour after each saline infusion. However, during these short-term periods only 20% of the infused water and less than 20% of the infused sodium were excreted. Furthermore, a significantly increased volume, sodium and cyclic GMP excretion was observed for over 48 hours after each fluid load experiment. These data suggest that HDT does not induce major alterations in the regulation of an acute saline infusion and plasma ANP does not play a major role in the diuretic/natriuretic effects of volume loading.

  19. Inactivation of cyclic Di-GMP binding protein TDE0214 affects the motility, biofilm formation, and virulence of Treponema denticola.

    PubMed

    Bian, Jiang; Liu, Xiangyang; Cheng, Yi-Qiang; Li, Chunhao

    2013-09-01

    As a ubiquitous second messenger, cyclic dimeric GMP (c-di-GMP) has been studied in numerous bacteria. The oral spirochete Treponema denticola, a periodontal pathogen associated with human periodontitis, has a complex c-di-GMP signaling network. However, its function remains unexplored. In this report, a PilZ-like c-di-GMP binding protein (TDE0214) was studied to investigate the role of c-di-GMP in the spirochete. TDE0214 harbors a PilZ domain with two signature motifs: RXXXR and DXSXXG. Biochemical studies showed that TDE0214 binds c-di-GMP in a specific manner, with a dissociation constant (Kd) value of 1.73 μM, which is in the low range compared to those of other reported c-di-GMP binding proteins. To reveal the role of c-di-GMP in T. denticola, a TDE0214 deletion mutant (TdΔ214) was constructed and analyzed in detail. First, swim plate and single-cell tracking analyses showed that TdΔ214 had abnormal swimming behaviors: the mutant was less motile and reversed more frequently than the wild type. Second, we found that biofilm formation of TdΔ214 was substantially repressed (∼6.0-fold reduction). Finally, in vivo studies using a mouse skin abscess model revealed that the invasiveness and ability to induce skin abscesses and host humoral immune responses were significantly attenuated in TdΔ214, indicative of the impact that TDE0214 has on the virulence of T. denticola. Collectively, the results reported here indicate that TDE0214 plays important roles in motility, biofilm formation, and virulence of the spirochete. This report also paves a way to further unveil the roles of the c-di-GMP signaling network in the biology and pathogenicity of T. denticola.

  20. Cyclosporine A-induced increase in glomerular cyclic GMP in rats and the involvement of the endothelinB receptor

    PubMed Central

    Tack, Ivan; Marin-Castano, Encarna; Bascands, Jean-Loup; Pecher, Christiane; Ader, Jean-Louis; Girolami, Jean-Pierre

    1997-01-01

    A transient two fold increase in the cyclic GMP content was observed in rat freshly isolated glomeruli 6 to 9 h after a single subcutaneous injection of 20 mg kg−1 cyclosporine A (CsA) in conscious animals. In vitro stimulation with endothelin 3 (ET-3) of isolated glomeruli obtained from CsA-untreated rats resulted in a dose-dependent increase in cyclic GMP content. The increase observed with 10 nM ET-3 was similar to that observed in glomeruli isolated 9 h after in vivo CsA administration. The rise in glomerular cyclic GMP content after in vivo CsA injection was prevented by in vivo treatment with L-NAME (10 mg kg−1) or by in vitro calcium deprivation of the incubation medium. The stimulating effects of CsA on glomerular cyclic GMP content were inhibited by in vivo administration of the ETB receptor antagonist BQ-788 (2 mg kg−1) but not by the ETA receptor antagonist BQ-123 (2 mg kg−1). The maximum increase in glomerular cyclic GMP content induced in vitro by acetylcholine (100 μM) and by ET-3 (100 nM) was slightly lower (approximately by 20–25%, P<0.05) in glomeruli from CsA-treated rats than in glomeruli from untreated rats. In contrast, the maximum increase achieved with 1 μM sodium nitroprusside was similar in both groups. A single subcutaneous injection of CsA did not significantly alter the glomerular mRNA expression of constitutive endothelial NO synthase (eNOS), as evaluated by RT–PCR, whereas the mRNA expression of the inducible NO synthase (iNOS), which follows pretreatment with lipopolysaccharide, was prevented. These results indicate that in vivo administration of a single dose of cyclosporine A transiently increases the cyclic GMP content of freshly isolated glomeruli, and that activation of ETB receptors and stimulation of the NO pathway are involved in this process. Furthermore, a single administration of CsA does not impair eNOS mRNA expression and only slightly reduces NO-dependent glomerular cyclic GMP

  1. Cyclic GMP-AMP Synthase is an Innate Immune Sensor of HIV and Other Retroviruses

    PubMed Central

    Gao, Daxing; Wu, Jiaxi; Wu, You-Tong; Du, Fenghe; Aroh, Chukwuemika; Yan, Nan; Sun, Lijun; Chen, Zhijian J.

    2013-01-01

    Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic-GMP-AMP (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type-I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse transcribed HIV DNA triggers the innate immune response. Knockout or knockdown of cGAS in mouse or human cell lines blocked cytokine induction by HIV, murine leukemia virus (MLV) and Simian immunodeficiency virus (SIV). These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses. PMID:23929945

  2. Pulmonary effects of type V cyclic GMP specific phosphodiesterase inhibition in the anaesthetized guinea-pig.

    PubMed Central

    Turner, N. C.; Dolan, J. S.; Grimsditch, D.; Lamb, J.; Worby, A.; Murray, K. J.; Coates, W. J.; Warrington, B. H.

    1994-01-01

    1. We have investigated the bronchodilator potential of type V phosphodiesterase (PDE V) inhibitors in anaesthetized ventilated guinea-pigs using the potent and selective PDE V inhibitor, SK&F 96231. We have compared its activity to that of salbutamol, the PDE III inhibitors, siguazodan and SK&F 95654 and to the PDE IV inhibitor rolipram. 2. Administered as an i.v. infusion SK&F 96231 (0.6 and 1 mg kg-1 min-1, i.v.) caused a slowly developing inhibition of histamine (100 nmol kg-1, i.v.)-induced bronchoconstriction and elevated tracheal cyclic GMP levels in the anaesthetized guinea-pig. SK&F 96231 (0.1 and 0.3 mg kg-1 min-1, i.v.) was without effect on histamine-induced bronchoconstriction. In the presence of a sub-threshold infusion of SNP (0.1 mumol kg-1 min-1, i.v.) there was a marked enhancement of SK&F 96231-induced inhibition of histamine responses such that at infusion rates that were ineffective alone, SK&F 96231 caused a > 50% inhibition of histamine responses. The stimulation of tracheal cyclic GMP accumulation by SK&F 96231 was also potentiated. 3. Administered directly into the airway, SK&F 96231 (300 micrograms in 5 mg lactose carrier) was largely without effect on histamine-induced bronchoconstriction (4.9 +/- 1.9% inhibition). In the presence of SNP (0.1 mumol kg-1 min-1, i.v.) or isosorbide dinitrate (200 micrograms administered by insufflation into the trachea) there was a marked potentiation of the inhibitory activity of SK&F 96231 (40 +/- 4% and 62 +/- 1.8% respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8032606

  3. Cyclic GMP-dependent vasodilatory properties of LASSBio 294 in rat aorta

    PubMed Central

    Silva, C L M; Noël, F; Barreiro, E J

    2002-01-01

    The effects of LASSBio 294, a new 3,4-methylenedioxybenzoyl-2-thienylhydrazone, on vascular tonus were investigated in isolated rat aortic rings.LASSBio 294 induced a concentration-dependent relaxation of intact rat aortic rings with an inhibitory concentration (IC50) of 74 μM (95% confidence limits: 59 – 92). The mechanical removal of the endothelium abolished this effect.In aortic rings with intact endothelium the effect of 100 μM LASSBio 294 was not altered by the pharmacological inhibition of NOS and cyclo-oxygenase pathways with 500 μM L-NAME and 10 μM indomethacin, respectively.LASSBio 294 (100 μM) was able to relax aortic rings pre-contracted with high extracellular K+ (KCl 100 mM).The relaxant effect of LASSBio 294 was fully reversed (and prevented) by the addition of 1 μM ODQ (1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one), a selective inhibitor of soluble guanylate cyclase.LASSBio 294 (100 μM) had no direct effect on PDE3 and PDE4 activities, however, it increased by 150% cyclic GMP content in aortic rings pre-treated with 100 μM L-NAME and 10 μM indomethacin, as did 1 μM zaprinast, a selective PDE5 inhibitor.In conclusion, LASSBio 294 induced relaxation of isolated rat aorta probably by directly increasing cyclic GMP content, possibly as a consequence of PDE5 inhibition. PMID:11786506

  4. Cyclic GMP-dependent vasodilatory properties of LASSBio 294 in rat aorta.

    PubMed

    Silva, C L M; Noël, F; Barreiro, E J

    2002-01-01

    . The effects of LASSBio 294, a new 3,4-methylenedioxybenzoyl-2-thienylhydrazone, on vascular tonus were investigated in isolated rat aortic rings. 2. LASSBio 294 induced a concentration-dependent relaxation of intact rat aortic rings with an inhibitory concentration (IC(50)) of 74 microM (95% confidence limits: 59 - 92). The mechanical removal of the endothelium abolished this effect. 3. In aortic rings with intact endothelium the effect of 100 microM LASSBio 294 was not altered by the pharmacological inhibition of NOS and cyclo-oxygenase pathways with 500 microM L-NAME and 10 microM indomethacin, respectively. 4. LASSBio 294 (100 microM) was able to relax aortic rings pre-contracted with high extracellular K(+) (KCl 100 mM). 5. The relaxant effect of LASSBio 294 was fully reversed (and prevented) by the addition of 1 microM ODQ (1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one), a selective inhibitor of soluble guanylate cyclase. 6. LASSBio 294 (100 microM) had no direct effect on PDE3 and PDE4 activities, however, it increased by 150% cyclic GMP content in aortic rings pre-treated with 100 microM L-NAME and 10 microM indomethacin, as did 1 microM zaprinast, a selective PDE5 inhibitor. 7. In conclusion, LASSBio 294 induced relaxation of isolated rat aorta probably by directly increasing cyclic GMP content, possibly as a consequence of PDE5 inhibition.

  5. Identification and molecular characterization of a cyclic-di-GMP effector protein, PlzA (BB0733): additional evidence for the existence of a functional cyclic-di-GMP regulatory network in the Lyme disease spirochete, Borrelia burgdorferi

    PubMed Central

    Freedman, John C.; Rogers, Elizabeth A.; Kostick, Jessica L.; Zhang, Hongming; Iyer, Radha; Schwartz, Ira; Marconi, Richard T.

    2010-01-01

    The Borrelia burgdorferi Rrp1 protein is a diguanylate cyclase that controls a regulon consisting of ~10% of the total genome. Because Rrp1 lacks a DNA-binding domain, its regulatory capability is most likely mediated through the production of bis-(3′–5′)-cyclic dimeric GMP (c-di-GMP). C-di-GMP binds to and activates the regulatory activity of proteins that harbor a PilZ domain. The occurrence of a PilZ domain within a protein is not in and of itself sufficient to convey c-di-GMP binding, as other structural aspects of the protein are important in the interaction. In this study, we have assessed the expression and c-di-GMP binding ability of the sole PilZ domain-containing protein of B. burgdorferi B31, PlzA. PlzA was determined to be upregulated by tick feeding and to be expressed during mammalian infection. The gene is highly conserved and present in all Borrelia species. Analyses of recombinant PlzA demonstrated its ability to bind c-di-GMP and site-directed mutagenesis revealed that this interaction is highly specific and dependent on Arg residues contained within the PilZ domain. In summary, this study is the first to identify a c-di-GMP effector molecule in a spirochete and provides additional evidence for the existence of a complete c-di-GMP regulatory network in the Lyme disease spirochete, B. burgdorferi. PMID:20030712

  6. Effects of 5-hydroxytryptamine, dopamine, and acetylcholine on accumulation of cyclic AMP and cyclic GMP in the anterior byssus retractor muscle of Mytilus edulis L. (Mollusca).

    PubMed

    Köhler, G; Lindl, T

    1980-02-01

    We investigated in vitro accumulation of adenosine 3',5'-monophosphate (induced by 5-hydroxytryptamine and dopamine) and of guanosine 3',5'-monophosphate (induced by acetylcholine) in the anterior byssus retractor muscle of Mytilus. The response to 5-hydroxytryptamine exceeded that induced by equimolar concentrations of dopamine. 1-methyl lysergic acid, a 5-hydroxytryptamine-blocking agent, diminished the 5-hydroxytryptamine-induced increase of cyclic AMP level. This parallels the effect of this amine on the contracted muscle. Acetylcholine, which causes a tonic contraction of the muscle, increased intracellular levels of cyclic GMP in a dose-dependent (max. 45-fold at 10(-4) M ACh) manner. The time course of the rise in cyclic GMP level was rapid and transient (peak concentration of cyclic GMP at 2 min). Mytolon was the most effective of all cholinergic blockers tested. It was concluded that cyclic nucleotides may play a role in the modulatory process of the transmitters. A direct relation to the relaxation-contraction process could not be established.

  7. Cocaine self-administration by rats is inhibited by cyclic GMP-elevating agents: involvement of epigenetic markers.

    PubMed

    Deschatrettes, Elodie; Romieu, Pascal; Zwiller, Jean

    2013-08-01

    The C-type natriuretic peptide (CNP) exerts its action via stimulation of the cyclic GMP (cGMP) signalling pathway, which includes the activation of cGMP-dependent protein kinases. The pathway can also be activated by inhibitors of phosphodiesterases (PDE) that hydrolyse cGMP. The present report shows that activation of the cGMP pathway by CNP, by bromo-cGMP, a cell-permeant cGMP analogue, or by the PDE inhibitor zaprinast dose dependently reduces intravenous cocaine self-administration by rats. The effect was found when the compounds were injected in situ into the prefrontal cortex, but not when they were injected into the nucleus accumbens. A decrease in the number of cocaine infusions performed by rats was obtained under the fixed ratio-1 schedule of reinforcement as well as under a progressive ratio schedule, which evaluates the motivation of the animals for the drug. Decrease in cocaine self-administration was accompanied with reduced expression of the epigenetic markers methyl-CpG-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2) in dopaminergic projection areas. An increase in the acetylation level of histone H3, but not of histone H4, was also noticed. Since MeCP2 and HDAC2 are known to modulate dynamic functions in the adult brain, such as synaptic plasticity, our results showing that activation of the cGMP signal transduction pathway decreased both cocaine intake and expression of the epigenetic markers strongly suggest that the MeCP2/HDAC2 complex is involved in the analysis of the reinforcing properties of cocaine in the prefrontal cortex.

  8. Stress-dependent dilated cardiomyopathy in mice with cardiomyocyte-restricted inactivation of cyclic GMP-dependent protein kinase I

    PubMed Central

    Frantz, Stefan; Klaiber, Michael; Baba, Hideo A.; Oberwinkler, Heike; Völker, Katharina; Gaβner, Birgit; Bayer, Barbara; Abeβer, Marco; Schuh, Kai; Feil, Robert; Hofmann, Franz; Kuhn, Michaela

    2013-01-01

    Aims Cardiac hypertrophy is a common and often lethal complication of arterial hypertension. Elevation of myocyte cyclic GMP levels by local actions of endogenous atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) or by pharmacological inhibition of phosphodiesterase-5 was shown to counter-regulate pathological hypertrophy. It was suggested that cGMP-dependent protein kinase I (cGKI) mediates this protective effect, although the role in vivo is under debate. Here, we investigated whether cGKI modulates myocyte growth and/or function in the intact organism. Methods and results To circumvent the systemic phenotype associated with germline ablation of cGKI, we inactivated the murine cGKI gene selectively in cardiomyocytes by Cre/loxP-mediated recombination. Mice with cardiomyocyte-restricted cGKI deletion exhibited unaltered cardiac morphology and function under resting conditions. Also, cardiac hypertrophic and contractile responses to β-adrenoreceptor stimulation by isoprenaline (at 40 mg/kg/day during 1 week) were unaltered. However, angiotensin II (Ang II, at 1000 ng/kg/min for 2 weeks) or transverse aortic constriction (for 3 weeks) provoked dilated cardiomyopathy with marked deterioration of cardiac function. This was accompanied by diminished expression of the [Ca2+]i-regulating proteins SERCA2a and phospholamban (PLB) and a reduction in PLB phosphorylation at Ser16, the specific target site for cGKI, resulting in altered myocyte Ca2+i homeostasis. In isolated adult myocytes, CNP, but not ANP, stimulated PLB phosphorylation, Ca2+i-handling, and contractility via cGKI. Conclusion These results indicate that the loss of cGKI in cardiac myocytes compromises the hypertrophic program to pathological stimulation, rendering the heart more susceptible to dysfunction. In particular, cGKI mediates stimulatory effects of CNP on myocyte Ca2+i handling and contractility. PMID:22199120

  9. GEMM-I riboswitches from Geobacter sense the bacterial second messenger cyclic AMP-GMP.

    PubMed

    Kellenberger, Colleen A; Wilson, Stephen C; Hickey, Scott F; Gonzalez, Tania L; Su, Yichi; Hallberg, Zachary F; Brewer, Thomas F; Iavarone, Anthony T; Carlson, Hans K; Hsieh, Yu-Fang; Hammond, Ming C

    2015-04-28

    Cyclic dinucleotides are an expanding class of signaling molecules that control many aspects of bacterial physiology. A synthase for cyclic AMP-GMP (cAG, also referenced as 3'-5', 3'-5' cGAMP) called DncV is associated with hyperinfectivity of Vibrio cholerae but has not been found in many bacteria, raising questions about the prevalence and function of cAG signaling. We have discovered that the environmental bacterium Geobacter sulfurreducens produces cAG and uses a subset of GEMM-I class riboswitches (GEMM-Ib, Genes for the Environment, Membranes, and Motility) as specific receptors for cAG. GEMM-Ib riboswitches regulate genes associated with extracellular electron transfer; thus cAG signaling may control aspects of bacterial electrophysiology. These findings expand the role of cAG beyond organisms that harbor DncV and beyond pathogenesis to microbial geochemistry, which is important to environmental remediation and microbial fuel cell development. Finally, we have developed an RNA-based fluorescent biosensor for live-cell imaging of cAG. This selective, genetically encodable biosensor will be useful to probe the biochemistry and cell biology of cAG signaling in diverse bacteria.

  10. Increase in nitric oxide and cyclic GMP of rat cerebellum by radio frequency burst-type electromagnetic field radiation.

    PubMed Central

    Miura, M; Takayama, K; Okada, J

    1993-01-01

    1. Using rat cerebellum supernatant, the effects of radio frequency (RF) burst-type electromagnetic (EM) field radiation on the production of cyclic GMP were examined under various conditions. The radiation was generated by a generator coil, and set at a 10 MHz radiation frequency, a 50% burst time, a 10 kHz burst rate and a 5 V peak-to-peak generator voltage. 2. When the cerebellum supernatant was incubated with both exogenous L-arginine (nitric oxide (NO) donor) and NADPH, and irradiated by an RF burst-type EM field, the production of cyclic GMP was increased significantly from a level of 21-22 nmol min-1 (g tissue)-1 to 25-26 nmol min-1 (g tissue)-1. By contrast, such an effect was not found when the cerebellum supernatant was irradiated by an RF volley-type EM field. 3. When neither L-arginine nor NADPH were added to the cerebellum supernatant, the production of cyclic GMP was lowered to a level of 6 nmol min-1 (g tissue)-1 and the radiation effect was not found. When the cerebellum supernatant was chelated with EDTA, the production of cyclic GMP was lowered to a level of 7 nmol min-1 (g tissue)-1 and the radiation effect was not found. 4. Incubation with Methylene Blue, a guanylate cyclase inhibitor, lowered the production of cyclic GMP to a level of 10-12 nmol min-1 (g tissue)-1, and the radiation effect did not occur. On incubation with a NO synthase inhibitor, either NG-methyl-L-arginine or N omega-nitro-L-arginine methyl ester, the production of cyclic GMP was lowered to a level of 10-12 nmol min-1 (g tissue)-1 or 5-9 nmol min-1 (g tissue)-1 respectively, and the radiation effect was not observed. 5. Using electrochemical NO probes, the production of NO in the cerebellum supernatant was detected. The concentration of NO increased gradually after the onset of the EM field radiation. The radiation effect persisted, and reached a maximum after the cessation of the radiation. 6. In an in vivo study, the arterioles of the frog web were dilated by the radiation

  11. Downregulation of nitrovasodilator-induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin-1 beta.

    PubMed Central

    Papapetropoulos, A.; Abou-Mohamed, G.; Marczin, N.; Murad, F.; Caldwell, R. W.; Catravas, J. D.

    1996-01-01

    1. Induction of nitric oxide synthase (iNOS) results in overproduction of nitric oxide (NO), which may be a principal cause of the massive vasodilatation and hypotension observed in septic shock. Since NO-induced vasorelaxation is mediated via the soluble isoform of guanylate cyclase (sGC), the regulation of sGC activity during shock is of obvious importance, but yet poorly understood. The aim of the present study was to investigate the activation of sGC by sodium nitroprusside (SNP) before and after exposure of rat aortic smooth muscle cells to endotoxin (LPS) or interleukin-1 beta (IL-1 beta). 2. Exposure of rat aortic smooth muscle cells to SNP (10 microM) elicited up to 200 fold increases in cyclic GMP. This effect was attenuated by 30-70% in IL-1 beta- or LPS-pretreated cells, in a pretreatment time-and IL-1 beta- or LPS-concentration-dependent manner. When, however, cells were exposed to IL-1 beta or LPS and then stimulated with the particulate guanylate cyclase activator, atriopeptin II, no reduction in cyclic GMP accumulation was observed. 3. Pretreatment of rats with LPS (5 mg kg-1, i.v.) for 6 h led to a decrease in aortic ring SNP-induced cyclic GMP accumulation. 4. The IL-1 beta-induced reduction in SNP-stimulated cyclic GMP accumulation in cultured cells was dependent on NO production, as arginine depletion abolished the downregulation of cyclic GMP accumulation in response to SNP. 5. Reverse-transcriptase-polymerase chain reaction analysis revealed that the ratio of steady state mRNA for the alpha, subunit of sGC to glyceraldehyde phosphate dehydrogenase was decreased in LPS- or IL-1 beta-treated cells, as compared to vehicle-treated cells. 6. Protein levels of the alpha 1 sGC subunit remained unaltered upon exposure to LPS or IL-1 beta, suggesting that the early decreased cyclic GMP accumulation in IL-1 beta- or LPS-pretreated cells was probably due to reduced sGC activation. Thus, the observed decreased responsiveness of sGC to NO stimulation

  12. Vibrio cholerae VpsT Regulates Matrix Production and Motility by Directly Sensing Cyclic di-GMP

    SciTech Connect

    Krasteva, P.; Fong, J; Shikuma, N; Beyhan, S; Navarro, M; Yildiz, F; Sondermann, H

    2010-01-01

    Microorganisms can switch from a planktonic, free-swimming life-style to a sessile, colonial state, called a biofilm, which confers resistance to environmental stress. Conversion between the motile and biofilm life-styles has been attributed to increased levels of the prokaryotic second messenger cyclic di-guanosine monophosphate (c-di-GMP), yet the signaling mechanisms mediating such a global switch are poorly understood. Here we show that the transcriptional regulator VpsT from Vibrio cholerae directly senses c-di-GMP to inversely control extracellular matrix production and motility, which identifies VpsT as a master regulator for biofilm formation. Rather than being regulated by phosphorylation, VpsT undergoes a change in oligomerization on c-di-GMP binding.

  13. PdeB, a cyclic Di-GMP-specific phosphodiesterase that regulates Shewanella oneidensis MR-1 motility and biofilm formation.

    PubMed

    Chao, Lily; Rakshe, Shauna; Leff, Maija; Spormann, Alfred M

    2013-09-01

    Shewanella oneidensis MR-1, a gammaproteobacterium with respiratory versatility, forms biofilms on mineral surfaces through a process controlled by the cyclic dinucleotide messenger c-di-GMP. Cellular concentrations of c-di-GMP are maintained by proteins containing GGDEF and EAL domains, which encode diguanylate cyclases for c-di-GMP synthesis and phosphodiesterases for c-di-GMP hydrolysis, respectively. The S. oneidensis MR-1 genome encodes several GGDEF and EAL domain proteins (50 and 31, respectively), with a significant fraction (∼10) predicted to be multidomain (e.g., GGDEF-EAL) enzymes containing an additional Per-Arnt-Sim (PAS) sensor domain. However, the biochemical activities and physiological functions of these multidomain enzymes remain largely unknown. Here, we present genetic and biochemical analyses of a predicted PAS-GGDEF-EAL domain-containing protein, SO0437, here named PdeB. A pdeB deletion mutant exhibited decreased swimming motility and increased biofilm formation under rich growth medium conditions, which was consistent with an increase in intracellular c-di-GMP. A mutation inactivating the EAL domain also produced similar swimming and biofilm phenotypes, indicating that the increase in c-di-GMP was likely due to a loss in phosphodiesterase activity. Therefore, we also examined the enzymatic activity of purified PdeB and found that the protein exhibited phosphodiesterase activity via the EAL domain. No diguanylate cyclase activity was observed. In addition to the motility and biofilm phenotypes, transcriptional profiling by DNA microarray analysis of biofilms of pdeB (in-frame deletion and EAL) mutant cells revealed that expression of genes involved in sulfate uptake and assimilation were repressed. Addition of sulfate to the growth medium resulted in significantly less motile pdeB mutants. Together, these results indicate a link between c-di-GMP metabolism, S. oneidensis MR-1 biofilm development, and sulfate uptake/assimilation.

  14. Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

    PubMed

    Miner, Kyle D; Kurtz, Donald M

    2016-02-16

    HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

  15. Output Targets and Transcriptional Regulation by a Cyclic Dimeric GMP-Responsive Circuit in the Vibrio parahaemolyticus Scr Network

    PubMed Central

    Ferreira, Rosana B. R.; Chodur, Daniel M.; Antunes, Luis Caetano M.; Trimble, Michael J.

    2012-01-01

    The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP). In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ΔscrABC and wild-type strains revealed expression differences with respect to ∼100 genes. Elevated c-di-GMP repressed genes in the surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide. The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct, positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory module in the circuit. PMID:22194449

  16. Structural and Functional Analysis of STING Sheds New Light on Cyclic di-GMP Mediated Immune Signaling Mechanism

    PubMed Central

    Ouyang, Songying; Song, Xianqiang; Wang, Yaya; Ru, Heng; Shaw, Neil; Jiang, Yan; Niu, Fengfeng; Zhu, Yanping; Qiu, Weicheng; Parvatiyar, Kislay; Li, Yang; Zhang, Rongguang; Cheng, Genhong; Liu, Zhi-Jie

    2012-01-01

    SUMMARY STING is an essential signaling molecule for DNA and cyclic di-GMP (c-di-GMP)-mediated type I interferon (IFN) production via TANK-binding kinase 1 (TBK1) and Interferon regulatory factor 3 (IRF3) pathway. It contains an N-terminal transmembrane region and a cytosolic C-terminal domain (CTD). Here, we describe crystal structures of STING CTD alone and complexed with c-di-GMP in a unique binding mode. The strictly conserved AA153-173 region was shown to be cytosolic and participated in dimerization via hydrophobic interactions. The STING CTD functions as a dimer and the dimerization was independent of post-translational modifications. Binding of c-di-GMP enhanced interaction of a shorter construct of STING CTD (residues 139-344) with TBK1. This suggests an extra TBK1 binding site, other than Ser358. This study provides a glimpse into the unique architecture of STING and sheds new light on the mechanism of c-di-GMP-mediated TBK1 signaling. PMID:22579474

  17. ChIP-seq reveals the global regulator AlgR mediating cyclic di-GMP synthesis in Pseudomonas aeruginosa

    PubMed Central

    Kong, Weina; Zhao, Jingru; Kang, Huaping; Zhu, Miao; Zhou, Tianhong; Deng, Xin; Liang, Haihua

    2015-01-01

    AlgR is a key transcriptional regulator required for the expression of multiple virulence factors, including type IV pili and alginate in Pseudomonas aeruginosa. However, the regulon and molecular regulatory mechanism of AlgR have yet to be fully elucidated. Here, among 157 loci that were identified by a ChIP-seq assay, we characterized a gene, mucR, which encodes an enzyme that synthesizes the intracellular second messenger cyclic diguanylate (c-di-GMP). A ΔalgR strain produced lesser biofilm than did the wild-type strain, which is consistent with a phenotype controlled by c-di-GMP. AlgR positively regulates mucR via direct binding to its promoter. A ΔalgRΔmucR double mutant produced lesser biofilm than did the single ΔalgR mutant, demonstrating that c-di-GMP is a positive regulator of biofilm formation. AlgR controls the levels of c-di-GMP synthesis via direct regulation of mucR. In addition, the cognate sensor of AlgR, FimS/AlgZ, also plays an important role in P. aeruginosa virulence. Taken together, this study provides new insights into the AlgR regulon and reveals the involvement of c-di-GMP in the mechanism underlying AlgR regulation. PMID:26206672

  18. Cyclic di-GMP mediates a histidine kinase/phosphatase switch by noncovalent domain cross-linking

    PubMed Central

    Dubey, Badri N.; Lori, Christian; Ozaki, Shogo; Fucile, Geoffrey; Plaza-Menacho, Ivan; Jenal, Urs; Schirmer, Tilman

    2016-01-01

    Histidine kinases are key components of regulatory networks in bacteria. Although many of these enzymes are bifunctional, mediating both phosphorylation and dephosphorylation of downstream targets, the molecular details of this central regulatory switch are unclear. We showed recently that the universal second messenger cyclic di–guanosine monophosphate (c-di-GMP) drives Caulobacter crescentus cell cycle progression by forcing the cell cycle kinase CckA from its default kinase into phosphatase mode. We use a combination of structure determination, modeling, and functional analysis to demonstrate that c-di-GMP reciprocally regulates the two antagonistic CckA activities through noncovalent cross-linking of the catalytic domain with the dimerization histidine phosphotransfer (DHp) domain. We demonstrate that both c-di-GMP and ADP (adenosine diphosphate) promote phosphatase activity and propose that c-di-GMP stabilizes the ADP-bound quaternary structure, which allows the receiver domain to access the dimeric DHp stem for dephosphorylation. In silico analyses predict that c-di-GMP control is widespread among bacterial histidine kinases, arguing that it can replace or modulate canonical transmembrane signaling. PMID:27652341

  19. PilZ Domain Protein FlgZ Mediates Cyclic Di-GMP-Dependent Swarming Motility Control in Pseudomonas aeruginosa

    PubMed Central

    Baker, Amy E.; Diepold, Andreas; Kuchma, Sherry L.; Scott, Jessie E.; Ha, Dae Gon; Orazi, Giulia

    2016-01-01

    ABSTRACT The second messenger cyclic diguanylate (c-di-GMP) is an important regulator of motility in many bacterial species. In Pseudomonas aeruginosa, elevated levels of c-di-GMP promote biofilm formation and repress flagellum-driven swarming motility. The rotation of P. aeruginosa's polar flagellum is controlled by two distinct stator complexes, MotAB, which cannot support swarming motility, and MotCD, which promotes swarming motility. Here we show that when c-di-GMP levels are elevated, swarming motility is repressed by the PilZ domain-containing protein FlgZ and by Pel polysaccharide production. We demonstrate that FlgZ interacts specifically with the motility-promoting stator protein MotC in a c-di-GMP-dependent manner and that a functional green fluorescent protein (GFP)-FlgZ fusion protein shows significantly reduced polar localization in a strain lacking the MotCD stator. Our results establish FlgZ as a c-di-GMP receptor affecting swarming motility by P. aeruginosa and support a model wherein c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator. IMPORTANCE The regulation of surface-associated motility plays an important role in bacterial surface colonization and biofilm formation. c-di-GMP signaling is a widespread means of controlling bacterial motility, and yet the mechanism whereby this signal controls surface-associated motility in P. aeruginosa remains poorly understood. Here we identify a PilZ domain-containing c-di-GMP effector protein that contributes to c-di-GMP-mediated repression of swarming motility by P. aeruginosa. We provide evidence that this effector, FlgZ, impacts swarming motility via its interactions with flagellar stator protein MotC. Thus, we propose a new mechanism for c-di-GMP-mediated regulation of motility for a bacterium with two flagellar stator sets, increasing our understanding of surface-associated behaviors, a key prerequisite to identifying ways to control the formation of biofilm communities. PMID

  20. Bacterial Rotary Export ATPases Are Allosterically Regulated by the Nucleotide Second Messenger Cyclic-di-GMP*

    PubMed Central

    Trampari, Eleftheria; Stevenson, Clare E. M.; Little, Richard H.; Wilhelm, Thomas; Lawson, David M.; Malone, Jacob G.

    2015-01-01

    The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins. PMID:26265469

  1. New fluorescent analogs of cAMP and cGMP available as substrates for cyclic nucleotide phosphodiesterase.

    PubMed

    Hiratsuka, T

    1982-11-25

    The synthesis of fluorescent derivatives of cAMP and cGMP, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding 2'-O-anthraniloyl derivatives of cyclic nucleotides, is here described. 2'-O-(N-Methylanthraniloyl) derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm, these derivatives exhibited maximum fluorescence emission at 430-445 nm in aqueous solution with quantum yields of 0.11-0.26. Their fluorescence was sensitive to the polarity of solvent; in N,N-dimethylformamide quantum yields of 0.8-0.95. The major differences between the two fluorophores were the longer wavelength of the emission maximum of the N-methylanthraniloyl group and its greater quantum yield. The derivatives were substrates for beef heart cyclic nucleotide phosphodiesterase, 15-24% as effective as the natural substrate cAMP. When combined with thin layer chromatography techniques, two apparent Km values (3-4 microM and 36-76 microM) for the cAMP derivatives and one value (10-18 microM) for the cGMP derivatives were obtained. The results indicate that these 2'-hydroxyl-modified cAMP and cGMP can be useful fluorescent substrate analogs for cyclic nucleotide phosphodiesterase.

  2. Participation of nitric oxide and cyclic GMP in the supersensitivity of acute diabetic rat myocardium by cholinergic stimuli.

    PubMed

    Wald, M R; Borda, E S; Sterin-Borda, L

    1998-06-15

    The purpose of this study was to explore the pharmacological and biochemical mechanisms involved in diabetic cardiomyopathy, with particular interest in the abnormal function of cholinergic neurotransmission at the onset of the pathology. The muscarinic acethylcholine agonist carbachol showed a negative inotropic response on both normal and diabetic isolated atria, but the latter showed a supersensitive response. No changes were found in muscarinic acethylcholine receptor (mAChR) expression. Measurements of mAChR-associated second messengers indicated no significant differences between normal and diabetic rat atria in the stimulatory effect of carbachol on protein kinase C activity and the production of inositol phosphates, or in the inhibitory effect induced by carbachol on cyclic AMP (cAMP) production. On the contrary, nitric oxide (NO) synthase activity and cyclic GMP production were higher in diabetic cardiac preparations than in normal ones. Moreover, in diabetic atria, nitric oxide synthase and guanylate cyclase inhibitors shifted the carbachol concentration-response curve on contractility to the right, reaching values similar to those of normal atria. These results suggest an early alteration in the mACh system during the diabetic state, associated with increased production of nitric oxide and cyclic GMP (cGMP). This, in turn, could increase the biological mechanical activity of the mAChR agonist, inducing in this way a higher pharmacological response, without changes in mAChR expression.

  3. Molecular cloning and functional characterization of porcine cyclic GMP-AMP synthase.

    PubMed

    Wang, Jiang; Chu, Beibei; Du, Lili; Han, Yingqian; Zhang, Xuemei; Fan, Shuangshuang; Wang, Yueying; Yang, Guoyu

    2015-06-01

    Cyclic GMP-AMP synthase (cGAS), which belongs to the nucleotidyltransferase family, recognizes cytosolic DNA and induces the type I interferon (IFN) pathway through the synthesis of the second messenger cGAMP. In this study, porcine cGAS (p-cGAS) was identified and its tissue distribution, subcellular localization, and functions in innate immunity were characterized. The coding sequence of p-cGAS is 1494 bp long, encodes 497 amino acids, and is most similar (74%) to Bos taurus cGAS. p-cGAS mRNA is abundant in the spleen, duodenum, jejunum, and ileum. The subcellular distribution of p-cGAS is not only in the cytosol, but also on the endoplasmic reticulum (ER) membrane. The overexpression of wild-type p-cGAS in porcine kidney epithelial cells, but not its catalytically inactive mutants, induced IFN-β expression, which was dependent on STING and IRF3. However, the downregulation of p-cGAS by RNA interference markedly reduced IFN-β expression after pseudorabies virus (PRV) infection or poly(dA:dT) transfection. These results demonstrate that p-cGAS is an important DNA sensor, required for IFN-β activation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Cyclic GMP levels in the rat brain and plasma during clinical death and after resuscitation.

    PubMed

    Kapuściński, A

    1993-01-01

    Changes of cGMP content in the rat brain and plasma have been evaluated by means of the radioimmunologic method after 5-min clinical death and up to 2 hours after resuscitation. Ischemia produced a decrease of cGMP in the brain, however, at the 15th min after resuscitation a reversible significant rise of nucleotide concentration was noted. In plasma at the end of ischemia and in the postischemic period a significant decrease of cGMP level was observed. The mechanisms of cGMP regulation in the central nervous system and the significance of the obtained results are discussed.

  5. Nitric oxide signaling in Pseudomonas aeruginosa biofilms mediates phosphodiesterase activity, decreased cyclic di-GMP levels, and enhanced dispersal.

    PubMed

    Barraud, Nicolas; Schleheck, David; Klebensberger, Janosch; Webb, Jeremy S; Hassett, Daniel J; Rice, Scott A; Kjelleberg, Staffan

    2009-12-01

    Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa. Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO.

  6. Genomic analysis of cyclic-di-GMP-related genes in rhizobial type strains and functional analysis in Rhizobium etli.

    PubMed

    Gao, Shanjun; Romdhane, Samir Ben; Beullens, Serge; Kaever, Volkhard; Lambrichts, Ivo; Fauvart, Maarten; Michiels, Jan

    2014-05-01

    Rhizobia are soil bacteria that can fix nitrogen in symbiosis with leguminous plants or exist free living in the rhizosphere. Crucial to their complex lifestyle is the ability to sense and respond to diverse environmental stimuli, requiring elaborate signaling pathways. In the majority of bacteria, the nucleotide-based second messenger cyclic diguanosine monophosphate (c-di-GMP) is involved in signal transduction. Surprisingly, little is known about the importance of c-di-GMP signaling in rhizobia. We have analyzed the genome sequences of six well-studied type species (Bradyrhizobium japonicum, Mesorhizobium loti, Rhizobium etli, Rhizobium leguminosarum, Sinorhizobium fredii, and Sinorhizobium meliloti) for proteins possibly involved in c-di-GMP signaling based on the presence of four domains: GGDEF (diguanylate cyclase), EAL and HD-GYP (phosphodiesterase), and PilZ (c-di-GMP sensor). We find that rhizobia possess a high number of these proteins. Conservation analysis suggests that c-di-GMP signaling proteins modulate species-specific pathways rather than ancient rhizobia-specific processes. Two hybrid GGDEF-EAL proteins were selected for functional analysis, R. etli RHE_PD00105 (CdgA) and RHE_PD00137 (CdgB). Expression of cdgA and cdgB is repressed by the alarmone (p)ppGpp. cdgB is significantly expressed on plant roots and free living. Mutation of cdgA, cdgB, or both does not affect plant root colonization, nitrogen fixation capacity, biofilm formation, motility, and exopolysaccharide production. However, heterologous expression of the individual GGDEF and EAL domains of each protein in Escherichia coli strongly suggests that CdgA and CdgB are bifunctional proteins, possessing both diguanylate cyclase and phosphodiesterase activities. Taken together, our results provide a platform for future studies of c-di-GMP signaling in rhizobia.

  7. c-di-GMP (3′-5′-Cyclic Diguanylic Acid) Inhibits Staphylococcus aureus Cell-Cell Interactions and Biofilm Formation

    PubMed Central

    Karaolis, David K. R.; Rashid, Mohammed H.; Chythanya, Rajanna; Luo, Wensheng; Hyodo, Mamoru; Hayakawa, Yoshihiro

    2005-01-01

    Staphylococcus aureus is an important pathogen of humans and animals, and antibiotic resistance is a public health concern. Biofilm formation is essential in virulence and pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. As such, novel antimicrobial approaches are of great interest to the scientific, medical, and agriculture communities. We recently proposed that modulating levels of the cyclic dinucleotide signaling molecule, c-di-GMP (cyclic diguanylate [3′,5′-cyclic diguanylic acid], cGpGp), has utility in regulating phenotypes of prokaryotes. We report that extracellular c-di-GMP shows activity against human clinical and bovine intramammary mastitis isolates of S. aureus, including methicillin-resistant S. aureus (MRSA) isolates. We show that chemically synthesized c-di-GMP is soluble and stable in water and physiological saline and stable following boiling and exposure to acid and alkali. Treatment of S. aureus with extracellular c-di-GMP inhibited cell-to-cell (intercellular) adhesive interactions in liquid medium and reduced (>50%) biofilm formation in human and bovine isolates compared to untreated controls. c-di-GMP inhibited the adherence of S. aureus to human epithelial HeLa cells. The cyclic nucleotide analogs cyclic GMP and cyclic AMP had a lesser inhibitory effect on biofilms, while 5′-GMP had no major effect. We propose that cyclic dinucleotides such as c-di-GMP, used either alone or in combination with other antimicrobial agents, represent a novel and attractive approach in the development of intervention strategies for the prevention of biofilms and the control and treatment of infection. PMID:15728899

  8. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix.

    PubMed

    Borlee, Bradley R; Goldman, Aaron D; Murakami, Keiji; Samudrala, Ram; Wozniak, Daniel J; Parsek, Matthew R

    2010-02-01

    Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a beta-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural

  9. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix

    PubMed Central

    Borlee, Bradley R; Goldman, Aaron D; Murakami, Keiji; Samudrala, Ram; Wozniak, Daniel J; Parsek, Matthew R

    2010-01-01

    Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a β-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural

  10. Cyclic di-GMP contributes to adaption and virulence of Bacillus thuringiensis through a riboswitch-regulated collagen adhesion protein

    PubMed Central

    Tang, Qing; Yin, Kang; Qian, Hongliang; Zhao, Youwen; Wang, Wen; Chou, Shan-Ho; Fu, Yang; He, Jin

    2016-01-01

    Cyclic di-GMP is a ubiquitous second messenger that regulates diverse cellular processes in bacteria by binding to various protein or riboswitch effectors. In Bacillus thuringiensis BMB171, a c-di-GMP riboswitch termed Bc2 RNA resides in the 5′-untranslated region (5′-UTR) of an mRNA that encodes a collagen adhesion protein (Cap). The expression of cap was strongly repressed in parent strain BMB171 because of the presence of Bc2 RNA but was significantly promoted in the Bc2 RNA markerless deletion mutant. Bc2 RNA acts as a genetic “on” switch, which forms an anti-terminator structure to promote cap read-through transcription upon c-di-GMP binding. As a result, cap transcription was de-repressed under high c-di-GMP levels. Therefore, Bc2 RNA regulates cap expression using a repression/de-repression model. Bc2 RNA-regulated Cap was also found to be tightly associated with motility, aggregation, exopolysaccharide secretion, biofilm formation, and virulence of B. thuringiensis BMB171 against its host insect Helicoverpa armigera. PMID:27381437

  11. [Phosphodiesterase 3 mediates cross-talk between the protein kinase- and cGMP- dependent pathways and cyclic AMP metabolism].

    PubMed

    Makuch, Edyta; Matuszyk, Janusz

    2012-07-20

    PDE3 is a dual-substrate phosphodiesterase responsible for hydrolyzing both cAMP and cGMP whilst being simultaneously inhibited by cGMP. This feature is related to presence of the 44 amino acid insert in the catalytic domain, which determines the mechanism of introduction of the cyclic nucleotide into the catalytic pocket of the enzyme. Once bound in the catalytic site cGMP results in steric hindrance for cAMP to enter the site. The regulatory domain of PDE3 consists of two hydrophobic regions: NHR1 and NHR2. Their presence defines the enzyme's intracellular localization, thus determining its participation in particular signaling cascades. Due to the properties of PDE3 this enzyme has exceptional importance for the cross-talk between cAMP-dependent signaling and other cascades. There are two different mechanisms of action of PDE3 enzymes in cell signaling pathways. In many signaling cascades assembly of a signalosome is necessary for phosphorylation and activation of the PDE3 proteins. In response to certain hormones and growth factors, PDE3 merges the metabolism of cAMP with protein kinase-dependent signaling pathways. PDE3 also controls the level of cAMP with regard to the alternating concentration of cGMP. This effect occurs in signaling cascades activated by natriuretic peptide.

  12. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases.

    PubMed

    Gao, Daxing; Li, Tuo; Li, Xiao-Dong; Chen, Xiang; Li, Quan-Zhen; Wight-Carter, Mary; Chen, Zhijian J

    2015-10-20

    TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi-Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1(-/-) mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1(-/-) mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1(-/-) mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII(-/-) mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1(-/-) and DNaseII(-/-) mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA.

  13. Inhibition of capacitative calcium entry is not obligatory for relaxation of the mouse anococcygeus by the NO/cyclic GMP signalling pathway

    PubMed Central

    Ayman, Sinem; Gibson, Alan; McFadzean, Ian; Reynolds, Martyn; Wallace, Pat

    2001-01-01

    The object of this study was to determine whether inhibition of capacitative calcium entry is essential for relaxation of the mouse anococcygeus via the NO/cyclic GMP signalling pathway. In intact muscles, thapsigargin (Tg; 100 nM)-induced tone was relaxed by NO, sodium nitroprusside (SNP), 8-Br-cyclic GMP, and nitrergic field stimulation. The relaxations were similar in magnitude to those observed against carbachol (50 μM) tone and, with the exception of those to 8-Br-cyclic GMP, were reduced by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 μM). In single smooth muscle cells, loaded with Fura-2, both carbachol and Tg produced sustained elevations in cytoplasmic calcium levels ([Ca2+]i). SNP inhibited the rise in [Ca2+]i produced by carbachol, an effect attenuated by ODQ. In contrast, neither SNP nor 8-Br-cyclic GMP reduced the elevated [Ca2+]i associated with Tg. In β-escin skinned preparations, NO had no effect on tone induced by calcium (1 μM in the presence of 100 μM GTP). Carbachol and Tg produced further increases in calcium/GTP-induced tone and, in both cases, this additional tone was relaxed by NO and 8-Br-cyclic GMP. The results support the hypothesis that the NO/cyclic GMP pathway inhibits capacitative calcium entry by refilling the internal stores, since reduction in [Ca2+]i was not observed in the presence of Tg. However, as muscle relaxation was still observed, impairment of capacitative calcium entry cannot be considered obligatory for relaxation. Results from skinned tissues suggest that inhibition of calcium sensitization processes, perhaps associated with store-depletion, may be an important mechanism of NO/cyclic GMP-induced relaxation. PMID:11181421

  14. Cyclic di-GMP-dependent Signaling Pathways in the Pathogenic Firmicute Listeria monocytogenes

    PubMed Central

    Chen, Li-Hong; Köseoğlu, Volkan K.; Güvener, Zehra T.; Myers-Morales, Tanya; Reed, Joseph M.; D'Orazio, Sarah E. F.; Miller, Kurt W.; Gomelsky, Mark

    2014-01-01

    We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence. PMID:25101646

  15. Cyclic di-GMP-dependent signaling pathways in the pathogenic Firmicute Listeria monocytogenes.

    PubMed

    Chen, Li-Hong; Köseoğlu, Volkan K; Güvener, Zehra T; Myers-Morales, Tanya; Reed, Joseph M; D'Orazio, Sarah E F; Miller, Kurt W; Gomelsky, Mark

    2014-08-01

    We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.

  16. Distinct sensory pathways in Vibrio cholerae El Tor and classical biotypes modulate cyclic dimeric GMP levels to control biofilm formation.

    PubMed

    Hammer, Brian K; Bassler, Bonnie L

    2009-01-01

    Quorum sensing (QS), or cell-cell communication in bacteria, is achieved through the production and subsequent response to the accumulation of extracellular signal molecules called autoinducers (AIs). To identify AI-regulated target genes in Vibrio cholerae El Tor (V. cholerae(El)), the strain responsible for the current cholera pandemic, luciferase expression was assayed in an AI(-) strain carrying a random lux transcriptional reporter library in the presence and absence of exogenously added AIs. Twenty-three genes were identified and shown to require the QS transcription factor, HapR, for their regulation. Several of the QS-dependent target genes, annotated as encoding hypothetical proteins, in fact encode HD-GYP proteins, phosphodiesterases that degrade the intracellular second messenger cyclic dimeric GMP (c-di-GMP), which is important for controlling biofilm formation. Indeed, overexpression of a representative QS-activated HD-GYP protein in V. cholerae(El) reduced the intracellular concentration of c-di-GMP, which in turn decreased exopolysaccharide production and biofilm formation. The V. cholerae classical biotype (V. cholerae(Cl)), which caused previous cholera pandemics and is HapR(-), controls c-di-GMP levels and biofilm formation by the VieA signaling pathway. We show that the VieA pathway is dispensable for biofilm formation in V. cholerae(El) but that restoring HapR in V. cholerae(Cl) reestablishes QS-dependent repression of exopolysaccharide production. Thus, different pandemic strains of V. cholerae modulate c-di-GMP levels and control biofilm formation in response to distinct sensory pathways.

  17. Currents carried by monovalent cations through cyclic GMP-activated channels in excised patches from salamander rods.

    PubMed Central

    Menini, A

    1990-01-01

    1. Ionic selectivity and affinity for monovalent cations of channels activated by guanosine 3',5'-cyclic monophosphate (cyclic GMP) were studied in excised inside-out patches of plasma membrane from retinal rods of the tiger salamander. Channels were activated by addition of cyclic GMP to the medium bathing the cytoplasmic side of the membrane. The ionic solution at the cytoplasmic side was rapidly changed using the method of Nunn (1987 a). 2. Permeability ratios were calculated with the Goldman-Hodgkin-Katz potential equation from reversal potential measurements for alkali monovalent cations in bi-ionic conditions. The permeability sequence was: Li+:Na+:K+:Rb+:Cs+ = 1.14:1:0.98:0.84:0.58. 3. The selectivity sequence obtained from macroscopic current measurements in bi-ionic conditions at +100 mV was: Na+:K+:Rb+:Li+:Cs+ = 1:1:0.67:0.36:0.25. 4. The organic cations tetramethylammonium (TMA+), choline and tetraethylammonium (TEA+) were not permeant through the cyclic GMP-activated channels and caused a reduction of the Na+ inward current. At -100 mV the current ratio for inward current was 1:0.75:0.58:0.2 in the presence, at the cytoplasmic side, of 110 mM-Na+, TMA+, choline or TEA+ respectively. 5. The concentration dependence of the macroscopic current and the reversal potential was studied by changing the internal concentration of Na+ or K+ or Li+ from 5 mM to 500 mM. The permeability ratios were nearly constant regardless of the permeant ion concentration. 6. The current as a function of internal ion activity could be described by a Michaelis-Menten relation with a half-saturating activity, Km, at +90 mV equal to 249, 203 and 160 mM for Na+, K+ and Li+ respectively. The ratio of the extrapolated saturating current Imax at +90 mV was 1:0.86:0.26 for Na+, K+ and Li+ respectively. 7. The outward currents and the reversal potentials measured in different mixtures of Na+ and Li+ were monotonic function of the mole fraction. 8. These results can be explained by

  18. Light adaption of the cyclic GMP phosphodiesterase of frog photoreceptor membranes mediated by ATP and calcium ions

    SciTech Connect

    Kawamura, S.; Bownds, M.D.

    1981-05-01

    The light-activated guanosine 3',5'-cyclic monophosphate (cyclic GMP) phosphodiesterase (PDE) of frog photoreceptor membranes has been assayed by measuring the evolution of protons that accompanies cyclic GMP hydrolysis. The validity of this assay has been confirmed by comparison with an isotope assay used in previous studies (Robinson et al. 1980. J. Gen. Physiol. 76: 631-645). The PDE activity elicited by either flash or continuous dim illumination is reduced if ATP is added to outer segment suspensions. This desensitization is most pronounced at low calcium levels. In 10(-9) M Ca/sup + +/, with 0.5 mM ATP and 0.5 mM GTP present, PDE activity remains almost constant as dim illumination and rhodopsin bleaching continue. At intermediate Ca/sup + +/ levels (10-7-10-5M) the activity slowly increases during illumination. Finally, in 10(-4) and PDE activity is more a reflection of the total number of rhodopsin molecules bleached than of the rate of the rhodopsin bleaching. At intermediate or low calcium levels a short-lived inhibitory process is revealed by observing a nonlinear summation of responses of the enzyme to closely spaced flashes of light. Each flash makes PDE activity less responsive to successive flashes, and a steady state is obtained in which activation and inactivation are balanced. It is suggested that calcium and ATP regulation of PDE play a role in the normal light adaption processes of frog photoreceptor membranes.

  19. Hypergravity modulates cyclic GMP efflux in nitric oxide-stimulated human melanocytic cells

    NASA Astrophysics Data System (ADS)

    Stieber, Christiane; Ivannova, Krassimira; Block, Ingrid; Gerzer, Rupert

    2005-08-01

    Gravity alteration is known to influence cell functions. We recently found that hypergravity may stimulate cGMP efflux in human melanocytic cells when cGMP hydrolysis is inhibited. Here we examined whether hypergravity modulates cGMP efflux in nitric oxide (NO)-stimulated melanocytes and melanoma cells (MCs) using NONOates as direct NO donors. In the presence of 0.1 mM DETA-NO and long-term application of hypergravity (up to 5g for 24 h) an elevated cGMP efflux in cultured melanocytes and non-metastatic MCs compared to 1g was observed, whereas short-term exposure was not effective. The hypergravity-stimulated cGMP efflux was inhibited by 1 μM trequinsin, a selective inhibitor of the multidrug resistance proteins 4 and 5 (MRP4/5). The results of the present study indicate that hypergravity may stimulate cGMP efflux in NO- stimulated human melanocytes and non-metastatic MCs most probably by an enhanced expression of MRP4/5. Thus, an altered acceleration vector may induce signaling events in human melanocytic cells.

  20. Expression and distribution of key enzymes of the cyclic GMP signaling in the human clitoris: relation to phosphodiesterase type 5 (PDE5).

    PubMed

    Ückert, S; Oelke, M; Albrecht, K; Breitmeier, D; Kuczyk, M A; Hedlund, P

    2011-01-01

    The clitoris contributes to the normal female sexual response cycle. A significance of cyclic guanosine monophosphate (GMP) has been assumed in the control of clitoral vascular smooth muscle. As only a few investigations on the physiology of the vascular and non-vascular clitoral tissue have been carried out, knowledge on the mechanisms controlling this particular female genital organ is still vague. It has been suggested that human clitoral corpus cavernosum smooth muscle is regulated by nitric oxide (NO)/cyclic GMP and related key enzymes, such as NO synthases (NOSs) and the phosphodiesterase type 5 (PDE5). The present study evaluated in the human clitoris, by means of immunohistochemistry, the expression and distribution of key enzymes of the cyclic GMP pathway, such as the endothelial NOS, PDE2, PDE11 and cyclic GMP-dependent protein kinase type I (cGKI) in relation to the PDE5. Immunohistochemistry revealed the presence of PDE2, PDE5 and cGKI in the smooth muscle wall of blood vessels transversing the supepithelial and stromal space. Immunosignals specific for PDE2 were also identified in interstitial-like cells located in the basal epithelial layer. Staining for PDE11A was observed in single nerve trunks located in the clitoral stroma. The results are in favor of a role of the cyclic GMP signaling in the control of clitoral blood flow. It seems likely that PDE2 and PDE11 are also involved in the mechanism of local (neuro)transmission in the clitoris.

  1. The catalytic subunit of protein kinase A triggers activation of the type V cyclic GMP-specific phosphodiesterase from guinea-pig lung.

    PubMed Central

    Burns, F; Rodger, I W; Pyne, N J

    1992-01-01

    The type V cyclic GMP phosphodiesterase was partially purified from the high-speed supernatant of guinea-pig lung. The isoenzyme displayed linear kinetics for cyclic GMP hydrolysis, with Km = 2.2 +/- 0.2 microM and Vmax. = 1.2 +/- 0.08 nmol/min per mg. The selective type V phosphodiesterase inhibitor Zaprinast inhibited cyclic GMP hydrolysis with IC50 (concn. giving 50% inhibition) = 0.45 +/- 0.08 microM. Isobutylmethylxanthine promoted a 3-fold increase in the binding of cyclic GMP to the isoenzyme. The addition of the catalytic subunit of protein kinase A to an activation cocktail containing the partially purified type V phosphodiesterase resulted in a marked increase in Vmax. for cyclic GMP hydrolysis (approximately 10-fold at 40 units of protein kinase A). We have suggested that protein kinase A triggers phosphorylation of the phosphodiesterase, which results in activation of phosphodiesterase activity. In addition, the sensitivity to inhibition by Zaprinast is severely decreased (the IC50 for inhibition is 7.5 +/- 1.1 microM), suggesting that the potency of phosphodiesterase inhibitors is effected by phosphorylation of the enzyme. PMID:1315515

  2. Evidence that additional mechanisms to cyclic GMP mediate the decrease in intracellular calcium and relaxation of rabbit aortic smooth muscle to nitric oxide

    PubMed Central

    Weisbrod, Robert M; Griswold, Mark C; Yaghoubi, Mohammad; Komalavilas, Padmini; Lincoln, Thomas M; Cohen, Richard A

    1998-01-01

    The role of cyclic GMP in the ability of nitric oxide (NO) to decrease intracellular free calcium concentration [Ca2+]i and divalent cation influx was studied in rabbit aortic smooth muscle cells in primary culture. In cells stimulated with angiotensin II (AII, 10−7 M), NO (10−10–10−6 M) increased cyclic GMP levels measured by radioimmunoassay and decreased [Ca2+]i and cation influx as indicated by fura-2 fluorimetry.Zaprinast (10−4 M), increased NO-stimulated levels of cyclic GMP by 3–20 fold. Although the phosphodiesterase inhibitor lowered the level of [Ca2+]i reached after administration of NO, the initial decreases in [Ca2+]i initiated by NO were not significantly different in magnitude or duration from those that occurred in the absence of zaprinast.The guanylyl cyclase inhibitor, H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (ODQ, 10−5 M), blocked cyclic GMP accumulation and activation of protein kinase G, as measured by back phosphorylation of the inositol trisphosphate receptor. ODQ and Rp-8-Br-cyclic GMPS, a protein kinase G inhibitor, decreased the effects of NO, 10−10–10−8 M, but the decrease in [Ca2+]i or cation influx caused by higher concentrations of NO (10−7–10−6 M) were unaffected. Relaxation of intact rabbit aorta rings to NO (10−7–10−5 M) also persisted in the presence of ODQ without a significant increase in cyclic GMP. Rp-8-Br-cyclic GMPS blocked the decreases in cation influx caused by a cell permeable cyclic GMP analog, but ODQ and/or the protein kinase G inhibitor had no significant effect on the decrease caused by NO.Although inhibitors of cyclic GMP, protein kinase G and phosphodiesterase can be shown to affect the decrease in [Ca2+]i and cation influx via protein kinase G, these studies indicate that when these mechanisms are blocked, cyclic GMP-independent mechanisms also contribute significantly to the decrease in [Ca2+]i and smooth muscle relaxation to NO. PMID:9886761

  3. A label-free and self-assembled electrochemical biosensor for highly sensitive detection of cyclic diguanylate monophosphate (c-di-GMP) based on RNA riboswitch.

    PubMed

    Xie, Qingyun; Zhao, Fulin; Liu, Hongrui; Shan, Yanke; Liu, Fei

    2015-07-02

    Cyclic diguanylate monophosphate (c-di-GMP) is an important second messenger that regulates a variety of complex physiological processes involved in motility, virulence, biofilm formation and cell cycle progression in several bacteria. Herein we report a simple label-free and self-assembled RNA riboswitch-based biosensor for sensitive and selective detection of c-di-GMP. The detectable concentration range of c-di-GMP is from 50 nM to 1 μM with a detection limit of 50 nM. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.

    PubMed

    Zhang, Xu; Shi, Heping; Wu, Jiaxi; Zhang, Xuewu; Sun, Lijun; Chen, Chuo; Chen, Zhijian J

    2013-07-25

    The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation.

  5. In vivo microdialysis study of a specific inhibitor of soluble guanylyl cyclase on the glutamate receptor/nitric oxide/cyclic GMP pathway.

    PubMed Central

    Fedele, E.; Jin, Y.; Varnier, G.; Raiteri, M.

    1996-01-01

    1. Nitric oxide (NO) is known to stimulate soluble guanylyl cyclase, thereby eliciting an elevation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) in target cells. Recently, a selective inhibitor of soluble guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), has been identified and characterized in vitro. We have investigated the in vivo effects of ODQ on the glutamate receptor/NO/ cyclic GMP pathway by monitoring extracellular cyclic GMP during microdialysis of the cerebellum or the hippocampus of freely-moving adult rats. 2. Intracerebellar administration of ODQ (1-100 microM) via the microdialysis probe inhibited, in a concentration-dependent manner, the basal extracellular level of cyclic GMP. The maximal inhibition, measured after a 20 min perfusion with 100 microM ODQ, amounted to 80% and persisted unchanged as long as ODQ was perfused. When ODQ was removed from the perfusion stream after 20 min, the levels of cyclic GMP started to recover, suggesting reversibility of guanylyl cyclase inhibition by ODQ. 3. The cyclic GMP response evoked in the cerebellum by NMDA (200 microM) or by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 100 microM) was largely attenuated by 100 microM ODQ. The pattern of the inhibition curves suggests competition for guanylyl cyclase between ODQ and the NO generated by NMDA or AMPA receptor activation. 4. ODQ (100 microM) prevented the elevation of extracellular cyclic GMP levels provoked by intracerebellar infusion of the NO generator S-nitroso-N-acetylpenicillamine (SNAP; 1 mM). The inhibition of the SNAP effect was rapidly relieved when ODQ was removed from the perfusion fluid. However, ODQ (100 microM) was unable to affect the cyclic GMP response elicited by 5 mM SNAP, in keeping with the proposed idea that ODQ binds to the "NO receptor' in a reversible and competitive manner. 5. Infusion of ODQ (10, 100 or 300 microM) into the hippocampus of freely-moving rats diminished the basal extracellular

  6. Effect of Ca2+, cyclic GMP, and cyclic AMP added to artificial solution perfusing lingual artery on frog gustatory nerve responses

    PubMed Central

    1982-01-01

    The lingual artery of the bullfrog was perfused with artificial solution and the effects of Ca2+, Ca-channel blockers (MnCl2 and verapamil), cGMP, and cAMP added to the perfusing solution of the gustatory nerve responses were examined. The responses to chemical stimuli of group 1 (CaCl2, NaCl, distilled water, D-galactose, and L- threonine) applied to the tongue surface were greatly decreased by a decrease in Ca2+ concentration in the perfusing solution, suppressed by the Ca-channel blockers, enhanced by cGMP, and suppressed by cAMP. The responses to chemical stimuli of group 2 (quinine hydrochloride, theophylline, ethanol, and HCl) were practically not affected by a decrease in Ca2+ concentration, the Ca-channel blockers, cGMP, and cAMP. The responses to the stimuli of group 1 seem to be induced by Ca influx into a taste cell that is triggered by depolarization and modulated by the cyclic nucleotides in a taste cell. The responses to group 2 seem to be induced without accompanying Ca influx. PMID:6294223

  7. 8-Mercapto-Cyclic GMP Mediates Hydrogen Sulfide-Induced Stomatal Closure in Arabidopsis.

    PubMed

    Honda, Kenji; Yamada, Naotake; Yoshida, Riichiro; Ihara, Hideshi; Sawa, Tomohiro; Akaike, Takaaki; Iwai, Sumio

    2015-08-01

    Plants are exposed to hydrogen sulfide (H2S) both exogenously, as it exists as a pollutant gas in the environment, and endogenously, as it is synthesized in cells. H2S has recently been found to function as a gaseous signaling molecule, but its signaling cascade remains unknown. Here, we examined H2S-mediated guard cell signaling in Arabidopsis. The H2S donor GYY4137 (morpholin-4-ium-4-methoxyphenyl [morpholino] phosphinodithioate) induced stomatal closure, which peaked after 150 min at 1 µM or after 90 min at 10 and 100 µM. After reaching maximal closure, stomatal apertures gradually increased in size in response to further exposure to GYY4137. GYY4137 induced nitric oxide (NO) generation in guard cells, and GYY4137-induced stomatal closure was reduced by an NO scavenger and inhibitors of NO-producing enzymes. Mass spectrometry analyses showed that GYY4137 induces the synthesis of 8-nitro-cGMP and 8-mercapto-cGMP and that this synthesis is mediated by NO. In addition, 8-mercapto-cGMP triggered stomatal closure. Moreover, inhibitor and genetic studies showed that calcium, cADP ribose and slow anion channel 1 act downstream of 8-mercapto-cGMP. This study therefore demonstrates that 8-mercapto-cGMP mediates the H2S signaling cascade in guard cells.

  8. IP3 Mediates Nitric Oxide-Guanosine 3',5'-Cyclic Monophosphate (NO-cGMP)-Induced Isoflavone Accumulation in Soybean Sprouts under UV-B Radiation.

    PubMed

    Jiao, Caifeng; Wang, Pei; Yang, Runqiang; Tian, Lu; Gu, Zhenxin

    2016-11-09

    In this study, to investigate the role of inositol 1,4,5-trisphosphate (IP3) in nitric oxide-guanosine 3',5'-cyclic monophosphate (NO-cGMP)-induced isoflavone accumulation in soybean sprouts under UV-B radiation, the sprouts were treated with donors and inhibitors of NO and cGMP as well as IP3 inhibitor. Results showed that NO, with cGMP as a second messenger, stimulates IP3 accumulation under UV-B radiation. Consistent with the increase in IP3 content, the up-regulation of gene and protein expression of phosphoinositide-specific phospholipase C (PI-PLC) in response to sodium nitroprusside (SNP) (exogenous NO donor) and 8-Br-cGMP (cGMP analogue) was also observed. In addition, protein kinase G (PKG) participated in NO-cGMP-induced IP3 production. IP3 induced by the NO-cGMP pathway was involved in isoflavone synthesis by elevating the activity and gene and protein expressions of chalcone synthase (CHS) and isoflavone synthase (IFS). Overall, IP3 mediates NO-cGMP-induced isoflavone accumulation in soybean sprouts under UV-B stress.

  9. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

    PubMed

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J

    2013-02-15

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-β in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-β induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

  10. Memantine inhibits serotonin-induced rise of cytosolic Ca2+ activity and of cyclic GMP level in a neuronal cell line.

    PubMed

    Reiser, G; Koch, R

    1989-05-11

    Serotonin (5-HT) evoked a rise of cytosolic Ca2+ activity in neuroblastoma X glioma hybrid cells, most probably due to the entry of extracellular Ca2+; cyclic GMP synthesis was also stimulated. The rise of both cytosolic Ca2+ activity and of cyclic GMP level was blocked by memantine (1-amino-3,5-dimethyladamantane). Memantine inhibited the rise of the cyclic GMP level non-competitively (Ki about 50 microM). Thus, memantine suppresses the effects of 5-HT in the neuronal cell line, most likely by blocking Ca2+-permeable ion channels. This interpretation is in line with the previously reported finding that memantine suppressed the 5-HT-induced depolarizing response in the same cell line.

  11. Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP)

    PubMed Central

    Hallberg, Zachary F.; Wang, Xin C.; Wright, Todd A.; Nan, Beiyan; Ad, Omer; Yeo, Jongchan; Hammond, Ming C.

    2016-01-01

    Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling. PMID:26839412

  12. Multigene Family Encoding 3′,5′-Cyclic-GMP-Dependent Protein Kinases in Paramecium tetraurelia Cells

    PubMed Central

    Kissmehl, Roland; Krüger, Tim P.; Treptau, Tilman; Froissard, Marine; Plattner, Helmut

    2006-01-01

    In the ciliate Paramecium tetraurelia, 3′,5′-cyclic GMP (cGMP) is one of the second messengers involved in several signal transduction pathways. The enzymes for its production and degradation are well established for these cells, whereas less is known about the potential effector proteins. On the basis of a current Paramecium genome project, we have identified a multigene family with at least 35 members, all of which encode cGMP-dependent protein kinases (PKGs). They can be classified into 16 subfamilies with several members each. Two of the genes, PKG1-1 and PKG2-1, were analyzed in more detail after molecular cloning. They encode monomeric enzymes of 770 and 819 amino acids, respectively, whose overall domain organization resembles that in higher eukaryotes. The enzymes contain a regulatory domain of two tandem cyclic nucleotide-binding sites flanked by an amino-terminal region for intracellular localization and a catalytic domain with highly conserved regions for ATP binding and catalysis. However, some Paramecium PKGs show a different structure. In Western blots, PKGs are detected both as cytosolic and as structure-bound forms. Immunofluorescence labeling shows enrichment in the cell cortex, notably around the dense-core secretory vesicles (trichocysts), as well as in cilia. Immunogold electron microscopy analysis reveals consistent labeling of ciliary membranes, of the membrane complex composed of cell membrane and cortical Ca2+ stores, and of regions adjacent to ciliary basal bodies, trichocysts, and trafficking vesicles. Since PKGs (re)phosphorylate the exocytosis-sensitive phosphoprotein pp63/pf upon stimulation, the role of PKGs during stimulated exocytosis is discussed, in addition to a role in ciliary beat regulation. PMID:16400170

  13. Control of Formation and Cellular Detachment from Shewanella oneidensis MR-1 Biofilms by Cyclic di-GMP

    SciTech Connect

    Thormann, Kai M.; Duttler, Stefanie; Saville, Renee; Hyodo, Mamoru; Shukla, Soni; Hayakawa, Yoshihiro; Spormann, Alfred M.

    2006-04-01

    Stability and resilience against environmental perturbations are critical properties of medical and environmental biofilms and pose important targets for their control. Biofilm stability is determined by two mutually exclusive processes: attachment of cells to and detachment from the biofilm matrix. Using Shewanella oneidensis MR-1, an environmentally versatile, Fe(III) and Mn(IV) mineral -reducing microorganism, we identified mxdABCD as a new set of genes essential for formation of a three-dimensional biofilm. Molecular analysis revealed that mxdA encodes a cyclic bis(3',5')guanylic acid (cyclic di-GMP)-forming enzyme with an unusual GGDEF motif, i.e., NVDEF, which is essential for its function. mxdB encodes a putative membrane-associated glycosyl transferase. Both genes are essential for matrix attachment. The attachment-deficient phenotype of a Delta mxdA mutant was rescued by ectopic expression of VCA0956, encoding another diguanylate cyclase. Interestingly, a rapid cellular detachment from the biofilm occurred upon induction of yhjH, a gene encoding an enzyme that has been shown to have phosphodiesterase activity. In this way, it was possible to bypass the previously identified sudden depletion of molecular oxygen as an environmental trigger to induce biofilm dissolution. We propose a model for c-di-GMP as a key intracellular regulator for controlling biofilm stability by shifting the state of a biofilm cell between attachment and detachment in a concentration-dependent manner.

  14. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.

    PubMed

    Orr, Mona W; Donaldson, Gregory P; Severin, Geoffrey B; Wang, Jingxin; Sintim, Herman O; Waters, Christopher M; Lee, Vincent T

    2015-09-08

    The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A from Pseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆orn strains of P. aeruginosa PA14 for pGpG stability. The lysates from ∆orn showed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆orn strain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP-regulated pel promoter. Additionally, the c-di-GMP-governed auto-aggregation and biofilm phenotypes were elevated in the ∆orn strain in a pel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆orn strain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.

  15. Cyclic di-GMP Modulates Gene Expression in Lyme Disease Spirochetes at the Tick-Mammal Interface To Promote Spirochete Survival during the Blood Meal and Tick-to-Mammal Transmission

    PubMed Central

    Dunham-Ems, Star; Allard, Anna M.; Cassera, Maria B.; Kenedy, Melisha; Radolf, Justin D.

    2015-01-01

    Borrelia burgdorferi, the Lyme disease spirochete, couples environmental sensing and gene regulation primarily via the Hk1/Rrp1 two-component system (TCS) and Rrp2/RpoN/RpoS pathways. Beginning with acquisition, we reevaluated the contribution of these pathways to spirochete survival and gene regulation throughout the enzootic cycle. Live imaging of B. burgdorferi caught in the act of being acquired revealed that the absence of RpoS and the consequent derepression of tick-phase genes impart a Stay signal required for midgut colonization. In addition to the behavioral changes brought on by the RpoS-off state, acquisition requires activation of cyclic di-GMP (c-di-GMP) synthesis by the Hk1/Rrp1 TCS; B. burgdorferi lacking either component is destroyed during the blood meal. Prior studies attributed this dramatic phenotype to a metabolic lesion stemming from reduced glycerol uptake and utilization. In a head-to-head comparison, however, the B. burgdorferi Δglp mutant had a markedly greater capacity to survive tick feeding than B. burgdorferi Δhk1 or Δrrp1 mutants, establishing unequivocally that glycerol metabolism is only one component of the protection afforded by c-di-GMP. Data presented herein suggest that the protective response mediated by c-di-GMP is multifactorial, involving chemotactic responses, utilization of alternate substrates for energy generation and intermediary metabolism, and remodeling of the cell envelope as a means of defending spirochetes against threats engendered during the blood meal. Expression profiling of c-di-GMP-regulated genes through the enzootic cycle supports our contention that the Hk1/Rrp1 TCS functions primarily, if not exclusively, in ticks. These data also raise the possibility that c-di-GMP enhances the expression of a subset of RpoS-dependent genes during nymphal transmission. PMID:25987708

  16. Systems Pharmacology and Rational Polypharmacy: Nitric Oxide−Cyclic GMP Signaling Pathway as an Illustrative Example and Derivation of the General Case

    PubMed Central

    Garmaroudi, Farshid S.; Handy, Diane E.; Liu, Yang-Yu; Loscalzo, Joseph

    2016-01-01

    Impaired nitric oxide (NO˙)-cyclic guanosine 3', 5'-monophosphate (cGMP) signaling has been observed in many cardiovascular disorders, including heart failure and pulmonary arterial hypertension. There are several enzymatic determinants of cGMP levels in this pathway, including soluble guanylyl cyclase (sGC) itself, the NO˙-activated form of sGC, and phosphodiesterase(s) (PDE). Therapies for some of these disorders with PDE inhibitors have been successful at increasing cGMP levels in both cardiac and vascular tissues. However, at the systems level, it is not clear whether perturbation of PDE alone, under oxidative stress, is the best approach for increasing cGMP levels as compared with perturbation of other potential pathway targets, either alone or in combination. Here, we develop a model-based approach to perturbing this pathway, focusing on single reactions, pairs of reactions, or trios of reactions as targets, then monitoring the theoretical effects of these interventions on cGMP levels. Single perturbations of all reaction steps within this pathway demonstrated that three reaction steps, including the oxidation of sGC, NO˙ dissociation from sGC, and cGMP degradation by PDE, exerted a dominant influence on cGMP accumulation relative to other reaction steps. Furthermore, among all possible single, paired, and triple perturbations of this pathway, the combined perturbations of these three reaction steps had the greatest impact on cGMP accumulation. These computational findings were confirmed in cell-based experiments. We conclude that a combined perturbation of the oxidatively-impaired NO˙-cGMP signaling pathway is a better approach to the restoration of cGMP levels as compared with corresponding individual perturbations. This approach may also yield improved therapeutic responses in other complex pharmacologically amenable pathways. PMID:26985825

  17. Comprehensive overexpression analysis of cyclic-di-GMP signalling proteins in the phytopathogen Pectobacterium atrosepticum reveals diverse effects on motility and virulence phenotypes.

    PubMed

    Tan, H; West, J A; Ramsay, J P; Monson, R E; Griffin, J L; Toth, I K; Salmond, G P C

    2014-07-01

    Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous bacterial signalling molecule produced by diguanylate cyclases of the GGDEF-domain family. Elevated c-di-GMP levels or increased GGDEF protein expression is frequently associated with the onset of sessility and biofilm formation in numerous bacterial species. Conversely, phosphodiesterase-dependent diminution of c-di-GMP levels by EAL- and HD-GYP-domain proteins is often accompanied by increased motility and virulence. In this study, we individually overexpressed 23 predicted GGDEF, EAL or HD-GYP-domain proteins encoded by the phytopathogen Pectobacterium atrosepticum strain SCRI1043. MS-based detection of c-di-GMP and 5'-phosphoguanylyl-(3'-5')-guanosine in these strains revealed that overexpression of most genes promoted modest 1-10-fold changes in cellular levels of c-di-GMP, with the exception of the GGDEF-domain proteins ECA0659 and ECA3374, which induced 1290- and 7660-fold increases, respectively. Overexpression of most EAL domain proteins increased motility, while overexpression of most GGDEF domain proteins reduced motility and increased poly-β-1,6-N-acetyl-glucosamine-dependent flocculation. In contrast to domain-based predictions, overexpression of the EAL protein ECA3549 or the HD-GYP protein ECA3548 increased c-di-GMP concentrations and reduced motility. Most overexpression constructs altered the levels of secreted cellulases, pectinases and proteases, confirming c-di-GMP regulation of virulence in Pe. atrosepticum. However, there was no apparent correlation between virulence-factor induction and the domain class expressed or cellular c-di-GMP levels, suggesting that regulation was in response to specific effectors within the network, rather than total c-di-GMP concentration. Finally, we demonstrated that the cellular localization patterns vary considerably for GGDEF/EAL/HD-GYP proteins, indicating it is a likely factor restricting specific interactions within the c-di-GMP

  18. Cyclic Di-GMP Phosphodiesterases RmdA and RmdB Are Involved in Regulating Colony Morphology and Development in Streptomyces coelicolor

    PubMed Central

    Hull, Travis D.; Ryu, Min-Hyung; Sullivan, Matthew J.; Johnson, Ryan C.; Klena, Nikolai T.; Geiger, Robert M.; Gomelsky, Mark

    2012-01-01

    Cyclic dimeric GMP (c-di-GMP) regulates numerous processes in Gram-negative bacteria, yet little is known about its role in Gram-positive bacteria. Here we characterize two c-di-GMP phosphodiesterases from the filamentous high-GC Gram-positive actinobacterium Streptomyces coelicolor, involved in controlling colony morphology and development. A transposon mutation in one of the two phosphodiesterase genes, SCO0928, hereby designated rmdA (regulator of morphology and development A), resulted in decreased levels of spore-specific gray pigment and a delay in spore formation. The RmdA protein contains GGDEF-EAL domains arranged in tandem and possesses c-di-GMP phosphodiesterase activity, as is evident from in vitro enzymatic assays using the purified protein. RmdA contains a PAS9 domain and is a hemoprotein. Inactivation of another GGDEF-EAL-encoding gene, SCO5495, designated rmdB, resulted in a phenotype identical to that of the rmdA mutant. Purified soluble fragment of RmdB devoid of transmembrane domains also possesses c-di-GMP phosphodiesterase activity. The rmdA rmdB double mutant has a bald phenotype and is impaired in aerial mycelium formation. This suggests that RmdA and RmdB functions are additive and at least partially overlapping. The rmdA and rmdB mutations likely result in increased local pools of intracellular c-di-GMP, because intracellular c-di-GMP levels in the single mutants did not differ significantly from those of the wild type, whereas in the double rmdA rmdB mutant, c-di-GMP levels were 3-fold higher than those in the wild type. This study highlights the importance of c-di-GMP-dependent signaling in actinomycete colony morphology and development and identifies two c-di-GMP phosphodiesterases controlling these processes. PMID:22753061

  19. Cyclic nucleotide phosphodiesterase-mediated integration of cGMP and cAMP signaling in cells of the cardiovascular system.

    PubMed

    Maurice, Donald H

    2005-05-01

    Numerous pharmacological and physiological agents acting via either cAMP- or cGMP-mediated impact the activities of cells of the cardiovascular system. While most define cAMP and cGMP signaling systems as separate and independent, recent advances in our understanding of cyclic nucleotide signaling, and more specifically, of the roles which cyclic nucleotide phosphodiesterases (PDEs) play in these events, have altered this view. In this short chapter, I will review the data identifying expression of several PDEs in cells of the cardiovascular system. In addition, I will review the data that identify PDEs as enzymes capable of allowing integration between cAMP and cGMP signaling in cells, and propose that cAMP and cGMP signaling systems can represent parallel and interdependent signaling systems. Moreover, I will propose that cGMP-mediated effects on the activities of variants of the Phosphodiesterase 2 (PDE2), PDE3 and PDE5 families may act to coordinate linkage between cAMP and cGMP signaling in these cells.

  20. Integration of Cyclic di-GMP and Quorum Sensing in the Control of vpsT and aphA in Vibrio cholerae ▿ †

    PubMed Central

    Srivastava, Disha; Harris, Rebecca C.; Waters, Christopher M.

    2011-01-01

    Vibrio cholerae transitions between aquatic environmental reservoirs and infection in the gastrointestinal tracts of human hosts. The second-messenger molecule cyclic di-GMP (c-di-GMP) and quorum sensing (QS) are important signaling systems that enable V. cholerae to alternate between these distinct environments by controlling biofilm formation and virulence factor expression. Here we identify a conserved regulatory mechanism in V. cholerae that integrates c-di-GMP and QS to control the expression of two transcriptional regulators: aphA, an activator of virulence gene expression and an important regulator of the quorum-sensing pathway, and vpsT, a transcriptional activator that induces biofilm formation. Surprisingly, aphA expression was induced by c-di-GMP. Activation of both aphA and vpsT by c-di-GMP requires the transcriptional activator VpsR, which binds to c-di-GMP. The VpsR binding site at each of these promoters overlaps with the binding site of HapR, the master QS regulator at high cell densities. Our results suggest that V. cholerae combines information conveyed by QS and c-di-GMP to appropriately respond and adapt to divergent environments by modulating the expression of key transcriptional regulators. PMID:21926235

  1. c-di-GMP enhances protective innate immunity in a murine model of pertussis.

    PubMed

    Elahi, Shokrollah; Van Kessel, Jill; Kiros, Tedele G; Strom, Stacy; Hayakawa, Yoshihiro; Hyodo, Mamoru; Babiuk, Lorne A; Gerdts, Volker

    2014-01-01

    Innate immunity represents the first line of defense against invading pathogens in the respiratory tract. Innate immune cells such as monocytes, macrophages, dendritic cells, NK cells, and granulocytes contain specific pathogen-recognition molecules which induce the production of cytokines and subsequently activate the adaptive immune response. c-di-GMP is a ubiquitous second messenger that stimulates innate immunity and regulates biofilm formation, motility and virulence in a diverse range of bacterial species with potent immunomodulatory properties. In the present study, c-di-GMP was used to enhance the innate immune response against pertussis, a respiratory infection mainly caused by Bordetella pertussis. Intranasal treatment with c-di-GMP resulted in the induction of robust innate immune responses to infection with B. pertussis characterized by enhanced recruitment of neutrophils, macrophages, natural killer cells and dendritic cells. The immune responses were associated with an earlier and more vigorous expression of Th1-type cytokines, as well as an increase in the induction of nitric oxide in the lungs of treated animals, resulting in significant reduction of bacterial numbers in the lungs of infected mice. These results demonstrate that c-di-GMP is a potent innate immune stimulatory molecule that can be used to enhance protection against bacterial respiratory infections. In addition, our data suggest that priming of the innate immune system by c-di-GMP could further skew the immune response towards a Th1 type phenotype during subsequent infection. Thus, our data suggest that c-di-GMP might be useful as an adjuvant for the next generation of acellular pertussis vaccine to mount a more protective Th1 phenotype immune response, and also in other systems where a Th1 type immune response is required.

  2. Role of soluble guanylyl cyclase-cyclic GMP signaling in tumor cell proliferation

    PubMed Central

    Mujoo, Kalpana; Sharin, Vladislav G.; Martin, Emil; Choi, Byung-Kwon; Sloan, Courtney; Nikonoff, Lubov E.; Kots, Alexander Y; Murad, Ferid

    2010-01-01

    Our previous studies demonstrate a differential expression of nitric oxide (NO) signaling components in ES cells and our recent study demonstrated an enhanced differentiation of ES cells into myocardial cells with NO donors and soluble guanylyl cyclase (sGC) activators. Since NO-cGMP pathway exhibits a diverse role in cancer, we were interested in evaluating the role of the NO receptor sGC and other components of the pathway in regulation of the tumor cell proliferation. Our results demonstrate a differential expression of the sGC subunits, NOS-1 and PKG mRNA and protein levels in various human cancer models. In contrast to sGCα1, robust levels of sGC β1 were observed in OVCAR-3 (ovarian) and MDA-MB-468 (breast) cancer cells which correlated well with the sGC activity and a marked increase in cGMP levels upon exposure to the combination of a NO donor and a sGC activator. NOC-18 (DETA NONOate; NO donor), BAY41-2272 (3-(4-Amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine); sGC activator), NOC-18+BAY41-2272, IBMX (3-Isobutyl-1-methylxanthine; phosphodiesterase inhibitor) and 8-bromo-cGMP (cGMP analog) caused growth inhibition and apoptosis in various cancer cell lines. To elucidate the molecular mechanisms involved in growth inhibition, we evaluated the effect of activators/inhibitors on ERK phosphorylation. Our studies indicate that BAY41-2272 or the combination NOC18+BAY41-2272 caused inhibition of the basal ERK1/2 phosphorylation in OVCAR-3 (high sGC activity), SK-OV-3 and SK-Br-3 (low sGC activity) cell lines and in some cases the inhibition was rescued by the sGC inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). These studies suggest that the effects of activators/inhibitors of NO-sGC-cGMP in tumor cell proliferation is mediated by both cGMP-dependent and independent mechanisms. PMID:19948239

  3. YfiBNR mediates cyclic di-GMP dependent small colony variant formation and persistence in Pseudomonas aeruginosa.

    PubMed

    Malone, Jacob G; Jaeger, Tina; Spangler, Christian; Ritz, Daniel; Spang, Anne; Arrieumerlou, Cécile; Kaever, Volkhard; Landmann, Regine; Jenal, Urs

    2010-03-12

    During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important

  4. Low-power laser irradiation of blood inhibits platelet function: role of cyclic GMP

    NASA Astrophysics Data System (ADS)

    Brill, Alexander G.; Brill, Gregory E.; Shenkman, Boris; Tamarin, Ilya; Dardik, Rima; Varon, David; Savion, Naphtali

    1998-12-01

    The aim of the present work was to investigate effect of low power laser irradiation (LPLI) on platelet function in vitro. He-Ne laser (Optronix, USA; (lambda) - 632.8 nm, output power - 7 mW) was employed. Platelet adhesion and aggregation in whole blood (WB) under defined shear conditions were assayed by a Cone and Plate(let) Analyzer. Platelet activation was evaluated by flow cytometry. Level of platelet cGMP was estimated by immunoenzyme assay. Experiments performed showed that LPLI of WB resulted in decrease of platelet deposition on extracellular matrix at high shear rate (1300 s-1). Similar results were obtained using surfaces precoated with either collagen type I or von Willebrand factor. LPLI inhibited fibrinogen binding as well as P-selectin expression on the platelet membrane, induced by thrombin analogue. It was found out that primary acceptor of laser energy responsible for the effect on platelets was located in platelets themselves and not in blood plasma or in other blood cells. LPLI of gel- filtered platelets resulted in increase of intracellular level of cGMP both in the absence and in presence of izobutylmethylxantine (phosphodiesterase inhibitor) suggesting stimulation of synthesis rather than destruction of cGMP under the influence of LPLI. It is suggested that guanylate cyclase and/or NO-synthase might serve as primary acceptors of He-Ne laser light in platelets.

  5. Cyclic GMP-dependent protein kinase and soluble guanylyl cyclase disappear in elicited rat neutrophils.

    PubMed

    Ciuman, Małgorzata; Siednienko, Jakub; Czyzyk, Rafał; Witwicka, Hanna; Kołosionek, Ewa; Kobiałka, Marcin; Gorczyca, Wojciech A

    2006-11-01

    The nitric oxide/soluble guanylyl cyclase/cGMP-dependent protein kinase (NO/sGC/PKG) cascade has been shown to affect important functions of circulating neutrophils. We demonstrate that neutrophils isolated from rats treated intraperitoneally with peptone protease cannot use this signaling pathway. Although PKG was detected at both the mRNA and protein levels in peripheral blood neutrophils (PBNs) of control rats, it was expressed neither in PBNs nor in peritoneal exudate neutrophils (PENs) of provoked rats. Also, mRNA of the alpha and beta chains of heterodimeric sGC was present in PBNs, but absent in PENs. Consistently, PBNs responded to activators of sGC with cGMP synthesis, while PENs did not. These results showed that neutrophils recruited by a provoking agent lost PKG and, in the case of PENs, also sGC and thus the capacity to respond to NO with cGMP signaling. We speculate that such downregulation of the sGC/PKG pathway is likely a result of the high activity of inducible NO synthase observed in inflammatory neutrophils.

  6. The Xanthomonas oryzae pv. oryzae PilZ Domain Proteins Function Differentially in Cyclic di-GMP Binding and Regulation of Virulence and Motility.

    PubMed

    Yang, Fenghuan; Tian, Fang; Chen, Huamin; Hutchins, William; Yang, Ching-Hong; He, Chenyang

    2015-07-01

    The PilZ domain proteins have been demonstrated to be one of the major types of receptors mediating cyclic di-GMP (c-di-GMP) signaling pathways in several pathogenic bacteria. However, little is known about the function of PilZ domain proteins in c-di-GMP regulation of virulence in the bacterial blight pathogen of rice Xanthomonas oryzae pv. oryzae. Here, the roles of PilZ domain proteins PXO_00049 and PXO_02374 in c-di-GMP binding, regulation of virulence and motility, and subcellular localization were characterized in comparison with PXO_02715, identified previously as an interactor with the c-di-GMP receptor Filp to regulate virulence. The c-di-GMP binding motifs in the PilZ domains were conserved in PXO_00049 and PXO_02374 but were less well conserved in PXO_02715. PXO_00049 and PXO_02374 but not PXO_02715 proteins bound to c-di-GMP with high affinity in vitro, and the R(141) and R(10) residues in the PilZ domains of PXO_00049 and PXO_02374, respectively, were crucial for c-di-GMP binding. Gene deletion of PXO_00049 and PXO_02374 resulted in significant increases in virulence and hrp gene transcription, indicating their negative regulation of virulence via type III secretion system expression. All mutants showed significant changes in sliding motility but not exopolysaccharide production and biofilm formation. In trans expression of the full-length open reading frame (ORF) of each gene in the relevant mutants led to restoration of the phenotype to wild-type levels. Moreover, PXO_00049 and PXO_02374 displayed mainly multisite subcellular localizations, whereas PXO_02715 showed nonpolar distributions in the X. oryzae pv. oryzae cells. Therefore, this study demonstrated the different functions of the PilZ domain proteins in mediation of c-di-GMP regulation of virulence and motility in X. oryzae pv. oryzae.

  7. Rrp1, a cyclic-di-GMP-producing response regulator, is an important regulator of Borrelia burgdorferi core cellular functions

    PubMed Central

    Rogers, Elizabeth A.; Terekhova, Darya; Zhang, Hong-Ming; Hovis, Kelley M.; Schwartz, Ira; Marconi, Richard T.

    2010-01-01

    Summary Two-component systems (TCS) are universal among bacteria and play critical roles in gene regulation. Our understanding of the contributions of TCS in the biology of the Borrelia is just now beginning to develop. Borrelia burgdorferi, a causative agent of Lyme disease, harbours a TCS comprised of open reading frames (ORFs) BB0419 and BB0420. BB0419 encodes a response regulator designated Rrp1, and BB0420 encodes a hybrid histidine kinase–response regulator designated Hpk1. Rrp1, which contains a conserved GGDEF domain, undergoes phosphorylation and produces the secondary messenger, cyclic diguanylate (c-di-GMP), a critical signaling molecule in numerous organisms. However, the regulatory role of the Rrp1–Hpk1 TCS and c-di-GMP signaling in Borrelia biology are unexplored. In this study, the distribution, conservation, expression and potential global regulatory capability of Rrp1 were assessed. rrp1 was found to be universal and highly conserved among isolates, co-transcribed with hpk1, constitutively expressed during in vitro cultivation, and significantly upregulated upon tick feeding. Allelic exchange replacement and microarray analyses revealed that the Rrp1 regulon consists of a large number of genes encoded by the core Borrelia genome (linear chromosome, linear plasmid 54 and circular plasmid 26) that encode for proteins involved in central metabolic processes and virulence mechanisms including immune evasion. PMID:19210621

  8. Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING

    PubMed Central

    Shi, Heping; Wu, Jiaxi; Chen, Zhijian J.; Chen, Chuo

    2015-01-01

    Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2′3′-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2′3′-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2′3′-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein–ligand interactions. PMID:26150511

  9. Aerobic granulation of aggregating consortium X9 isolated from aerobic granules and role of cyclic di-GMP.

    PubMed

    Wan, Chunli; Yang, Xue; Lee, Duu-Jong; Wang, Xin-Yue; Yang, Qiaoli; Pan, Xiangliang

    2014-01-01

    This study monitored the granulation process of an aggregating functional consortium X9 that was consisted of Pseudomonas putida X-1, Acinetobacter sp. X-2, Alcaligenes sp. X-3 and Comamonas testosteroni X-4 in shaken reactors. The growth curve of X9 was fit using logistic model as follows y=1.49/(1+21.3*exp(-0.33x)), the maximum specific cell growth rate for X9 was 0.33 h(-1). Initially X9 consumed polysaccharides (PS) and secreted proteins (PN) to trigger granulation. Then X9 grew in biomass and formed numerous micro-granules, driven by increasing hydrophobicity of cell membranes and of accumulated extracellular polymeric substances (EPS). In later stage the intracellular cyclic diguanylate (c-di-GMP) was at high levels for inhibiting bacteria swarming motility, thereby promotion formation of large aerobic granules. The findings reported herein advise the way to accelerate granule formation and to stabilize operation in aerobic granular reactors.

  10. Cyclic GMP-AMP Synthase is a Cytosolic DNA Sensor that Activates the Type-I Interferon Pathway

    PubMed Central

    Sun, Lijun; Wu, Jiaxi; Du, Fenghe; Chen, Xiang; Chen, Zhijian J.

    2013-01-01

    The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers the host immune responses such as the production of type-I interferons (IFN). Cytosolic DNA induces IFN through the production of cyclic-GMP-AMP (cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced IFNβ in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and IFNβ induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP. PMID:23258413

  11. Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING

    DOE PAGES

    Shi, Heping; Wu, Jiaxi; Chen, Zhijian J.; ...

    2015-07-06

    Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2'3'-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2'3'-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. In this paper, we show that 2'3'-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Finally, our results demonstrate that analysesmore » of free-ligand conformations can be as important as analyses of protein conformations in understanding protein–ligand interactions.« less

  12. Molecular basis for the specific recognition of the metazoan cyclic GMP-AMP by the innate immune adaptor protein STING

    SciTech Connect

    Shi, Heping; Wu, Jiaxi; Chen, Zhijian J.; Chen, Chuo

    2015-07-06

    Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2'3'-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2'3'-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. In this paper, we show that 2'3'-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Finally, our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein–ligand interactions.

  13. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover

    PubMed Central

    Orr, Mona W.; Donaldson, Gregory P.; Severin, Geoffrey B.; Wang, Jingxin; Sintim, Herman O.; Waters, Christopher M.; Lee, Vincent T.

    2015-01-01

    The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A from Pseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆orn strains of P. aeruginosa PA14 for pGpG stability. The lysates from ∆orn showed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆orn strain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP–regulated pel promoter. Additionally, the c-di-GMP–governed auto-aggregation and biofilm phenotypes were elevated in the ∆orn strain in a pel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆orn strain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway. PMID:26305945

  14. Effects of atrial and brain natriuretic peptides upon cyclic GMP levels, potassium transport, and receptor binding in rat astrocytes

    SciTech Connect

    Beaumont, K.; Tan, P.K. )

    1990-02-01

    The ability of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) to alter cyclic GMP levels and NaKCl cotransport in rat neocortical astrocytes was determined. At concentrations of 10(-9)-10(-6) M, rat ANP99-126 (rANF), rat ANP102-126 (auriculin B), and rat ANP103-126 (atriopeptin III) stimulated 6- to 100-fold increases in cyclic GMP levels. Porcine BNP (pBNP) and rat BNP (rBNP) were 20%-90% as effective as rANF over most of this concentration range, although 10(-6) M pBNP produced a greater effect than rANF. NaKCl cotransport as measured by bumetanide-sensitive 86Rb+ influx was not altered by exposure of astrocytes to 10(-6)M rANF, pBNP, or rBNP. Both pBNP and rBNP, as well as rat ANP103-123 (atriopeptin I) and des(gl18, ser19, gly20, leu21, gly22) ANF4-23-NH2 (C-ANF4-23) strongly competed for specific 125I-rANF binding sites in astrocyte membranes with affinities ranging from 0.03 to 0.4 nM, suggesting that virtually all binding sites measured at subnanomolar concentrations of 125I-rANF were of the ANP-C (ANF-R2) receptor subtype. These receptors are thought to serve a clearance function and may be linked to a guanylate cyclase activity that is chemically and pharmacologically distinct from that coupled to ANP-A (ANF-R1) receptors. ANP receptors on astrocytes may function in limiting the access of ANP and BNP to neurons involved in body fluid and cardiovascular regulation.

  15. Cyclic GMP, sodium nitroprusside and sodium azide reduce aqueous humour formation in the isolated arterially perfused pig eye.

    PubMed

    Shahidullah, Mohammad; Yap, Maurice; To, Chi-Ho

    2005-05-01

    The effect of nitric oxide (NO) on aqueous humour formation (AHF) and intraocular pressure (IOP) was studied using NO donors, sodium azide (AZ) and sodium nitroprusside (SNP). Using the porcine arterially perfused eye preparation, drug effects on AHF and IOP were measured by fluorescein dilution and manometry, respectively. Perfusion pressure of the ocular vasculature was also monitored using digital pressure transducer and pen recorder. L-Arginine (1.0 mM), a precursor of NO, but not D-arginine (1.0 mM), the inactive analogue, produced a significant reduction in AHF (28.5%) and IOP (21.1%). L-NAME (L-nitro-L-arginine) (10-100 microM), an NO synthase inhibitor, had no effect on AHF and IOP. However, L-NAME (100 microM) completely reversed L-arginine's effect. AZ and SNP reduced the AHF and IOP dose-dependently. AZ at 100 nM, 1 and 10 microM reduced AHF by 26.0, 39.7 and 51.7% and IOP by 10.8, 17.3 and 24.0%, respectively. SNP at 1, 10 and 100 microM reduced the AHF by 6.0, 24.2 and 35.4% and IOP by 3.5, 9.5 and 15.5%, respectively. 8-pCPT-cGMP (8-para-chlorophenyl-thioguanosine-3',5'-cyclic guanosine monophosphate, 10 microM), a cGMP analogue, also reduced the AHF (34.9%) and IOP (15.9%). The effects of AZ and SNP on the AHF and IOP were blocked by a soluble guanylate cyclase inhibitor ODQ (10 microM), whereas ODQ alone or combined with 8-pCPT-cGMP had no effect on the AHF and IOP. None of the drugs had any significant effect on ocular vasculature. The reduction of the AHF and IOP in the arterially perfused pig eye by nitrovasodilators is likely to involve the NO-cGMP pathway.

  16. Estradiol reduces depressive-like behavior through inhibiting nitric oxide/cyclic GMP pathway in ovariectomized mice.

    PubMed

    Heydarpour, Pouria; Salehi-Sadaghiani, Mohammad; Javadi-Paydar, Mehrak; Rahimian, Reza; Fakhfouri, Gohar; Khosravi, Mohsen; Khoshkish, Shayan; Gharedaghi, Mohammad Hadi; Ghasemi, Mehdi; Dehpour, Ahmad Reza

    2013-02-01

    Estradiol decline has been associated with depressive-like behavior in female mice and NO has been suggested to play a major role in the pathogenesis of major depression. This study was conducted to investigate the antidepressant-like effects of acute estradiol administration in female ovariectomized (OVX) mice and the possible role of nitric oxide (NO)/cyclic GMP (cGMP) pathway. To this end, bilateral ovariectomy was performed in female mice and different doses of estradiol were injected alone or in combination with non-specific NO synthase (NOS) inhibitor (L-NAME), selective neural NOS (nNOS) inhibitor (7-NI), an NO precursor (L-arginine) or selective phosphodiesterase type 5 inhibitor (sildenafil). The duration of immobility was recorded in the forced swimming test (FST) to assess the depressive behavior. Moreover, hippocampal levels of NO were determined in select groups. 10 days following the procedure, OVX mice showed significantly prolonged immobility time in comparison with the sham group. Estradiol (3, 10, and 30 μg/kg, s.c.), when injected 1 h prior to FST, exerted antidepressant-like effects in OVX mice. Both L-NAME (30 mg/kg, i.p.), and 7-NI (50 mg/kg, i.p.) significantly reduced the immobility times of OVX mice. Administration of a sub-effective dose of L-NAME (10mg/kg), 15 min after a sub-effective dose of estradiol (1 μg/kg, s.c.) had a robust antidepressant-like effect in OVX mice. Also a sub-effective dose of 7-NI (25 mg/kg), 30 min after a sub-effective dose of estradiol (1 μg/kg, s.c.) showed antidepressant-like effect in OVX mice. Both the NO precursor L-arginine (750 mg/kg, i.p.) and the cGMP-specific phosphodiesterase type 5 inhibitor sildenafil (5 mg/kg, i.p.), 30 min before estradiol treatment, prevented the antidepressant-like effect of a potent dose of estradiol (10 μg/kg, s.c.) in OVX mice. The present findings suggest that suppression of the NO synthase/NO/cGMP pathway may be involved in the antidepressant-like effects of estradiol

  17. Ammonia inhibits the C-type natriuretic peptide-dependent cyclic GMP synthesis and calcium accumulation in a rat brain endothelial cell line.

    PubMed

    Konopacka, Agnieszka; Zielińska, Magdalena; Albrecht, Jan

    2008-05-01

    Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielińska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260-1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca2+]i) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1h, 5mM NH4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca2+]i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca2+ accumulation in the absence of Na+. In cells incubated with ammonia in the absence of Ca2+ a slight CNP-dependent increase of [Ca2+]i was observed, most likely representing Ca2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca2+]i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy.

  18. Cyclic GMP-dependent protein kinase EGL-4 controls body size and lifespan in C elegans.

    PubMed

    Hirose, Takashi; Nakano, Yoshiya; Nagamatsu, Yasuko; Misumi, Takashi; Ohta, Hiromitsu; Ohshima, Yasumi

    2003-03-01

    We designed an automatic system to measure body length, diameters and volume of a C. elegans worm. By using this system, mutants with an increased body volume exceeding 50% were isolated. Four of them are grossly normal in morphology and development, grow longer to be almost twice as big, and have weak egg-laying defects and extended lifespan. All the four mutants have a mutation in the egl-4 gene. We show that the egl-4 gene encodes cGMP-dependent protein kinases. egl-4 promoter::gfp fusion genes are mainly expressed in head neurons, hypodermis, intestine and body wall muscles. Procedures to analyze morphology and volume of major organs were developed. The results indicate that volumes of intestine, hypodermis and muscle and cell volumes in intestine and muscle are increased in the egl-4 mutants, whereas cell numbers are not. Experiments on genetic interaction suggest that the cGMP-EGL-4 signaling pathway represses body size and lifespan through DBL-1/TGF-beta and insulin pathways, respectively.

  19. Host Immune Response to Bacterial Cyclic Diguanylic Acid (c-di-GMP)

    DTIC Science & Technology

    2009-07-01

    biofilm formation of Staphylococcus aureus, including human methicillin- resistant S. aureus strains and animal clinical isolates (6,19). We have also...Malouin. 2005. 3’,5’- cyclic diguanylic acid reduces the virulence of biofilm -forming Staphylococcus aureus strains in a mouse model of mastitis...8217- cyclic diguanylic acid reduces the virulence of biofilm -forming Staphylococcus aureus strains in a mouse model of mastitis infection 2. Antimicrob

  20. The nitric oxide donors, SNAP and DEA/NO, exert a negative inotropic effect in rat cardiomyocytes which is independent of cyclic GMP elevation.

    PubMed

    Sandirasegarane, L; Diamond, J

    1999-04-01

    The role of guanosine 3',5'-cyclic monophosphate (cGMP) in the regulation of cardiac contractility remains controversial. The present study has examined the effects of high concentrations of the nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine (SNAP) and 1,1-diethyl-2-hydroxy-2-nitroso-hydrazine (DEA/NO), on cGMP levels and isoproterenol-induced increases in contractility in rat cardiomyocytes before and after selective inhibition of soluble guanylyl cyclase with 1 H -[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In control myocytes, 100 microm SNAP or 100 microm DEA/NO increased cGMP levels by more than 15-fold at 2 and 6 min and produced marked attenuations of isoproterenol-mediated increases in maximal cell shortening over the same time period. The NO donors had no significant effect on basal cell shortening (in the absence of isoproterenol). Pretreatment of myocytes with 25 microm ODQ for 30 min resulted in a complete blockade of the SNAP- or DEA/NO-induced increases in cGMP with no reversal of negative inotropy. ODQ did not affect basal contractility, basal cGMP levels or isoproterenol-induced increases in cell shortening. Furthermore, myocytes exposed to the cGMP analog, 8-bromo-cGMP (100 microm), did not exhibit significant differences in basal contractility or isoproterenol-induced increases in cell shortening. These results suggest that attenuation of cardiac contractility by NO donors in rat cardiomyocytes occurs by a mechanism independent of increases in cGMP levels. Copyright 1999 Academic Press.

  1. Cyclic GMP protein kinase activity is reduced in thyroxine-induced hypertrophic cardiac myocytes.

    PubMed

    Yan, Lin; Zhang, Qihang; Scholz, Peter M; Weiss, Harvey R

    2003-12-01

    1. We tested the hypothesis that the cGMP-dependent protein kinase has major negative functional effects in cardiac myocytes and that the importance of this pathway is reduced in thyroxine (T4; 0.5 mg/kg per day for 16 days) hypertrophic myocytes. 2. Using isolated ventricular myocytes from control (n = 7) and T4-treated (n = 9) rabbit hypertrophic hearts, myocyte shortening was studied with a video edge detector. Oxygen consumption was measured using O2 electrodes. Protein phosphorylation was measured autoradiographically. 3. Data were collected following treatment with: (i) 8-(4-chlorophenylthio)guanosine-3',5'-monophosphate (PCPT; 10-7 or 10-5 mol/L); (ii) 8-bromo-cAMP (10-5 mol/L) followed by PCPT; (iii) beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-monophosphorothioate, SP-isomer (SP; 10-7 or 10-5 mol/L); or (iv) 8-bromo-cAMP (10-5 mol/L) followed by SP. 4. There were no significant differences between groups in baseline percentage shortening (Pcs; 4.9 +/- 0.2 vs 5.6 +/- 0.4% for control and T4 groups, respectively) and maximal rate of shortening (Rs; 64.8 +/- 5.9 vs 79.9 +/- 7.1 micro m/ s for control and T4 groups, respectively). Both SP and PCPT decreased Pcs (-43 vs-21% for control and T4 groups, respectively) and Rs (-36 vs-22% for control and T4 groups, respectively), but the effect was significantly reduced in T4 myocytes. 8-Bromo-cAMP similarly increased Pcs (28 vs 23% for control and T4 groups, respectively) and Rs (20 vs 19% for control and T4 groups, respectively). After 8-bromo-cAMP, SP and PCPT decreased Pcs (-34%) and Rs (-29%) less in the control group. However, the effects of these drugs were not altered in T4 myocytes (Pcs -24%; Rs -22%). Both PCPT and cAMP phosphorylated the same five protein bands. In T4 myocytes, these five bands were enhanced less. 5. We conclude that, in control ventricular myocytes, the cGMP-dependent protein kinase exerted major negative functional effects but, in T4-induced hypertrophic myocytes, the importance of

  2. Genome-Based Comparison of Cyclic Di-GMP Signaling in Pathogenic and Commensal Escherichia coli Strains

    PubMed Central

    Povolotsky, Tatyana L.

    2015-01-01

    ABSTRACT The ubiquitous bacterial second messenger cyclic di-GMP (c-di-GMP) has recently become prominent as a trigger for biofilm formation in many bacteria. It is generated by diguanylate cyclases (DGCs; with GGDEF domains) and degraded by specific phosphodiesterases (PDEs; containing either EAL or HD-GYP domains). Most bacterial species contain multiples of these proteins with some having specific functions that are based on direct molecular interactions in addition to their enzymatic activities. Escherichia coli K-12 laboratory strains feature 29 genes encoding GGDEF and/or EAL domains, resulting in a set of 12 DGCs, 13 PDEs, and four enzymatically inactive “degenerate” proteins that act by direct macromolecular interactions. We present here a comparative analysis of GGDEF/EAL domain-encoding genes in 61 genomes of pathogenic, commensal, and probiotic E. coli strains (including enteric pathogens such as enteroaggregative, enterohemorrhagic, enteropathogenic, enterotoxigenic, and adherent and invasive Escherichia coli and the 2011 German outbreak O104:H4 strain, as well as extraintestinal pathogenic E. coli, such as uropathogenic and meningitis-associated E. coli). We describe additional genes for two membrane-associated DGCs (DgcX and DgcY) and four PDEs (the membrane-associated PdeT, as well as the EAL domain-only proteins PdeW, PdeX, and PdeY), thus showing the pangenome of E. coli to contain at least 35 GGDEF/EAL domain proteins. A core set of only eight proteins is absolutely conserved in all 61 strains: DgcC (YaiC), DgcI (YliF), PdeB (YlaB), PdeH (YhjH), PdeK (YhjK), PdeN (Rtn), and the degenerate proteins CsrD and CdgI (YeaI). In all other GGDEF/EAL domain genes, diverse point and frameshift mutations, as well as small or large deletions, were discovered in various strains. IMPORTANCE Our analysis reveals interesting trends in pathogenic Escherichia coli that could reflect different host cell adherence mechanisms. These may either benefit from or be

  3. Normoxic Cyclic GMP-independent Oxidative Signaling by Nitrite Enhances Airway Epithelial Cell Proliferation and Wound Healing

    PubMed Central

    Wang, Ling; Frizzell, Sheila A.; Zhao, Xuejun; Gladwin, Mark T.

    2013-01-01

    The airway epithelium provides important barrier and host defense functions. Recent studies reveal that nitrite is an endocrine reservoir of nitric oxide (NO) bioactivity that is converted to NO by enzymatic reductases along the physiological oxygen gradient. Nitrite signaling has been described as NO dependent activation mediated by reactions with deoxygenated redox active hemoproteins, such as hemoglobin, myoglobin, neuroglobin, xanthine oxidoreductase (XO) and NO synthase at low pH and oxygen tension. However, nitrite can also be readily oxidized to nitrogen dioxide (NO2•) via heme peroxidase reactions, suggesting the existence of alternative oxidative signaling pathways for nitrite under normoxic conditions. In the present study we examined normoxic signaling effects of sodium nitrite on airway epithelial cell wound healing. In an in vitro scratch injury model under normoxia, we exposed cultured monolayers of human airway epithelial cells to various concentrations of sodium nitrite and compared responses to NO donor. We found sodium nitrite potently enhanced airway epithelium wound healing at physiological concentrations (from 1uM). The effect of nitrite was blocked by the NO and NO2• scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (c-PTIO). Interestingly, nitrite treatment did not increase cyclic guanosine monophosphate (cGMP) levels under these normoxic conditions, even in the presence of a phosphodiesterase 5 inhibitor, suggesting cGMP independent signaling. Consistent with an oxidative signaling pathway requiring hydrogen peroxide (H2O2)/heme peroxidase/NO2• signaling, the effects of nitrite were potentiated by superoxide dismutase (SOD) and low concentration H2O2, whereas inhibited completely by catalase, followed by downstream extracellular-signal-regulated kinase (ERK) 1/2 activation. Our data represent the first description of normoxic nitrite signaling on lung epithelial cell proliferation and wound healing and suggest

  4. Role of cyclic GMP-dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes

    PubMed Central

    Layland, Joanne; Li, Jian-Mei; Shah, Ajay M

    2002-01-01

    Nitric oxide (NO) can directly modulate cardiac contractility by accelerating relaxation and reducing diastolic tone. The intracellular mechanisms underlying these contractile effects are poorly understood. Here we investigate the role of cyclic GMP-dependent protein kinase (PKG) in the contractile response to exogenous NO in rat ventricular myocytes. Isolated ventricular myocytes were stimulated electrically and contractility was assessed by measuring cell shortening. Some cells were loaded with the fluorescent Ca2+ probe indo-1 AM for simultaneous assessment of the intracellular Ca2+ transient. The NO donor diethylamine NONOate (DEA/NO, 10 μm) significantly increased resting cell length, reduced twitch amplitude and accelerated time to 50 % relaxation (to 100.8 ± 0.2, 83.7 ± 3.0 and 88.9 ± 3.7 % of control values, respectively). The contractile effects of DEA/NO occurred without significant changes in the amplitude or kinetics of the intracellular Ca2+ transient, suggesting that the myofilament response to Ca2+ was reduced. These effects were abolished by inhibition of either guanylyl cyclase (with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ, 10 μm) or PKG (with Rp-8-Br-cGMPs, 10 μm) suggesting that, at the concentration investigated, the effects of DEA/NO were mediated exclusively by PKG, following activation of guanylyl cyclase and elevation of cGMP. Direct activation of PKG with 8-pCPT-cGMP (10 μm) mimicked the effects of DEA/NO (resting cell length and time to 50 % relaxation were 100.6 ± 0.1 and 90.5 ± 1.5 % of control values, respectively).The reduced myofilament Ca2+ responsiveness was not attributable to an intracellular acidosis since the small reduction in pHi induced by DEA/NO was found to be uncoupled from its contractile effects. However, hearts treated with DEA/NO (10 μm) showed a significant increase (1.4-fold; P < 0.01) in troponin I phosphorylation compared to control, untreated hearts. These results suggest that the reduction in

  5. Identification of cyclic GMP-activated nonselective Ca2+-permeable cation channels and associated CNGC5 and CNGC6 genes in Arabidopsis guard cells.

    PubMed

    Wang, Yong-Fei; Munemasa, Shintaro; Nishimura, Noriyuki; Ren, Hui-Min; Robert, Nadia; Han, Michelle; Puzõrjova, Irina; Kollist, Hannes; Lee, Stephen; Mori, Izumi; Schroeder, Julian I

    2013-10-01

    Cytosolic Ca(2+) in guard cells plays an important role in stomatal movement responses to environmental stimuli. These cytosolic Ca(2+) increases result from Ca(2+) influx through Ca(2+)-permeable channels in the plasma membrane and Ca(2+) release from intracellular organelles in guard cells. However, the genes encoding defined plasma membrane Ca(2+)-permeable channel activity remain unknown in guard cells and, with some exceptions, largely unknown in higher plant cells. Here, we report the identification of two Arabidopsis (Arabidopsis thaliana) cation channel genes, CNGC5 and CNGC6, that are highly expressed in guard cells. Cytosolic application of cyclic GMP (cGMP) and extracellularly applied membrane-permeable 8-Bromoguanosine 3',5'-cyclic monophosphate-cGMP both activated hyperpolarization-induced inward-conducting currents in wild-type guard cells using Mg(2+) as the main charge carrier. The cGMP-activated currents were strongly blocked by lanthanum and gadolinium and also conducted Ba(2+), Ca(2+), and Na(+) ions. cngc5 cngc6 double mutant guard cells exhibited dramatically impaired cGMP-activated currents. In contrast, mutations in CNGC1, CNGC2, and CNGC20 did not disrupt these cGMP-activated currents. The yellow fluorescent protein-CNGC5 and yellow fluorescent protein-CNGC6 proteins localize in the cell periphery. Cyclic AMP activated modest inward currents in both wild-type and cngc5cngc6 mutant guard cells. Moreover, cngc5 cngc6 double mutant guard cells exhibited functional abscisic acid (ABA)-activated hyperpolarization-dependent Ca(2+)-permeable cation channel currents, intact ABA-induced stomatal closing responses, and whole-plant stomatal conductance responses to darkness and changes in CO2 concentration. Furthermore, cGMP-activated currents remained intact in the growth controlled by abscisic acid2 and abscisic acid insensitive1 mutants. This research demonstrates that the CNGC5 and CNGC6 genes encode unique cGMP-activated nonselective Ca(2

  6. Cyclic GMP signaling in rat urinary bladder, prostate, and epididymis: tissue-specific changes with aging and in response to Leydig cell depletion.

    PubMed

    Müller, Dieter; Mukhopadhyay, Amal K; Davidoff, Michail S; Middendorff, Ralf

    2011-08-01

    Aging of the male reproductive system leads to changes in endocrine signaling and is frequently associated with the emergence of prostate hyperplasia and bladder dysfunctions. Recent reports highlight prostate and bladder as promising targets for therapeutic interventions with inhibitors of the cyclic GMP (cGMP)-degrading phosphodiesterase 5 (PDE5). However, the cGMP signaling system in these organs is as yet poorly characterized, and the possibility of age-related alterations has not been addressed. This study investigates key proteins of cGMP pathways in bladder, prostate, and epididymis of young (3 months) and old (23-24 months) Wistar rats. Local differences in the abundance of PDE5, soluble guanylyl cyclase (sGC) and particulate guanylyl cyclases (GC-A, GC-B), endothelial nitric oxide synthase, and cGMP-dependent protein kinase I (PRKG1 (cGKI)) revealed pronounced tissue-specific peculiarities. Although cGMP-generating enzymes were not affected by age in all organs, we recognized age-related decreases of PDE5 expression in bladder and a selective diminishment of membrane-associated PRKG1 in epididymis. In disagreement with published data, all cGMP pathway proteins including PDE5 are poorly expressed in prostate. However, prostatic PRKG1 expression increases with aging. Androgen withdrawal during temporary Leydig cell elimination induced a massive (>12-fold) upregulation of PRKG1 in prostate but not in other (penis and epididymis) androgen-dependent organs. These findings identify PRKG1 as a key androgen-sensitive signaling protein in prostate of possible importance for growth regulation. The elucidated effects may have significance for age-associated pathologies in the male lower-urinary tract.

  7. Gating Kinetics of the Cyclic-GMP-Activated Channel of Retinal Rods: Flash Photolysis and Voltage-Jump Studies

    NASA Astrophysics Data System (ADS)

    Karpen, Jeffrey W.; Zimmerman, Anita L.; Stryer, Lubert; Baylor, Denis A.

    1988-02-01

    The gating kinetics of the cGMP-activated cation channel of salamander retinal rods have been studied in excised membrane patches. Relaxations in patch current were observed after two kinds of perturbation: (i) fast jumps of cGMP concentration, generated by laser flash photolysis of a cGMP ester (``caged'' cGMP), and (ii) membrane voltage jumps, which perturb activation of the channel by cGMP. In both methods the speed of activation increased with the final cGMP concentration. The results are explained by a simple kinetic model in which activation involves three sequential cGMP binding steps with bimolecular rate constants close to the diffusion-controlled limit; fully liganded channels undergo rapid open-closed transitions. Voltage perturbs activation by changing the rate constant for channel closing, which increases with hyperpolarization. Intramolecular transitions of the fully liganded channel limit the kinetics of activation at high cGMP concentrations (>50 μ M), whereas at physiological cGMP concentrations (<5 μ M), the kinetics of activation are limited by the third cGMP binding step. The channel appears to be optimized for rapid responses to changes in cytoplasmic cGMP concentration.

  8. Modification of Tau by 8-Nitroguanosine 3',5'-Cyclic Monophosphate (8-Nitro-cGMP): EFFECTS OF NITRIC OXIDE-LINKED CHEMICAL MODIFICATION ON TAU AGGREGATION.

    PubMed

    Yoshitake, Jun; Soeda, Yoshiyuki; Ida, Tomoaki; Sumioka, Akio; Yoshikawa, Misato; Matsushita, Kenji; Akaike, Takaaki; Takashima, Akihiko

    2016-10-21

    Neurofibrillar tangles caused by intracellular hyperphosphorylated tau inclusion and extracellular amyloid β peptide deposition are hallmarks of Alzheimer's disease. Tau contains one or two cysteine residues in three or four repeats of the microtubule binding region following alternative splicing of exon 10, and formation of intermolecular cysteine disulfide bonds accelerates tau aggregation. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) acts as a novel second messenger of nitric oxide (NO) by covalently binding cGMP to cysteine residues by electrophilic properties, a process termed protein S-guanylation. Here we studied S-guanylation of tau and its effects on tau aggregation. 8-Nitro-cGMP exposure induced S-guanylation of tau both in vitro and in tau-overexpressed HEK293T cells. S-guanylated tau inhibited heparin-induced tau aggregation in a thioflavin T assay. Atomic force microscopy observations indicated that S-guanylated tau could not form tau granules and fibrils. Further biochemical analyses showed that S-guanylated tau was inhibited at the step of tau oligomer formation. In P301L tau-expressing Neuro2A cells, 8-nitro-cGMP treatment significantly reduced the amount of sarcosyl-insoluble tau. NO-linked chemical modification on cysteine residues of tau could block tau aggregation, and therefore, increasing 8-nitro-cGMP levels in the brain could become a potential therapeutic strategy for Alzheimer's disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Clearance of Pseudomonas aeruginosa foreign-body biofilm infections through reduction of the cyclic Di-GMP level in the bacteria.

    PubMed

    Christensen, Louise D; van Gennip, Maria; Rybtke, Morten T; Wu, Hong; Chiang, Wen-Chi; Alhede, Morten; Høiby, Niels; Nielsen, Thomas E; Givskov, Michael; Tolker-Nielsen, Tim

    2013-08-01

    Opportunistic pathogenic bacteria can engage in biofilm-based infections that evade immune responses and develop into chronic conditions. Because conventional antimicrobials cannot efficiently eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. It has recently been established that the secondary messenger cyclic diguanosine monophosphate (c-di-GMP) functions as a positive regulator of biofilm formation in several different bacteria. In the present study we investigated whether manipulation of the c-di-GMP level in bacteria potentially can be used for biofilm control in vivo. We constructed a Pseudomonas aeruginosa strain in which a reduction in the c-di-GMP level can be achieved via induction of the Escherichia coli YhjH c-di-GMP phosphodiesterase. Initial experiments showed that induction of yhjH expression led to dispersal of the majority of the bacteria in in vitro-grown P. aeruginosa biofilms. Subsequently, we demonstrated that P. aeruginosa biofilms growing on silicone implants, located in the peritoneal cavity of mice, dispersed after induction of the YhjH protein. Bacteria accumulated temporarily in the spleen after induction of biofilm dispersal, but the mice tolerated the dispersed bacteria well. The present work provides proof of the concept that modulation of the c-di-GMP level in bacteria is a viable strategy for biofilm control.

  10. Luteinizing Hormone Reduces the Activity of the NPR2 Guanylyl Cyclase in Mouse Ovarian Follicles, Contributing to the Cyclic GMP Decrease that Promotes Resumption of Meiosis in Oocytes

    PubMed Central

    Robinson, Jerid W.; Zhang, Meijia; Shuhaibar, Leia C.; Norris, Rachael P.; Geerts, Andreas; Wunder, Frank; Eppig, John J.; Potter, Lincoln R.; Jaffe, Laurinda A.

    2012-01-01

    In preovulatory ovarian follicles of mice, meiotic prophase arrest in the oocyte is maintained by cyclic GMP from the surrounding granulosa cells that diffuses into the oocyte through gap junctions. The cGMP is synthesized in the granulosa cells by the transmembrane guanylyl cyclase natriuretic peptide receptor 2 (NPR2) in response to the agonist C-type natriuretic peptide (CNP). In response to luteinizing hormone (LH), cGMP in the granulosa cells decreases, and as a consequence, oocyte cGMP decreases and meiosis resumes. Here we report that within 20 minutes, LH treatment results in decreased guanylyl cyclase activity of NPR2, as determined in the presence of a maximally activating concentration of CNP. This occurs by a process that does not reduce the amount of NPR2 protein. We also show that by a slower process, first detected at 2 hours, LH decreases the amount of CNP available to bind to the receptor. Both of these LH actions contribute to decreasing cGMP in the follicle, thus signaling meiotic resumption in the oocyte. PMID:22546688

  11. Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its gamma subunit and transducin.

    PubMed

    Wensel, T G; Stryer, L

    1986-09-01

    The switching on of the cGMP phosphodiesterase (PDE) in retinal rod outer segments by activated transducin (T alpha-GTP) is a key step in visual excitation. The finding that trypsin activates PDE (alpha beta gamma) by degrading its gamma subunit and the reversal of this activation by gamma led to the proposal that T alpha-GTP activates PDE by relieving an inhibitory constraint imposed by gamma (Hurley and Stryer: J. Biol. Chem. 257:11094-11099, 1982). We report here studies showing that the addition of gamma subunit also reverses the activation of PDE by T alpha-GTP-gamma S. A procedure for preparing gamma in high yield (50-80%) is presented. Analyses of SDS polyacrylamide gel slices confirmed that inhibitory activity resides in the gamma subunit. Nanomolar gamma blocks the activation of PDE by micromolar T alpha-GTP gamma S. The degree of activation of PDE depends reciprocally on the concentrations of gamma and T alpha-GTP gamma S. gamma remains bound to the disk membrane during the activation of PDE by transducin. The binding of gamma to the alpha beta subunits of native PDE is very tight; the dissociation constant is less than 10 pM, indicating that fewer than 1 in 1,700 PDE molecules in rod outer segments are activated in the absence of T alpha-GTP.

  12. Selective inhibitory effect of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and 2'-nor-cyclic GMP on adenovirus replication in vitro.

    PubMed

    Baba, M; Mori, S; Shigeta, S; De Clercq, E

    1987-02-01

    The inhibitory effects of 20 selected antiviral compounds on the replication of adenoviruses (types 1 to 8) in vitro were investigated. While 18 compounds were ineffective, 2 compounds, namely (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] and 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide (2'-nor-cyclic GMP), were highly effective against all adenovirus types assayed in human embryonic fibroblast cultures. Their 50% inhibitory doses were 1.1 microgram/ml for (S)-HPMPA and 4.1 micrograms/ml for 2'-nor-cyclic GMP. They were nontoxic for the host cells at the effective antiviral doses.

  13. Selective inhibitory effect of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and 2'-nor-cyclic GMP on adenovirus replication in vitro.

    PubMed Central

    Baba, M; Mori, S; Shigeta, S; De Clercq, E

    1987-01-01

    The inhibitory effects of 20 selected antiviral compounds on the replication of adenoviruses (types 1 to 8) in vitro were investigated. While 18 compounds were ineffective, 2 compounds, namely (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] and 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide (2'-nor-cyclic GMP), were highly effective against all adenovirus types assayed in human embryonic fibroblast cultures. Their 50% inhibitory doses were 1.1 microgram/ml for (S)-HPMPA and 4.1 micrograms/ml for 2'-nor-cyclic GMP. They were nontoxic for the host cells at the effective antiviral doses. PMID:3566256

  14. Reactive oxygen species drive evolution of pro-biofilm variants in pathogens by modulating cyclic-di-GMP levels

    PubMed Central

    Ding, Yichen; Liu, Yang; Cai, Zhao; Zhou, Jianuan; Swarup, Sanjay; Drautz-Moses, Daniela I.; Schuster, Stephan Christoph; Kjelleberg, Staffan; Givskov, Michael; Yang, Liang

    2016-01-01

    The host immune system offers a hostile environment with antimicrobials and reactive oxygen species (ROS) that are detrimental to bacterial pathogens, forcing them to adapt and evolve for survival. However, the contribution of oxidative stress to pathogen evolution remains elusive. Using an experimental evolution strategy, we show that exposure of the opportunistic pathogen Pseudomonas aeruginosa to sub-lethal hydrogen peroxide (H2O2) levels over 120 generations led to the emergence of pro-biofilm rough small colony variants (RSCVs), which could be abrogated by l-glutathione antioxidants. Comparative genomic analysis of the RSCVs revealed that mutations in the wspF gene, which encodes for a repressor of WspR diguanylate cyclase (DGC), were responsible for increased intracellular cyclic-di-GMP content and production of Psl exopolysaccharide. Psl provides the first line of defence against ROS and macrophages, ensuring the survival fitness of RSCVs over wild-type P. aeruginosa. Our study demonstrated that ROS is an essential driving force for the selection of pro-biofilm forming pathogenic variants. Understanding the fundamental mechanism of these genotypic and phenotypic adaptations will improve treatment strategies for combating chronic infections. PMID:27881736

  15. Analysis of the Borrelia burgdorferi Cyclic-di-GMP-Binding Protein PlzA Reveals a Role in Motility and Virulence ▿

    PubMed Central

    Pitzer, Joshua E.; Sultan, Syed Z.; Hayakawa, Yoshihiro; Hobbs, Gerry; Miller, Michael R.; Motaleb, Md A.

    2011-01-01

    The cyclic-dimeric-GMP (c-di-GMP)-binding protein PilZ has been implicated in bacterial motility and pathogenesis. Although BB0733 (PlzA), the only PilZ domain-containing protein in Borrelia burgdorferi, was reported to bind c-di-GMP, neither its role in motility or virulence nor it's affinity for c-di-GMP has been reported. We determined that PlzA specifically binds c-di-GMP with high affinity (dissociation constant [Kd], 1.25 μM), consistent with Kd values reported for c-di-GMP-binding proteins from other bacteria. Inactivation of the monocistronically transcribed plzA resulted in an opaque/solid colony morphology, whereas the wild-type colonies were translucent. While the swimming pattern of mutant cells appeared normal, on swarm plates, mutant cells exhibited a significantly reduced swarm diameter, demonstrating a role of plzA in motility. Furthermore, the plzA mutant cells were significantly less infectious in experimental mice (as determined by 50% infectious dose [ID50]) relative to wild-type spirochetes. The mutant also had survival rates in fed ticks lower than those of the wild type. Consequently, plzA mutant cells failed to complete the mouse-tick-mouse infection cycle, indicating plzA is essential for the enzootic life cycle of B. burgdorferi. All of these defects were corrected when the mutant was complemented in cis. We propose that failure of plzA mutant cells to infect mice was due to altered motility; however, the possibility that an unidentified factor(s) contributed to interruption of the B. burgdorferi enzootic life cycle cannot yet be excluded. PMID:21357718

  16. Identification of a negative feedback loop between cyclic di-GMP-induced levels of IFI16 and p202 cytosolic DNA sensors and STING.

    PubMed

    Panchanathan, Ravichandran; Liu, Hongzhu; Xin, Duan; Choubey, Divaker

    2014-10-01

    A host type I IFN response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP (c-di-GMP) by STING (stimulator of IFN genes). Because the STING, an adaptor protein, links the cytosolic detection of DNA by the cytosolic DNA sensors such as the IFN-inducible human IFI16 and murine p202 proteins to the TBK1/IRF3 axis, we investigated whether c-di-GMP-induced signaling could regulate expression of IFI16 and p202 proteins. Here, we report that activation of c-di-GMP-induced signaling in human and murine cells increased steady-state levels of IFI16 and p202 proteins. The increase was c-di-GMP concentration- and time-dependent. Unexpectedly, treatment of cells with type I IFN decreased levels of the adaptor protein STING. Therefore, we investigated whether the IFI16 or p202 protein could regulate the expression of STING and activation of the TBK1/IRF3 axis. We found that constitutive knockdown of IFI16 or p202 expression in cells increased steady-state levels of STING. Additionally, the knockdown of IFI16 resulted in activation of the TBK1/IRF3 axis. Accordingly, increased levels of the IFI16 or p202 protein in cells decreased STING levels. Together, our observations identify a novel negative feedback loop between c-di-GMP-induced levels of IFI16 and p202 cytosolic DNA sensors and the adaptor protein STING. © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  17. Crystal Structures of YkuI and Its Complex with Second Messenger Cyclic Di-GMP Suggest Catalytic Mechanism of Phosphodiester Bond Cleavage by EAL Domains*

    PubMed Central

    Minasov, George; Padavattan, Sivaraman; Shuvalova, Ludmilla; Brunzelle, Joseph S.; Miller, Darcie J.; Baslé, Arnaud; Massa, Claudia; Collart, Frank R.; Schirmer, Tilman; Anderson, Wayne F.

    2009-01-01

    Cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that is involved in the regulation of cell surface-associated traits and the persistence of infections. Omnipresent GGDEF and EAL domains, which occur in various combinations with regulatory domains, catalyze c-di-GMP synthesis and degradation, respectively. The crystal structure of full-length YkuI from Bacillus subtilis, composed of an EAL domain and a C-terminal PAS-like domain, has been determined in its native form and in complex with c-di-GMP and Ca2+. The EAL domain exhibits a triose-phosphate isomerase-barrel fold with one antiparallel β-strand. The complex with c-di-GMP-Ca2+ defines the active site of the putative phosphodiesterase located at the C-terminal end of the β-barrel. The EAL motif is part of the active site with Glu-33 of the motif being involved in cation coordination. The structure of the complex allows the proposal of a phosphodiesterase mechanism, in which the divalent cation and the general base Glu-209 activate a catalytic water molecule for nucleophilic in-line attack on the phosphorus. The C-terminal domain closely resembles the PAS-fold. Its pocket-like structure could accommodate a yet unknown ligand. YkuI forms a tight dimer via EAL-EAL and trans EAL-PAS-like domain association. The possible regulatory significance of the EAL-EAL interface and a mechanism for signal transduction between sensory and catalytic domains of c-di-GMP-specific phosphodiesterases are discussed. PMID:19244251

  18. Analysis of the HD-GYP domain cyclic dimeric GMP phosphodiesterase reveals a role in motility and the enzootic life cycle of Borrelia burgdorferi.

    PubMed

    Sultan, Syed Z; Pitzer, Joshua E; Boquoi, Tristan; Hobbs, Gerry; Miller, Michael R; Motaleb, M A

    2011-08-01

    HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a K(m) of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.

  19. Analysis of the HD-GYP Domain Cyclic Dimeric GMP Phosphodiesterase Reveals a Role in Motility and the Enzootic Life Cycle of Borrelia burgdorferi ▿ †

    PubMed Central

    Sultan, Syed Z.; Pitzer, Joshua E.; Boquoi, Tristan; Hobbs, Gerry; Miller, Michael R.; Motaleb, M. A.

    2011-01-01

    HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a Km of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi. PMID:21670168

  20. Cyclic GMP-independent mechanisms contribute to the inhibition of platelet adhesion by nitric oxide donor: A role for α-actinin nitration

    PubMed Central

    Marcondes, Sisi; Cardoso, Marcia H. M.; Morganti, Rafael P.; Thomazzi, Sara M.; Lilla, Sergio; Murad, Ferid; De Nucci, Gilberto; Antunes, Edson

    2006-01-01

    The nitric oxide-mediated actions are mostly due to cyclic GMP (cGMP) formation, but cGMP-independent mechanisms, such as tyrosine nitration, have been suggested as potential signaling pathways modulating the NO-induced responses. However, the mechanisms that lead to tyrosine nitration in platelets are poorly studied, and the protein targets of nitration have not been identified in these cells. Therefore, we have used the model of platelet adhesion to fibrinogen-coated plates to investigate the cGMP-independent mechanisms of the NO-donor sodium nitroprusside (SNP) that leads to inhibition of platelet adhesion. SNP concentration-dependently inhibited platelet adhesion, as observed at 15-min and 60-min adhesion. Additionally, SNP markedly increased the cGMP levels, and the soluble guanylate inhibitor ODQ nearly abolished the SNP-mediated cGMP elevations in all experimental conditions used. Nevertheless, ODQ failed to affect the adhesion inhibition obtained with 1.0 mM SNP at 15 min. On the other hand, superoxide dismutase or peroxynitrite (ONOO−) scavenger epigallocatechin gallate significantly reversed the inhibition of platelet adhesion by SNP (1 mM, 15 min). Western blot analysis in SNP (1 mM, 15 min)-treated platelets showed a single tyrosine-nitrated protein with an apparent mass of ≈105 kDa. Nanospray LC-MS/MS identified the human α-actinin 1 cytoskeletal isoform (P12814) as the protein contained in the nitrated SDS gel band. Thus, tyrosine nitration of α-actinin, through ONOO− formation, may be a key modulatory mechanism to control platelet adhesion. PMID:16492779

  1. Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.

    PubMed

    Peltier, Johann; Shaw, Helen A; Couchman, Edward C; Dawson, Lisa F; Yu, Lu; Choudhary, Jyoti S; Kaever, Volkhard; Wren, Brendan W; Fairweather, Neil F

    2015-10-02

    In Gram-positive pathogens, surface proteins may be covalently anchored to the bacterial peptidoglycan by sortase, a cysteine transpeptidase enzyme. In contrast to other Gram-positive bacteria, only one single sortase enzyme, SrtB, is conserved between strains of Clostridium difficile. Sortase-mediated peptidase activity has been reported in vitro, and seven potential substrates have been identified. Here, we demonstrate the functionality of sortase in C. difficile. We identify two sortase-anchored proteins, the putative adhesins CD2831 and CD3246, and determine the cell wall anchor structure of CD2831. The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues, and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem. We show that CD2831 protein levels are elevated in the presence of high intracellular cyclic diGMP (c-diGMP) concentrations, in agreement with the control of CD2831 expression by a c-diGMP-dependent type II riboswitch. Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells. This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch. These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.

  2. ChIP-Seq and RNA-Seq Reveal an AmrZ-Mediated Mechanism for Cyclic di-GMP Synthesis and Biofilm Development by Pseudomonas aeruginosa

    PubMed Central

    Jones, Christopher J.; Newsom, David; Kelly, Benjamin; Irie, Yasuhiko; Jennings, Laura K.; Xu, Binjie; Limoli, Dominique H.; Harrison, Joe J.; Parsek, Matthew R.; White, Peter; Wozniak, Daniel J.

    2014-01-01

    The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for Amr

  3. Cyclic GMP-AMP Synthase Is an Innate Immune DNA Sensor for Mycobacterium tuberculosis.

    PubMed

    Collins, Angela C; Cai, Haocheng; Li, Tuo; Franco, Luis H; Li, Xiao-Dong; Nair, Vidhya R; Scharn, Caitlyn R; Stamm, Chelsea E; Levine, Beth; Chen, Zhijian J; Shiloh, Michael U

    2015-06-10

    Activation of the DNA-dependent cytosolic surveillance pathway in response to Mycobacterium tuberculosis infection stimulates ubiquitin-dependent autophagy and inflammatory cytokine production, and plays an important role in host defense against M. tuberculosis. However, the identity of the host sensor for M. tuberculosis DNA is unknown. Here we show that M. tuberculosis activated cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) in macrophages to produce cGAMP, a second messenger that activates the adaptor protein stimulator of interferon genes (STING) to induce type I interferons and other cytokines. cGAS localized with M. tuberculosis in mouse and human cells and in human tuberculosis lesions. Knockdown or knockout of cGAS in human or mouse macrophages blocked cytokine production and induction of autophagy. Mice deficient in cGAS were more susceptible to lethality caused by infection with M. tuberculosis. These results demonstrate that cGAS is a vital innate immune sensor of M. tuberculosis infection.

  4. The structure of an unconventional HD-GYP protein from Bdellovibrio reveals the roles of conserved residues in this class of cyclic-di-GMP phosphodiesterases.

    PubMed

    Lovering, Andrew L; Capeness, Michael J; Lambert, Carey; Hobley, Laura; Sockett, R Elizabeth

    2011-01-01

    Cyclic-di-GMP is a near-ubiquitous bacterial second messenger that is important in localized signal transmission during the control of various processes, including virulence and switching between planktonic and biofilm-based lifestyles. Cyclic-di-GMP is synthesized by GGDEF diguanylate cyclases and hydrolyzed by EAL or HD-GYP phosphodiesterases, with each functional domain often appended to distinct sensory modules. HD-GYP domain proteins have resisted structural analysis, but here we present the first structural representative of this family (1.28 Å), obtained using the unusual Bd1817 HD-GYP protein from the predatory bacterium Bdellovibrio bacteriovorus. Bd1817 lacks the active-site tyrosine present in most HD-GYP family members yet remains an excellent model of their features, sharing 48% sequence similarity with the archetype RpfG. The protein structure is highly modular and thus provides a basis for delineating domain boundaries in other stimulus-dependent homologues. Conserved residues in the HD-GYP family cluster around a binuclear metal center, which is observed complexed to a molecule of phosphate, providing information on the mode of hydroxide ion attack on substrate. The fold and active site of the HD-GYP domain are different from those of EAL proteins, and restricted access to the active-site cleft is indicative of a different mode of activity regulation. The region encompassing the GYP motif has a novel conformation and is surface exposed and available for complexation with binding partners, including GGDEF proteins. It is becoming apparent that many bacteria use the signaling molecule cyclic-di-GMP to regulate a variety of processes, most notably, transitions between motility and sessility. Importantly, this regulation is central to several traits implicated in chronic disease (adhesion, biofilm formation, and virulence gene expression). The mechanisms of cyclic-di-GMP synthesis via GGDEF enzymes and hydrolysis via EAL enzymes have been suggested by

  5. Regulation of Growth, Cell Shape, Cell Division, and Gene Expression by Second Messengers (p)ppGpp and Cyclic Di-GMP in Mycobacterium smegmatis

    PubMed Central

    Gupta, Kuldeepkumar Ramnaresh; Baloni, Priyanka; Indi, Shantinath S.

    2016-01-01

    ABSTRACT The alarmone (p)ppGpp regulates transcription, translation, replication, virulence, lipid synthesis, antibiotic sensitivity, biofilm formation, and other functions in bacteria. Signaling nucleotide cyclic di-GMP (c-di-GMP) regulates biofilm formation, motility, virulence, the cell cycle, and other functions. In Mycobacterium smegmatis, both (p)ppGpp and c-di-GMP are synthesized and degraded by bifunctional proteins RelMsm and DcpA, encoded by relMsm and dcpA genes, respectively. We have previously shown that the ΔrelMsm and ΔdcpA knockout strains are antibiotic resistant and defective in biofilm formation, show altered cell surface properties, and have reduced levels of glycopeptidolipids and polar lipids in their cell wall (K. R. Gupta, S. Kasetty, and D. Chatterji, Appl Environ Microbiol 81:2571–2578, 2015, http://dx.doi.org/10.1128/AEM.03999-14). In this work, we have explored the phenotypes that are affected by both (p)ppGpp and c-di-GMP in mycobacteria. We have shown that both (p)ppGpp and c-di-GMP are needed to maintain the proper growth rate under stress conditions such as carbon deprivation and cold shock. Scanning electron microscopy showed that low levels of these second messengers result in elongated cells, while high levels reduce the cell length and embed the cells in a biofilm-like matrix. Fluorescence microscopy revealed that the elongated ΔrelMsm and ΔdcpA cells are multinucleate, while transmission electron microscopy showed that the elongated cells are multiseptate. Gene expression analysis also showed that genes belonging to functional categories such as virulence, detoxification, lipid metabolism, and cell-wall-related processes were differentially expressed. Our results suggests that both (p)ppGpp and c-di-GMP affect some common phenotypes in M. smegmatis, thus raising a possibility of cross talk between these two second messengers in mycobacteria. IMPORTANCE Our work has expanded the horizon of (p)ppGpp and c-di-GMP signaling in

  6. Constitutive cyclic GMP accumulation in Arabidopsis thaliana compromises systemic acquired resistance induced by an avirulent pathogen by modulating local signals.

    PubMed

    Hussain, Jamshaid; Chen, Jian; Locato, Vittoria; Sabetta, Wilma; Behera, Smrutisanjita; Cimini, Sara; Griggio, Francesca; Martínez-Jaime, Silvia; Graf, Alexander; Bouneb, Mabrouk; Pachaiappan, Raman; Fincato, Paola; Blanco, Emanuela; Costa, Alex; De Gara, Laura; Bellin, Diana; de Pinto, Maria Concetta; Vandelle, Elodie

    2016-11-04

    The infection of Arabidopsis thaliana plants with avirulent pathogens causes the accumulation of cGMP with a biphasic profile downstream of nitric oxide signalling. However, plant enzymes that modulate cGMP levels have yet to be identified, so we generated transgenic A. thaliana plants expressing the rat soluble guanylate cyclase (GC) to increase genetically the level of cGMP and to study the function of cGMP in plant defence responses. Once confirmed that cGMP levels were higher in the GC transgenic lines than in wild-type controls, the GC transgenic plants were then challenged with bacterial pathogens and their defence responses were characterized. Although local resistance was similar in the GC transgenic and wild-type lines, differences in the redox state suggested potential cross-talk between cGMP and the glutathione redox system. Furthermore, large-scale transcriptomic and proteomic analysis highlighted the significant modulation of both gene expression and protein abundance at the infection site, inhibiting the establishment of systemic acquired resistance. Our data indicate that cGMP plays a key role in local responses controlling the induction of systemic acquired resistance in plants challenged with avirulent pathogens.

  7. Constitutive cyclic GMP accumulation in Arabidopsis thaliana compromises systemic acquired resistance induced by an avirulent pathogen by modulating local signals

    PubMed Central

    Hussain, Jamshaid; Chen, Jian; Locato, Vittoria; Sabetta, Wilma; Behera, Smrutisanjita; Cimini, Sara; Griggio, Francesca; Martínez-Jaime, Silvia; Graf, Alexander; Bouneb, Mabrouk; Pachaiappan, Raman; Fincato, Paola; Blanco, Emanuela; Costa, Alex; De Gara, Laura; Bellin, Diana; de Pinto, Maria Concetta; Vandelle, Elodie

    2016-01-01

    The infection of Arabidopsis thaliana plants with avirulent pathogens causes the accumulation of cGMP with a biphasic profile downstream of nitric oxide signalling. However, plant enzymes that modulate cGMP levels have yet to be identified, so we generated transgenic A. thaliana plants expressing the rat soluble guanylate cyclase (GC) to increase genetically the level of cGMP and to study the function of cGMP in plant defence responses. Once confirmed that cGMP levels were higher in the GC transgenic lines than in wild-type controls, the GC transgenic plants were then challenged with bacterial pathogens and their defence responses were characterized. Although local resistance was similar in the GC transgenic and wild-type lines, differences in the redox state suggested potential cross-talk between cGMP and the glutathione redox system. Furthermore, large-scale transcriptomic and proteomic analysis highlighted the significant modulation of both gene expression and protein abundance at the infection site, inhibiting the establishment of systemic acquired resistance. Our data indicate that cGMP plays a key role in local responses controlling the induction of systemic acquired resistance in plants challenged with avirulent pathogens. PMID:27811978

  8. BifA, a Cyclic-Di-GMP Phosphodiesterase, Inversely Regulates Biofilm Formation and Swarming Motility by Pseudomonas aeruginosa PA14▿ †

    PubMed Central

    Kuchma, Sherry L.; Brothers, Kimberly M.; Merritt, Judith H.; Liberati, Nicole T.; Ausubel, Frederick M.; O'Toole, George A.

    2007-01-01

    The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the ΔbifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the ΔbifA mutant but does not contribute to the observed swarming defect. The ΔbifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming. PMID:17586641

  9. The RNA binding protein CsrA controls cyclic di-GMP metabolism by directly regulating the expression of GGDEF proteins.

    PubMed

    Jonas, Kristina; Edwards, Adrianne N; Simm, Roger; Romeo, Tony; Römling, Ute; Melefors, Ojar

    2008-10-01

    The carbon storage regulator CsrA is an RNA binding protein that controls carbon metabolism, biofilm formation and motility in various eubacteria. Nevertheless, in Escherichia coli only five target mRNAs have been shown to be directly regulated by CsrA at the post-transcriptional level. Here we identified two new direct targets for CsrA, ycdT and ydeH, both of which encode proteins with GGDEF domains. A csrA mutation caused mRNA levels of ycdT and ydeH to increase more than 10-fold. RNA mobility shift assays confirmed the direct and specific binding of CsrA to the mRNA leaders of ydeH and ycdT. Overexpression of ycdT and ydeH resulted in a more than 20-fold increase in the cellular concentration of the second messenger cyclic di-GMP (c-di-GMP), implying that both proteins possess diguanylate cyclase activity. Phenotypic characterization revealed that both proteins are involved in the regulation of motility in a c-di-GMP-dependent manner. CsrA was also found to regulate the expression of five additional GGDEF/EAL proteins and a csrA mutation led to modestly increased cellular levels of c-di-GMP. All together, these data demonstrate a global role for CsrA in the regulation of c-di-GMP metabolism by regulating the expression of GGDEF proteins at the post-transcriptional level.

  10. Three cdg Operons Control Cellular Turnover of Cyclic Di-GMP in Acetobacter xylinum: Genetic Organization and Occurrence of Conserved Domains in Isoenzymes

    PubMed Central

    Tal, Rony; Wong, Hing C.; Calhoon, Roger; Gelfand, David; Fear, Anna Lisa; Volman, Gail; Mayer, Raphael; Ross, Peter; Amikam, Dorit; Weinhouse, Haim; Cohen, Avital; Sapir, Shai; Ohana, Patricia; Benziman, Moshe

    1998-01-01

    Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of β-1,4-glucan (cellulose) synthase in Acetobacter xylinum. The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate, cdg1, cdg2, and cdg3. Within each cdg operon, a pdeA gene lies upstream of a dgc gene. cdg1 contains two additional flanking genes, cdg1a and cdg1d. cdg1a encodes a putative transcriptional activator, similar to AadR of Rhodopseudomonas palustris and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the cdg operons, such that cdg1 contributes 80% of cellular DGC and PDEA activities and cdg2 and cdg3 contribute 15 and 5%, respectively. Disruption of dgc genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process. PMID:9721278

  11. Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes.

    PubMed

    Tal, R; Wong, H C; Calhoon, R; Gelfand, D; Fear, A L; Volman, G; Mayer, R; Ross, P; Amikam, D; Weinhouse, H; Cohen, A; Sapir, S; Ohana, P; Benziman, M

    1998-09-01

    Cyclic di-GMP (c-di-GMP) is the specific nucleotide regulator of beta-1,4-glucan (cellulose) synthase in Acetobacter xylinum. The enzymes controlling turnover of c-di-GMP are diguanylate cyclase (DGC), which catalyzes its formation, and phosphodiesterase A (PDEA), which catalyzes its degradation. Following biochemical purification of DGC and PDEA, genes encoding isoforms of these enzymes have been isolated and found to be located on three distinct yet highly homologous operons for cyclic diguanylate, cdg1, cdg2, and cdg3. Within each cdg operon, a pdeA gene lies upstream of a dgc gene. cdg1 contains two additional flanking genes, cdg1a and cdg1d. cdg1a encodes a putative transcriptional activator, similar to AadR of Rhodopseudomonas palustris and FixK proteins of rhizobia. The deduced DGC and PDEA proteins have an identical motif structure of two lengthy domains in their C-terminal regions. These domains are also present in numerous bacterial proteins of undefined function. The N termini of the DGC and PDEA deduced proteins contain putative oxygen-sensing domains, based on similarity to domains on bacterial NifL and FixL proteins, respectively. Genetic disruption analyses demonstrated a physiological hierarchy among the cdg operons, such that cdg1 contributes 80% of cellular DGC and PDEA activities and cdg2 and cdg3 contribute 15 and 5%, respectively. Disruption of dgc genes markedly reduced in vivo cellulose production, demonstrating that c-di-GMP controls this process.

  12. Control of the light-regulated current in rod photoreceptors by cyclic GMP, calcium, and l-cis-diltiazem.

    PubMed Central

    Stern, J H; Kaupp, U B; MacLeish, P R

    1986-01-01

    The effect of calcium ions on the cGMP-activated current of outer segment membrane was examined by the excised-patch technique. Changes in the extracellular calcium concentration had marked effects on the cGMP-activated current, while changes in intracellular calcium concentration were ineffective. Changes in calcium concentration in the absence of cGMP had little, if any, effect on membrane conductance. These results suggest that both intracellular cGMP and extracellular calcium can directly affect the conductance underlying the light response in rod cells. The pharmacological agent l-cis-diltiazem reversibly inhibited the cGMP-activated current when applied to the intracellular side of an excised patch. When superfused over intact rod cells, l-cis-diltiazem reversibly blocked much of the normal light response. The isomer, d-cis-diltiazem, did not significantly affect either patches or intact rod cells. Thus, the light-regulated conductance has binding sites for both calcium and cGMP that may interact during the normal light response in rod cells and a site specific for l-cis-diltiazem that can be used to identify and further study the conductance mechanism. PMID:3006029

  13. Control of the light-regulated current in rod photoreceptors by cyclic GMP, calcium, and l-cis-diltiazem.

    PubMed

    Stern, J H; Kaupp, U B; MacLeish, P R

    1986-02-01

    The effect of calcium ions on the cGMP-activated current of outer segment membrane was examined by the excised-patch technique. Changes in the extracellular calcium concentration had marked effects on the cGMP-activated current, while changes in intracellular calcium concentration were ineffective. Changes in calcium concentration in the absence of cGMP had little, if any, effect on membrane conductance. These results suggest that both intracellular cGMP and extracellular calcium can directly affect the conductance underlying the light response in rod cells. The pharmacological agent l-cis-diltiazem reversibly inhibited the cGMP-activated current when applied to the intracellular side of an excised patch. When superfused over intact rod cells, l-cis-diltiazem reversibly blocked much of the normal light response. The isomer, d-cis-diltiazem, did not significantly affect either patches or intact rod cells. Thus, the light-regulated conductance has binding sites for both calcium and cGMP that may interact during the normal light response in rod cells and a site specific for l-cis-diltiazem that can be used to identify and further study the conductance mechanism.

  14. Pharmacological manipulation of cyclic GMP levels in brain restores learning ability in animal models of hepatic encephalopathy: therapeutic implications

    PubMed Central

    Rodrigo, Regina; Monfort, Pilar; Cauli, Omar; Erceg, Slaven; Felipo, Vicente

    2006-01-01

    Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome present in patients with liver disease that includes impaired intellectual function. To develop therapeutic treatments to restore cognitive function, it is important to understand the molecular mechanisms that impair cognitive function in HE. This review summarizes data showing that: (a) cognitive function and learning are impaired in patients with liver disease and in animal models of chronic liver failure or hyperammonemia; (b) the glutamate–NO–cGMP pathway modulates some forms of learning; and (c) the function of this pathway is impaired in brain in vivo in rats with chronic hyperammonemia or liver failure and from patients who died from HE. Learning ability of hyperammonemic rats was restored by increasing cGMP by: (1) continuous intracerebral administration of zaprinast, an inhibitor of the cGMP-degrading phosphodiesterase; (2) chronic oral administration of sildenafil, an inhibitor of the phosphodiesterase that crosses the blood–brain barrier; and (3) continuous intracerebral administration of cGMP. The data summarized indicate that impairment of learning ability in rats with chronic liver failure or hyperammonemia is due to impairment of the glutamate–NO–cGMP pathway. Moreover, increasing extracellular cGMP by pharmacological means may be a new therapeutic approach to improve cognitive function in patients with HE. PMID:19412446

  15. Nitric oxide stimulates human sperm motility via activation of the cyclic GMP/protein kinase G signaling pathway.

    PubMed

    Miraglia, Erica; De Angelis, Federico; Gazzano, Elena; Hassanpour, Hossain; Bertagna, Angela; Aldieri, Elisabetta; Revelli, Alberto; Ghigo, Dario

    2011-01-01

    Nitric oxide (NO), a modulator of several physiological processes, is involved in different human sperm functions. We have investigated whether NO may stimulate the motility of human spermatozoa via activation of the soluble guanylate cyclase (sGC)/cGMP pathway. Sperm samples obtained by masturbation from 70 normozoospermic patients were processed by the swim-up technique. The kinetic parameters of the motile sperm-rich fractions were assessed by computer-assisted sperm analysis. After a 30-90  min incubation, the NO donor S-nitrosoglutathione (GSNO) exerted a significant enhancing effect on progressive motility (77, 78, and 78% vs 66, 65, and 62% of the control at the corresponding time), straight linear velocity (44, 49, and 48 μm/s vs 34, 35, and 35.5 μm/s), curvilinear velocity (81, 83, and 84 μm/s vs 68 μm/s), and average path velocity (52, 57, and 54 μm/s vs 40, 42, and 42 μm/s) at 5 μM but not at lower concentrations, and in parallel increased the synthesis of cGMP. A similar effect was obtained with the NO donor spermine NONOate after 30 and 60  min. The GSNO-induced effects on sperm motility were abolished by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (a specific sGC inhibitor) and mimicked by 8-bromo-cGMP (8-Br-cGMP; a cell-permeating cGMP analog); the treatment with Rp-8-Br-cGMPS (an inhibitor of cGMP-dependent protein kinases) prevented both the GSNO- and the 8-Br-cGMP-induced responses. On the contrary, we did not observe any effect of the cGMP/PRKG1 (PKG) pathway modulators on the onset of hyperactivated sperm motility. Our results suggest that NO stimulates human sperm motility via the activation of sGC, the subsequent synthesis of cGMP, and the activation of cGMP-dependent protein kinases.

  16. Regulation of cyclic GMP metabolism in toad photoreceptors. Definition of the metabolic events subserving photoexcited and attenuated states

    SciTech Connect

    Dawis, S.M.; Graeff, R.M.; Heyman, R.A.; Walseth, T.F.; Goldberg, N.D.

    1988-06-25

    Photoreceptor metabolism of cGMP and its regulation were characterized in isolated toad retinas by determining the intensity and time dependence of light-induced changes in the following metabolic parameters: cGMP hydrolytic flux determined by the rate of 18O incorporation from 18O-water into retinal guanine nucleotide alpha-phosphoryls; changes in the total concentrations of the guanine nucleotide metabolic intermediates; and changes in the concentration of metabolic GDP calculated from the fraction of the alpha-GDP that undergoes labeling with 18O. With narrow band 500 nm light that preferentially stimulates red rod photoreceptors, a range of intensities covering approximately 5 log units produced increases of over 10-fold in cGMP metabolic flux. However, the characteristics of the cGMP metabolic response over the first 2.5 log units of intensity are readily distinguishable from those at higher intensities which exhibit progressive attenuation by an intensity- and time-dependent process. Over the range of low intensities the metabolic response is characterized by 1) increases in cGMP hydrolytic flux of up to 8-fold as a logarithmic function of intensity of photic stimulation that are sustained for at least 200 s; 2) small increases or no change in the concentration of total cGMP; 3) large increases of up to 10-fold in the concentration of metabolically active GDP as a linear function of intensity with no significant change in the tissue concentrations of total GDP or GTP; and 4) amplification of the photosignal by the metabolism of approximately 10,000 molecules of cGMP per photoisomerization with the major site of amplification at the level of the interaction of bleached rhodopsin with G-protein.

  17. The cyclic-di-GMP diguanylate cyclase CdgA has a role in biofilm formation and exopolysaccharide production in Azospirillum brasilense.

    PubMed

    Ramírez-Mata, Alberto; López-Lara, Lilia I; Xiqui-Vázquez, Ma Luisa; Jijón-Moreno, Saúl; Romero-Osorio, Angelica; Baca, Beatriz E

    2016-04-01

    In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway.

  18. Crystal structure of cGMP-dependent protein kinase Iβ cyclic nucleotide-binding-B domain : Rp-cGMPS complex reveals an apo-like, inactive conformation.

    PubMed

    Campbell, James C; VanSchouwen, Bryan; Lorenz, Robin; Sankaran, Banumathi; Herberg, Friedrich W; Melacini, Giuseppe; Kim, Choel

    2017-01-01

    The R-diastereomer of phosphorothioate analogs of cGMP, Rp-cGMPS, is one of few known inhibitors of cGMP-dependent protein kinase I (PKG I); however, its mechanism of inhibition is currently not fully understood. Here, we determined the crystal structure of the PKG Iβ cyclic nucleotide-binding domain (PKG Iβ CNB-B), considered a 'gatekeeper' for cGMP activation, bound to Rp-cGMPS at 1.3 Å. Our structural and NMR data show that PKG Iβ CNB-B bound to Rp-cGMPS displays an apo-like structure with its helical domain in an open conformation. Comparison with the cAMP-dependent protein kinase regulatory subunit (PKA RIα) showed that this conformation resembles the catalytic subunit-bound inhibited state of PKA RIα more closely than the apo or Rp-cAMPS-bound conformations. These results suggest that Rp-cGMPS inhibits PKG I by stabilizing the inactive conformation of CNB-B. © 2016 Federation of European Biochemical Societies.

  19. Rat Hippocampal Neurons Express Genes for Both Rod Retinal and Olfactory Cyclic Nucleotide-Gated Channels: Novel Targets for cAMP/cGMP Function

    NASA Astrophysics Data System (ADS)

    Kingston, Paul A.; Zufall, Frank; Barnstable, Colin J.

    1996-09-01

    Cyclic nucleotide-gated (CNG) channels are Ca2+-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

  20. T-1032, a cyclic GMP phosphodiesterase-5 inhibitor, acutely blocks physiologic insulin-mediated muscle haemodynamic effects and glucose uptake in vivo.

    PubMed

    Mahajan, Hema; Richards, Stephen M; Rattigan, Stephen; Clark, Michael G

    2003-12-01

    1. Cyclic GMP phosphodiesterase-5 inhibitors have been shown to alter blood flow in specific tissues by potentiating local NO-dependent vasodilatory mechanisms. Since the haemodynamic effects of physiologic insulin, particularly capillary recruitment, may be critical for muscle glucose uptake in vivo and are blocked by inhibitors of nitric oxide synthase, we have explored the acute effects of the specific cGMP phosphodiesterase-5 inhibitor T-1032 on physiologic insulin action in anaesthetized healthy rats in vivo. 2. Whole-body glucose infusion (GIR), femoral blood flow (FBF), hind leg vascular resistance (VR), hind leg glucose uptake (HGU), 2-deoxyglucose uptake into muscles of the lower leg (R'g), hind leg metabolism of infused 1-methylxanthine (1-MX), a measure of capillary recruitment, and muscle cGMP were determined. The experimental groups were T-1032 (10 microg min-1 kg-1) infused for 1 h before and during a euglycaemic insulin clamp (3 mU min-1 kg-1 x 2 h), T-1032 infused for 3 h with saline, T-1032 during a 2 h clamp, T-1032 with saline for 2 h, and a 2 h saline control. 3. Insulin increased GIR from zero to 13 mg min-1 kg-1, HGU from 0.1+/-0.01 to 0.43+/-0.05 micromol min-1, R'g and 1-MX, marginally increased FBF, and had no effect on blood pressure or heart rate. T-1032 alone had no effect on blood pressure, heart rate, FBF, VR, HGU, R'g or 1-MX, but increased muscle cGMP. T-1032 1 h before and during insulin completely blocked GIR (1 h), HGU (2 h), R'g (2 h), and 1-MX (2 h). T-1032 commenced with insulin had only partial blocking activity against insulin. 4. We conclude that T-1032 is a potent acutely acting inhibitor of the muscle effects of physiologic insulin on capillary recruitment and glucose uptake in vivo. These, together with inhibition of whole-body glucose infusion during insulin, may caution against the use of isoenzyme-5-specific cyclic GMP phosphodiesterase inhibitors as therapeutic agents.

  1. Host cell contact induces expression of virulence factors and VieA, a cyclic di-GMP phosphodiesterase, in Vibrio cholerae.

    PubMed

    Dey, Amit K; Bhagat, Abha; Chowdhury, Rukhsana

    2013-05-01

    Vibrio cholerae, a noninvasive bacterium, colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes the severe fluid loss characteristic of the disease cholera. In this study, we demonstrate that adherence of V. cholerae to the intestinal epithelial cell line INT 407 strongly induces the expression of the major virulence genes ctxAB and tcpA and the virulence regulatory gene toxT. No induction of toxR and tcpP, which encode transcriptional activators of toxT, was observed in adhered bacteria, and the adherence-dependent upregulation of toxT expression was independent of ToxR and TcpP. A sharp increase in the expression of the vieA gene, which encodes a cyclic di-GMP (c-di-GMP) phosphodiesterase, was observed in INT 407-adhered V. cholerae immediately after infection. Induction of toxT, ctxAB, and tcpA in INT 407-adhered vieA mutant strain O395 ΔvieA was consistently lower than in the parent strain, although no effect was observed in unadhered bacteria, suggesting that VieA has a role in the upregulation of toxT expression specifically in host cell-adhered V. cholerae. Furthermore, though VieA has both a DNA binding helix-turn-helix domain and an EAL domain conferring c-di-GMP phosphodiesterase activity, the c-di-GMP phosphodiesterase activity of VieA is necessary and sufficient for the upregulation of toxT expression.

  2. Cyclic unequal error protection codes constructed from cyclic codes of composite length

    NASA Technical Reports Server (NTRS)

    Lin, Shu

    1987-01-01

    The distance structure of cyclic codes of composite length was investigated. A lower bound on the minimum distance for this class of codes is derived. In many cases, the lower bound gives the true minimum distance of a code. Then the distance structure of the direct sum of two cyclic codes of composite length were investigated. It was shown that, under certain conditions, the direct-sum code provides two levels of error correcting capability, and hence is a two-level unequal error protection (UEP) code. Finally, a class of two-level UEP cyclic direct-sum codes and a decoding algorithm for a subclass of these codes are presented.

  3. Voltage-gated calcium channel currents in human coronary myocytes. Regulation by cyclic GMP and nitric oxide.

    PubMed Central

    Quignard, J F; Frapier, J M; Harricane, M C; Albat, B; Nargeot, J; Richard, S

    1997-01-01

    Voltage-gated Ca2+ channels contribute to the maintenance of contractile tone in vascular myocytes and are potential targets for vasodilating agents. There is no information available about their nature and regulation in human coronary arteries. We used the whole-cell voltage-clamp technique to characterize Ca2+-channel currents immediately after enzymatic dissociation and after primary culture of coronary myocytes taken from heart transplant patients. We recorded a dihydropyridine-sensitive L-type current in both freshly isolated and primary cultured cells. A T-type current was recorded only in culture. The L- (but not the T-) type current was inhibited by permeable analogues of cGMP in a dose-dependent manner. This effect was mimicked by the nitric oxide-generating agents S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholinosydnonimine which increased intracellular cGMP. Methylene blue, known to inhibit guanylate cyclase, antagonized the effect of SNAP. Inhibitions by SNAP and cGMP were not additive and seemed to occur through a common pathway. We conclude that (a) L-type Ca2+ channels are the major pathway for voltage-gated Ca2+ entry in human coronary myocytes; (b) their inhibition by agents stimulating nitric oxide and/or intracellular cGMP production is expected to contribute to vasorelaxation and may be involved in the therapeutic effect of nitrovasodilators; and (c) the expression of T-type Ca2+ channels in culture may be triggered by cell proliferation. PMID:9005986

  4. A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide

    PubMed Central

    Klaiber, Michael; Dankworth, Beatrice; Kruse, Martin; Hartmann, Michael; Nikolaev, Viacheslav O.; Yang, Ruey-Bing; Völker, Katharina; Gaßner, Birgit; Oberwinkler, Heike; Feil, Robert; Freichel, Marc; Groschner, Klaus; Skryabin, Boris V.; Frantz, Stefan; Birnbaumer, Lutz; Pongs, Olaf; Kuhn, Michaela

    2011-01-01

    Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca2+]i increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca2+ levels. This pathway involves the activation of Ca2+‐permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca2+ channels and ultimately increases myocyte Ca2+i levels. These observations reveal a dual role of the ANP/GC-A–signaling pathway in the regulation of cardiac myocyte Ca2+i homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca2+i-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca2+]i might increase the propensity to cardiac hypertrophy and arrhythmias. PMID:22027011

  5. Study on the cyclic GMP-dependency of relaxations to endogenous and exogenous nitric oxide in the mouse gastrointestinal tract.

    PubMed

    De Man, J G; De Winter, B Y; Herman, A G; Pelckmans, P A

    2007-01-01

    cGMP mediates nitrergic relaxations of intestinal smooth muscle, but several studies have indicated that cGMP-independent mechanisms may also be involved. We addressed this contention by studying the effect of ODQ and ns2028, specific inhibitors of soluble guanylate cyclase, on nitrergic relaxations of the mouse gut. Mouse gastric fundus and small intestinal muscle preparations were mounted in organ baths to study relaxations to exogenous NO, NO donors and electrical field stimulation (EFS) of enteric nerves. In gastric fundus longitudinal muscle strips, ODQ and NS2028 abolished the L-nitroarginine-sensitive relaxations to EFS and the relaxations to NO and NO donors, glyceryl trinitrate (GTN), SIN-1 and sodium nitroprusside (SNP). EFS of intestinal segments and muscle strips showed L-nitroarginine-resistant relaxations, which were abolished by the purinoceptor blocker suramin. In the presence of suramin, ODQ and NS2028 abolished all relaxations to EFS in intestinal segments and strips. ODQ and NS2028 abolished the relaxations to exogenous NO and to the NO donors GTN, SIN-1 and SNP in circular and longitudinal intestinal muscle strips. Intestinal segments showed residual relaxations to NO and GTN. Our results indicate that relaxations to endogenous NO in the mouse gastric fundus and small intestine are completely dependent on cGMP. ODQ and NS2028 incompletely blocked nitrergic relaxations to exogenous NO in intact intestinal segments. However, it is unlikely that this is due to the involvement of cGMP-independent pathways because ODQ and NS2028 abolished all relaxations to endogenous and exogenous NO in intestinal muscle strips.

  6. Sodium depletion enhances renal expression of (pro)renin receptor via cyclic GMP-protein kinase G signaling pathway.

    PubMed

    Huang, Jiqian; Siragy, Helmy M

    2012-02-01

    (Pro)renin receptor (PRR) is expressed in renal vasculature, glomeruli, and tubules. The physiological regulation of this receptor is not well established. We hypothesized that sodium depletion increases PRR expression through cGMP- protein kinase G (PKG) signaling pathway. Renal PRR expressions were evaluated in Sprague-Dawley rats on normal sodium or low-sodium diet (LS) and in cultured rat proximal tubular cells and mouse renal inner medullary collecting duct cells exposed to LS concentration. LS augmented PRR expression in renal glomeruli, proximal tubules, distal tubules, and collecting ducts. LS also increased cGMP production and PKG activity. In cells exposed to normal sodium, cGMP analog increased PKG activity and upregulated PRR expression. In cells exposed to LS, blockade of guanylyl cyclase with 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one decreased PKG activity and downregulated PRR expression. PKG inhibition decreased phosphatase protein phosphatase 2A activity; suppressed LS-mediated phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, c-Jun, and nuclear factor-κB p65; and attenuated LS-mediated PRR upregulation. LS also enhanced DNA binding of cAMP response element binding protein 1 to cAMP response elements, nuclear factor-κB p65 to nuclear factor-κB elements, and c-Jun to activator protein 1 elements in PRR promoter in proximal tubular cells. We conclude that sodium depletion upregulates renal PRR expression via the cGMP-PKG signaling pathway by enhancing binding of cAMP response element binding protein 1, nuclear factor-κB p65, and c-Jun to PRR promotor.

  7. Stimulation of cyclic GMP production via AT2 and B2 receptors in the pressure-overloaded aorta after banding.

    PubMed

    Hiyoshi, Hiromi; Yayama, Katsutoshi; Takano, Masaoki; Okamoto, Hiroshi

    2004-06-01

    Abdominal aortic banding induces upregulation of the angiotensin II (Ang II) type-2 (AT2) receptor, thereby decreasing the contractile response to Ang II in the thoracic aorta of the rat. The aim of this study was to use a mouse model to clarify the mechanisms by which the banding elicits upregulation of the aortic AT2 receptor and the subsequent attenuation of Ang II responsiveness. Concomitantly with the elevation in blood pressure and plasma renin concentration after banding, AT2-receptor mRNA levels in the thoracic aorta rapidly increased in mice within 4 days. Upregulation of the AT2 receptor, as well as blood pressure elevation after banding, was abolished by losartan administration. The contractile response to Ang II was depressed in aortic rings of banding mice but not of sham mice, and was restored by either the AT2-receptor antagonist PD123319 or the bradykinin B2-receptor antagonist icatibant. cGMP content in the thoracic aorta of banding mice was 9-fold greater than that of sham mice, and the elevation was reduced to sham levels 1 hour after intravenous injection of PD123319 or icatibant. When aortic rings were incubated with Ang II, cGMP content increased in banding rings but not in sham rings; the pretreatment with PD123319 or icatibant inhibited Ang II-induced cGMP production. These results suggest that aortic banding induces upregulation of the AT2 receptor through increased circulating Ang II via the AT1 receptor, thereby activating a vasodilatory pathway in vessels through the AT2 receptor via the kinin/cGMP system.

  8. Role of cyclic GMP in cells with the properties of smooth muscle cultured from the rat myometrium

    SciTech Connect

    Krall, J.F.; Morin, A.

    1986-11-01

    Cells growing in culture with previously described properties of rat uterine smooth muscle accumulated /sup 45/Ca/sup 2 +/ from the medium. Ca/sup 2 +/ uptake by these cells was stimulated by the addition to the medium of 8-bromo-cGMP but not by 8-bromo-cAMP. Ca/sup 2 +/ uptake was also stimulated by carbachol and by the nitro-vasodilator nitroprusside. Although cholinergic agonists have been shown previously to stimulate contraction but not cGMP synthesis in the rat myometrium, both carbachol and nitroprusside stimulated cGMP production by the cultured cells. These results suggested the cells had cholinergic receptor-medicated functions that reflected some neurotransmitter-sensitive properties of uterine smooth muscle in situ. When determined by a specific radioligand binding assay, subcellular fractions of the cultured cells bound muscarinic cholinergic agonists and antagonists with affinities expected of the muscarinic receptor. The cells were also sensitive to the ..beta..-adrenergic catecholamine agonist isoproterenol, which stimulated cAMP production but not Ca/sup 2 +/ uptake. Carbachol failed to inhibit isoproterenol-dependent cAMP production, which is an important property of the cholinergic receptor in uterine smooth muscle in situ. These results suggest some but not all acetylcholine-sensitive properties of uterine smooth muscle may be retained in cell culture.

  9. Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens.

    PubMed

    Hendrick, William A; Orr, Mona W; Murray, Samantha R; Lee, Vincent T; Melville, Stephen B

    2017-05-15

    The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens, called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium.IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in

  10. Phenotype Overlap in Xylella fastidiosa Is Controlled by the Cyclic Di-GMP Phosphodiesterase Eal in Response to Antibiotic Exposure and Diffusible Signal Factor-Mediated Cell-Cell Signaling

    PubMed Central

    de Souza, Alessandra A.; Ionescu, Michael; Baccari, Clelia; da Silva, Aline M.

    2013-01-01

    Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3′-5′)-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates. PMID:23542613

  11. Phenotype overlap in Xylella fastidiosa is controlled by the cyclic di-GMP phosphodiesterase Eal in response to antibiotic exposure and diffusible signal factor-mediated cell-cell signaling.

    PubMed

    de Souza, Alessandra A; Ionescu, Michael; Baccari, Clelia; da Silva, Aline M; Lindow, Steven E

    2013-06-01

    Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3'-5')-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.

  12. The nitric oxide-cyclic GMP-protein kinase G-K+ channel pathway participates in the antiallodynic effect of spinal gabapentin.

    PubMed

    Mixcoatl-Zecuatl, Teresa; Flores-Murrieta, Francisco J; Granados-Soto, Vinicio

    2006-02-15

    The possible participation of the nitric oxide (NO)-cyclic GMP-protein kinase G (PKG) pathway on gabapentin-induced spinal antiallodynic activity was assessed in spinal nerve injured rats. Intrathecal gabapentin, diazoxide or pinacidil reduced tactile allodynia in a dose-dependent manner. Pretreatment with NG-L-nitro-arginine methyl ester (L-NAME, non-specific inhibitor of NO synthase NOS), 7-nitroindazole (neuronal NO synthase inhibitor), 1H-[1,2,4] -oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, guanylyl cyclase inhibitor) or (9S, 10R, 12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT-5823, specific PKG inhibitor), but not NG-D-nitro-arginine methyl ester (D-NAME) or okadaic acid (protein phosphatase 1 and 2 inhibitor) prevented gabapentin-induced antiallodynia. Pinacidil activity was not blocked by L-NAME, D-NAME, 7-nitroindazole, ODQ, KT-5823 or okadaic acid. Moreover, KT-5823, glibenclamide (ATP-sensitive K+ channel blocker), apamin and charybdotoxin (small- and large-conductance Ca2+-activated K+ channel blockers, respectively), but not margatoxin (voltage-gated K+ channel blocker), L-NAME, 7-nitroindazole, ODQ or okadaic acid, reduced diazoxide-induced antiallodynia. Data suggest that gabapentin-induced spinal antiallodynia could be due to activation of the NO-cyclic GMP-PKG-K+ channel pathway.

  13. Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

    PubMed

    Holt, Andrew W; Martin, Danielle N; Shaver, Patti R; Adderley, Shaquria P; Stone, Joshua D; Joshi, Chintamani N; Francisco, Jake T; Lust, Robert M; Weidner, Douglas A; Shewchuk, Brian M; Tulis, David A

    2016-09-01

    Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls

  14. Positive autoregulation of mrkHI by the cyclic di-GMP-dependent MrkH protein in the biofilm regulatory circuit of Klebsiella pneumoniae.

    PubMed

    Tan, Jason W H; Wilksch, Jonathan J; Hocking, Dianna M; Wang, Nancy; Srikhanta, Yogitha N; Tauschek, Marija; Lithgow, Trevor; Robins-Browne, Roy M; Yang, Ji; Strugnell, Richard A

    2015-05-01

    Klebsiella pneumoniae is an important cause of nosocomial infections, primarily through the formation of surface-associated biofilms to promote microbial colonization on host tissues. Expression of type 3 fimbriae by K. pneumoniae facilitates surface adherence, a process strongly activated by the cyclic di-GMP (c-di-GMP)-dependent transcriptional activator MrkH. In this study, we demonstrated the critical importance of MrkH in facilitating K. pneumoniae attachment on a variety of medically relevant materials and demonstrated the mechanism by which bacteria activate expression of type 3 fimbriae to colonize these materials. Sequence analysis revealed a putative MrkH recognition DNA sequence ("MrkH box"; TATCAA) located in the regulatory region of the mrkHI operon. Mutational analysis, electrophoretic mobility shift assay, and quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA sequence to autoregulate mrkHI expression in a c-di-GMP-dependent manner. A half-turn deletion, but not a full-turn deletion, between the MrkH box and the -35 promoter element rendered MrkH ineffective in activating mrkHI expression, implying that a direct interaction between MrkH and RNA polymerase exists. In vivo analyses showed that residues L260, R265, N268, C269, E273, and I275 in the C-terminal domain of the RNA polymerase α subunit are involved in the positive control of mrkHI expression by MrkH and revealed the regions of MrkH required for DNA binding and transcriptional activation. Taken together, the data suggest a model whereby c-di-GMP-dependent MrkH recruits RNA polymerase to the mrkHI promoter to autoactivate mrkH expression. Increased MrkH production subsequently drives mrkABCDF expression when activated by c-di-GMP, leading to biosynthesis of type 3 fimbriae and biofilm formation. Bacterial biofilms can cause persistent infections that are refractory to antimicrobial treatments. This study investigated how a commonly encountered hospital

  15. Positive Autoregulation of mrkHI by the Cyclic Di-GMP-Dependent MrkH Protein in the Biofilm Regulatory Circuit of Klebsiella pneumoniae

    PubMed Central

    Tan, Jason W. H.; Hocking, Dianna M.; Wang, Nancy; Srikhanta, Yogitha N.; Tauschek, Marija; Lithgow, Trevor; Robins-Browne, Roy M.; Yang, Ji; Strugnell, Richard A.

    2015-01-01

    ABSTRACT Klebsiella pneumoniae is an important cause of nosocomial infections, primarily through the formation of surface-associated biofilms to promote microbial colonization on host tissues. Expression of type 3 fimbriae by K. pneumoniae facilitates surface adherence, a process strongly activated by the cyclic di-GMP (c-di-GMP)-dependent transcriptional activator MrkH. In this study, we demonstrated the critical importance of MrkH in facilitating K. pneumoniae attachment on a variety of medically relevant materials and demonstrated the mechanism by which bacteria activate expression of type 3 fimbriae to colonize these materials. Sequence analysis revealed a putative MrkH recognition DNA sequence (“MrkH box”; TATCAA) located in the regulatory region of the mrkHI operon. Mutational analysis, electrophoretic mobility shift assay, and quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA sequence to autoregulate mrkHI expression in a c-di-GMP-dependent manner. A half-turn deletion, but not a full-turn deletion, between the MrkH box and the −35 promoter element rendered MrkH ineffective in activating mrkHI expression, implying that a direct interaction between MrkH and RNA polymerase exists. In vivo analyses showed that residues L260, R265, N268, C269, E273, and I275 in the C-terminal domain of the RNA polymerase α subunit are involved in the positive control of mrkHI expression by MrkH and revealed the regions of MrkH required for DNA binding and transcriptional activation. Taken together, the data suggest a model whereby c-di-GMP-dependent MrkH recruits RNA polymerase to the mrkHI promoter to autoactivate mrkH expression. Increased MrkH production subsequently drives mrkABCDF expression when activated by c-di-GMP, leading to biosynthesis of type 3 fimbriae and biofilm formation. IMPORTANCE Bacterial biofilms can cause persistent infections that are refractory to antimicrobial treatments. This study investigated how a commonly

  16. Expression and action of cyclic GMP-dependent protein kinase Ialpha in inflammatory hyperalgesia in rat spinal cord.

    PubMed

    Tao, Y X; Hassan, A; Haddad, E; Johns, R A

    2000-01-01

    Several lines of evidence have shown a role for the nitric oxide/cyclic guanosine monophosphate signaling pathway in the development of spinal hyperalgesia. However, the roles of effectors for cyclic guanosine monophosphate are not fully understood in the processing of pain in the spinal cord. The present study showed that cyclic guanosine monophosphate-dependent protein kinase Ialpha but not Ibeta was localized in the neuronal bodies and processes, and was distributed primarily in the superficial laminae of the spinal cord. Intrathecal administration of a selective inhibitor of cyclic guanosine monophosphate-dependent protein kinase Ialpha, Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine, produced a significant antinociception demonstrated by the decrease in the number of flinches and shakes in the formalin test. This was accompanied by a marked reduction in formalin-induced c-fos expression in the spinal dorsal horn. Moreover, cyclic guanosine monophosphate-dependent protein kinase Ialpha protein expression was dramatically increased in the lumbar spinal cord 96 h after injection of formalin into a hindpaw, which occurred mainly in the superficial laminae on the ipsilateral side of a formalin-injected hindpaw. This up-regulation of cyclic guanosine monophosphate-dependent protein kinase Ialpha expression was completely blocked not only by a neuronal nitric oxide synthase inhibitor, 7-nitroindazole, and a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, but also by an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate (MK-801). The present results indicate that noxious stimulation not only initially activates but also later up-regulates cyclic guanosine monophosphate-dependent protein kinase Ialpha expression in the superficial laminae via an N-methyl-D-aspartate-nitric oxide-cyclic guanosine monophosphate signaling pathway, suggesting that cyclic guanosine monophosphate-dependent protein kinase Ialpha may play an

  17. cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice*

    PubMed Central

    Ma, Hongwei; Butler, Michael R.; Thapa, Arjun; Belcher, Josh; Yang, Fan; Baehr, Wolfgang; Biel, Martin; Michalakis, Stylianos; Ding, Xi-Qin

    2015-01-01

    Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca2+ channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3−/−/Nrl−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca2+ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency. PMID:26124274

  18. Presynaptically Localized Cyclic GMP-Dependent Protein Kinase 1 Is a Key Determinant of Spinal Synaptic Potentiation and Pain Hypersensitivity

    PubMed Central

    Luo, Ceng; Gangadharan, Vijayan; Bali, Kiran Kumar; Xie, Rou-Gang; Agarwal, Nitin; Kurejova, Martina; Tappe-Theodor, Anke; Tegeder, Irmgard; Feil, Susanne; Lewin, Gary; Polgar, Erika; Todd, Andrew J.; Schlossmann, Jens; Hofmann, Franz; Liu, Da-Lu; Hu, San-Jue; Feil, Robert; Kuner, Thomas; Kuner, Rohini

    2012-01-01

    Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere (SNS-PKG-I−/− mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I−/− mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4,5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I−/− mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are

  19. Ghrelin protects human umbilical vein endothelial cells against advanced glycation end products-induced apoptosis via NO/cGMP signaling

    PubMed Central

    Li, Pengjie; Liu, Ying; Xiang, Ying; Lin, Miao; Gao, Jinling

    2015-01-01

    Objectives: The aim of this study was to investigate the intracellular mechanism involved in the anti-apoptotic effect of ghrelin on human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were pretreated with ghrelin before exposure to 200 μg/ml advanced glycation end products (AGEs)-BSA for 48 h. Cell viability and apoptosis were determined by MTT assay and Annexin V/PI staining. Intracellular cGMP levels evaluation and cGMP analogs were employed to explore possible mechanisms. Results: The inhibitory effect on AGEs induced HUVECs apoptosis could be exerted by ghrelin and co-incubation with growth hormone secretagogue receptor (GHSR)-1a antagonist [D-Lys3]-GHRP-6 abolished this inhibition. Decreased cGMP level in AGEs induced HUVECs apoptosis was restored by ghrelin pretreatment and abolished by [D-Lys3]-GHRP-6 co-incubation. cGMP analogs (8 Br-cGMP and DB-cGMP) pretreatment also exhibited inhibitory effect on AGEs induced HUVECs apoptosis. Conclusions: Our results demonstrated that ghrelin produces a protective effect on HUVECs through GHS-R1a and cGMP/NO signaling pathway mediates the effect of ghrelin. These observations suggest a novel intracellular mechanism in the process of AGEs induced HUVECs apoptosis. PMID:26629013

  20. Increased O2 consumption and positive inotropy caused by cyclic GMP reduction are not altered after L-type calcium channel blockade.

    PubMed

    Leone, R J; Naim, K L; Scholz, P M; Weiss, H R

    1998-01-01

    We tested the hypothesis that increased O2 consumption and inotropy after reduction of myocardial cyclic guanosine monophosphate (cGMP) are mediated through L-type calcium channels. Anesthetized, open-chest New Zealand white rabbits were divided into four groups. Hearts were exposed to control vehicle (n = 8); LY83583 (LY, 10(-3) mol/l, guanylate cyclase inhibitor, (n = 9); nifedipine (nif, 10(-4) mol/l, L-type calcium channel blocker, n = 8), or nif+LY (n = 6). Vehicle or compound was applied topically to the epicardium for 15 min. Subepicardial (EPI) blood flow increased (from 213 +/- 22 to 323 +/- 24 ml/ min/100 g) in the presence of LY, as did subendocardial (ENDO) blood flow (from 238 +/- 20 to 333 +/- 38 ml/min/ 100 g). O2 consumption increased in the presence of LY:18.0 +/- 1.0 (EPI) and 17.0 +/- 0.6 (ENDO) ml O2/min/100 g as compared with 9.5 +/- 2.0 (EPI) and 10.6 +/- 2.5 (ENDO) in the control group. The increase in O2 consumption with LY was undiminished in the presence of nif (nif+LY group 21.0 +/- 3.0 ml O2/min/100 g EPI and 22.1 +/- 3.8 ENDO). Nif alone decreased left ventricular dP/dtmax from (2,762 +/- 197 to 2,413 +/- 316 mm Hg/s) and maximal rate of change in wall thickness (dW/dtmax from 13.5 +/- 2.0 to 9.5 +/- 0.8 mm/s), while percent change of wall thickness (from 21.3 +/- 3.3 to 31.3 +/- 7.2) and dW/dtmax (from 13.3 +/- 3.0 to 15.3 +/- 2.3 mm/s) increased in the nif+LY group. Thus, the positive O2 consumption and inotropic effects of decreasing cGMP were undiminished by nif. These results suggest that the cGMP reduction induced increases in O2 consumption and that inotropy may not be mediated through L-type calcium channels.

  1. Cyclic-di-GMP binding induces structural rearrangements in the PlzA and PlzC proteins of the Lyme disease and relapsing fever spirochetes: a possible switch mechanism for c-di-GMP-mediated effector functions.

    PubMed

    Mallory, Katherine L; Miller, Daniel P; Oliver, Lee D; Freedman, John C; Kostick-Dunn, Jessica L; Carlyon, Jason A; Marion, James D; Bell, Jessica K; Marconi, Richard T

    2016-11-01

    The c-di-GMP network of Borrelia burgdorferi, a causative agent of Lyme disease, consists of Rrp1, a diguanylate cyclase/response regulator; Hpk1, a histidine kinase; PdeA and PdeB, c-di-GMP phosphodiesterases; and PlzA, a PilZ domain c-di-GMP receptor. Borrelia hermsii, a causative agent of tick-borne relapsing fever, possesses a putative c-di-GMP regulatory network that is uncharacterized. While B. burgdorferi requires c-di-GMP to survive within ticks, the associated effector mechanisms are poorly defined. Using site-directed mutagenesis, size exclusion chromatography, isothermal titration calorimetry and fluorescence resonance energy transfer, we investigate the interaction of c-di-GMP with the Borrelia PilZ domain-containing Plz proteins: B. burgdorferi PlzA and B. hermsii PlzC. The Plz proteins were determined to be monomeric in their apo and holo forms and to bind c-di-GMP with high affinity with a 1:1 stoichiometry. C-di-GMP binding induced structural rearrangements in PlzA and PlzC. C-di-GMP binding proved to be dependent on positive charge at R145 of the PilZ domain motif, R145xxxR. Comparative sequence analyses led to the identification of Borrelia consensus sequences for the PilZ domain signature motifs. This study provides insight into c-di-GMP:Plz receptor interaction and identifies a possible switch mechanism that may regulate Plz protein effector functions. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Cyclic GMP-dependent protein kinase-I localized in nociceptors modulates nociceptive cortical neuronal activity and pain hypersensitivity

    PubMed Central

    Gangadharan, Vijayan; Wang, Xu

    2017-01-01

    Chronic pain represents a frequent and poorly understood public health issue. Numerous studies have documented the key significance of plastic changes along the somatosensory pain pathways in chronic pain states. Our recent study demonstrated that the cGMP-dependent protein kinase I (PKG-I) specifically localized in nociceptors constitutes a key mediator of hyperexcitability of primary sensory neurons and spinal synaptic plasticity after inflammation. However, whether PKG-I in nociceptors further affects the cortical plasticity in the ascending pain pathways under pathological states has remained elusive. The immediate-early gene c-fos and phosphorylated ERK1/2 (pERK1/2) are considered reliable indicators for the neuronal activation status and it permits a comprehensive and large-scale observation of nociceptive neuronal activity along the ascending pain pathways subjected to tissue injury. In the present study, we systemically demonstrated that peripheral injury in PKG-Ifl/fl mice produced a significant upregulation of c-Fos or pERK1/2 over from the periphery to the cortex along the pain pathways, including dorsal root ganglion, spinal dorsal horn, ventral posterolateral thalamus, primary somatosensory hindlimb cortex, anterior cingulate cortex, basolateral amygdala, periaqueductal gray, and parabrachial nucleus. In contrast, very few cells in the above regions showed c-Fos or pERK1/2 induction in nociceptor-specific knockout mice lacking PKG-I (SNS-PKG-I−/− mice). Our results indicate that PKG-I expressed in nociceptors is not only a key determinant of dorsal root ganglion hyperexcitability and spinal synaptic plasticity but also an important modulator of cortical neuronal activity in pathological pain states and represent what we believe to be novel targets in the periphery for pain therapeutics. PMID:28326941

  3. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection

    PubMed Central

    Diner, Benjamin A.; Lum, Krystal K.; Toettcher, Jared E.

    2016-01-01

    ABSTRACT The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. PMID:27935834

  4. AMPK and Endothelial Nitric Oxide Synthase Signaling Regulates K-Ras Plasma Membrane Interactions via Cyclic GMP-Dependent Protein Kinase 2.

    PubMed

    Cho, Kwang-Jin; Casteel, Darren E; Prakash, Priyanka; Tan, Lingxiao; van der Hoeven, Dharini; Salim, Angela A; Kim, Choel; Capon, Robert J; Lacey, Ernest; Cunha, Shane R; Gorfe, Alemayehu A; Hancock, John F

    2016-12-15

    K-Ras must localize to the plasma membrane and be arrayed in nanoclusters for biological activity. We show here that K-Ras is a substrate for cyclic GMP-dependent protein kinases (PKGs). In intact cells, activated PKG2 selectively colocalizes with K-Ras on the plasma membrane and phosphorylates K-Ras at Ser181 in the C-terminal polybasic domain. K-Ras phosphorylation by PKG2 is triggered by activation of AMP-activated protein kinase (AMPK) and requires endothelial nitric oxide synthase and soluble guanylyl cyclase. Phosphorylated K-Ras reorganizes into distinct nanoclusters that retune the signal output. Phosphorylation acutely enhances K-Ras plasma membrane affinity, but phosphorylated K-Ras is progressively lost from the plasma membrane via endocytic recycling. Concordantly, chronic pharmacological activation of AMPK → PKG2 signaling with mitochondrial inhibitors, nitric oxide, or sildenafil inhibits proliferation of K-Ras-positive non-small cell lung cancer cells. The study shows that K-Ras is a target of a metabolic stress-signaling pathway that can be leveraged to inhibit oncogenic K-Ras function.

  5. N-3-Oxo-Decanoyl-l-Homoserine-Lactone Activates Auxin-Induced Adventitious Root Formation via Hydrogen Peroxide- and Nitric Oxide-Dependent Cyclic GMP Signaling in Mung Bean1[C][W][OA

    PubMed Central

    Bai, Xuegui; Todd, Christopher D.; Desikan, Radhika; Yang, Yongping; Hu, Xiangyang

    2012-01-01

    N-Acyl-homoserine-lactones (AHLs) are bacterial quorum-sensing signaling molecules that regulate population density. Recent evidence demonstrates their roles in plant defense responses and root development. Hydrogen peroxide (H2O2), nitric oxide (NO), and cyclic GMP (cGMP) are essential messengers that participate in various plant physiological processes, but how these messengers modulate the plant response to N-acyl-homoserine-lactone signals remains poorly understood. Here, we show that the N-3-oxo-decanoyl-homoserine-lactone (3-O-C10-HL), in contrast to its analog with an unsubstituted branch chain at the C3 position, efficiently stimulated the formation of adventitious roots and the expression of auxin-response genes in explants of mung bean (Vigna radiata) seedlings. This response was mimicked by the exogenous application of auxin, H2O2, NO, or cGMP homologs but suppressed by treatment with scavengers or inhibitors of H2O2, NO, or cGMP metabolism. The 3-O-C10-HL treatment enhanced auxin basipetal transport; this effect could be reversed by treatment with H2O2 or NO scavengers but not by inhibitors of cGMP synthesis. Inhibiting 3-O-C10-HL-induced H2O2 or NO accumulation impaired auxin- or 3-O-C10-HL-induced cGMP synthesis; however, blocking cGMP synthesis did not affect auxin- or 3-O-C10-HL-induced H2O2 or NO generation. Additionally, cGMP partially rescued the inhibitory effect of H2O2 or NO scavengers on 3-O-C10-HL-induced adventitious root development and auxin-response gene expression. These results suggest that 3-O-C10-HL, unlike its analog with an unmodified branch chain at the C3 position, can accelerate auxin-dependent adventitious root formation, possibly via H2O2- and NO-dependent cGMP signaling in mung bean seedlings. PMID:22138973

  6. [Progress in c-di-GMP inhibitors].

    PubMed

    Xiang, Xuwen; Liu, Xingyu; Tao, Hui; Cui, Zining; Zhang, Lianhui

    2017-09-25

    The cyclic dinucleotide c-di-GMP is known as an important second messenger in bacteria, which controls various important cellular processes, such as cell differentiation, biofilm formation and virulence factors production. It is extremely vital for the development of new antibacterial agents by virtue of blocking c-di-GMP signal conduction. Current research indicates that there are three potential targets for discovering new antibacterial agents based on c-di-GMP regulated signal pathway, which are c-di-GMP synthases, c-di-GMP degrading enzymes and c-di-GMP receptors. Herein, we review small molecules that have been developed to inhibit c-di-GMP related enzymes and indicate perspectives of c-di-GMP inhibitors.

  7. Nocturnal accumulation of cyclic 3',5'-guanosine monophosphate (cGMP) in the chick pineal organ is dependent on activation of guanylyl cyclase-B.

    PubMed

    Olcese, J; Majora, C; Stephan, A; Müller, D

    2002-01-01

    The role of cGMP in the avian pineal is not well understood. Although the light-sensitive secretion of melatonin is a well-known output of the circadian oscillator, pharmacologically elevated cGMP levels do not result in altered melatonin secretory amplitude or phase. This suggests that pineal cGMP signalling does not couple the endogenous circadian oscillator to the expression of melatonin rhythms. Nonetheless, the free-running rhythm of cGMP signalling implies a link to the circadian oscillator in chick pinealocytes. As the circadian rhythm of cGMP levels in vitro is not altered by pharmacological inhibition of phosphodiesterase activity, we infer that the synthesis, rather than the degradation of cGMP, is under circadian control. In vitro experiments with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine as well as with an inhibitor of the NO-sensitive soluble guanylyl cyclase (sGC), showed that the NOS-sGC pathway does not play a major role in the circadian control of cGMP generation. In organ culture experiments, we demonstrated that C-type natriuretic peptide (CNP), but not atrial natriuretic peptide (ANP), elevated daytime levels of cGMP. As CNP acts on the membrane guanylyl cyclase isoform B (GC-B), which is expressed at very high levels in mammalian pineals, we investigated the effect of the membrane GC-specific inhibitor HS-142 on chick pineal cGMP levels. CNP-induced daytime cGMP levels were reduced by HS-142. More importantly, 'spontaneously' high nocturnal levels of cGMP in vitro were reduced to daytime levels by acute addition of HS-142. These data implicate endogenous nocturnal CNP release and subsequent activation of GC-B as the major input driving cGMP synthesis in the chick pineal organ.

  8. Cellulose production, activated by cyclic di-GMP through BcsA and BcsZ, is a virulence factor and an essential determinant of the three-dimensional architectures of biofilms formed by Erwinia amylovora Ea1189.

    PubMed

    Castiblanco, Luisa F; Sundin, George W

    2016-10-18

    Bacterial biofilms are multicellular aggregates encased in an extracellular matrix mainly composed of exopolysaccharides (EPSs), protein and nucleic acids, which determines the architecture of the biofilm. Erwinia amylovora Ea1189 forms a biofilm inside the xylem of its host, which results in vessel plugging and water transport impairment. The production of the EPSs amylovoran and levan is critical for the formation of a mature biofilm. In addition, cyclic dimeric GMP (c-di-GMP) has been reported to positively regulate amylovoran biosynthesis and biofilm formation in E. amylovora Ea1189. In this study, we demonstrate that cellulose is synthesized by E. amylovora Ea1189 and is a major modulator of the three-dimensional characteristics of biofilms formed by this bacterium, and also contributes to virulence during systemic host invasion. In addition, we demonstrate that the activation of cellulose biosynthesis in E. amylovora is a c-di-GMP-dependent process, through allosteric binding to the cellulose catalytic subunit BcsA. We also report that the endoglucanase BcsZ is a key player in c-di-GMP activation of cellulose biosynthesis. Our results provide evidence of the complex composition of the extracellular matrix produced by E. amylovora and the implications of cellulose biosynthesis in shaping the architecture of the biofilm and in the expression of one of the main virulence phenotypes of this pathogen.

  9. Nitric oxide synthesis leads to vascular endothelial growth factor synthesis via the NO/cyclic guanosine 3',5'-monophosphate (cGMP) pathway in human corpus cavernosal smooth muscle cells.

    PubMed

    Komori, Kazuhiko; Tsujimura, Akira; Takao, Tetsuya; Matsuoka, Yasuhiro; Miyagawa, Yasushi; Takada, Shingo; Nonomura, Norio; Okuyama, Akihiko

    2008-07-01

    Vascular smooth muscle cells express endothelial nitric oxide synthase (eNOS) and produce nitric oxide (NO). Recently, increased NO production has been reported to induce the synthesis and secretion of vascular endothelial growth factor (VEGF) via the NO/cyclic guanosine 3',5'-monophosphate (cGMP) pathway. L-arginine (L-arg), the precursor of NO, and selective phosphodiesterase type 5 (PDE-5) inhibitors that increase levels of intracellular cGMP may complementarily enhance VEGF synthesis in corpus cavernosal smooth muscle cells (CCSMCs), and may consequently restore impaired endothelial function. Expression of eNOS in corpus cavernosal smooth muscle has also been reported. However, it is unclear whether CCSMCs can generate NO. To elucidate whether CCSMCs can synthesize NO and whether NO synthesis enhances VEGF synthesis via the NO/cGMP pathway. Corpus cavernosal cells were cultured and characterized by immunocytochemistry and immunoblotting. CCSMCs were treated with L-arg. CCSMCs were also incubated with L-arg and with vardenafil, an inhibitor of PDE-5. Release of NO from cells was confirmed by assay of NO metabolites (NOx). Intracellular cGMP concentration and VEGF concentration in the medium were measured. Isolated cells were determined to be CCSMCs. The expression of eNOS by CCSMCs was also identified. NOx and cGMP levels in the L-arg-treated group were significantly greater than those in the control group. VEGF and cGMP levels in the L-arg-treated group were also significantly greater than those in the control group. VEGF and cGMP levels in the L-arg + vardenafil-treated group were significantly greater than those in the L-arg-treated group and the control group. CCSMCs express eNOS and synthesize NO. NO synthesis leads to enhancement of VEGF synthesis via the NO/cGMP pathway. Combined L-arg and vardenafil treatment, which can enhance VEGF production, may provide a novel therapeutic strategy for the treatment of erectile dysfunction as well as endothelial

  10. E88, a new cyclic-di-GMP class I riboswitch aptamer from Clostridium tetani, has a similar fold to the prototypical class I riboswitch, Vc2, but differentially binds to c-di-GMP analogs.

    PubMed

    Luo, Yiling; Chen, Bin; Zhou, Jie; Sintim, Herman O; Dayie, T Kwaku

    2014-03-04

    C-di-GMP has emerged as a ubiquitous second messenger, which regulates the transition between sessile and motile lifestyles and virulence factor expression in many pathogenic bacteria using both RNA riboswitches and protein effectors. We recently showed that two additional class I c-di-GMP riboswitch aptamers (Ct-E88 and Cb-17B) bind c-di-GMP with nanomolar affinity, and that Ct-E88 RNA binds 2'-F-c-di-GMP 422 times less tightly than class I Vc2 RNA. Based on sequence comparison, it was concluded that the global folds of Ct-E88 and Vc2 RNAs were similar and that differences in ligand binding were probably due to differences in binding site architectures. Herein, we utilized EMSA, aptamer sensing spinach modules, SAXS and 1D NMR titration to study the conformational transitions of Ct-E88. We conclude that whereas the global folds of the bound states of Vc2 and Ct-E88 RNAs are similar, the unbound states are different and this could explain differences in ligand affinities between these class I c-di-GMP riboswitches.

  11. Viral DNA Sensors IFI16 and Cyclic GMP-AMP Synthase Possess Distinct Functions in Regulating Viral Gene Expression, Immune Defenses, and Apoptotic Responses during Herpesvirus Infection.

    PubMed

    Diner, Benjamin A; Lum, Krystal K; Toettcher, Jared E; Cristea, Ileana M

    2016-11-15

    The human interferon-inducible protein IFI16 is an important antiviral factor that binds nuclear viral DNA and promotes antiviral responses. Here, we define IFI16 dynamics in space and time and its distinct functions from the DNA sensor cyclic dinucleotide GMP-AMP synthase (cGAS). Live-cell imaging reveals a multiphasic IFI16 redistribution, first to viral entry sites at the nuclear periphery and then to nucleoplasmic puncta upon herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. Optogenetics and live-cell microscopy establish the IFI16 pyrin domain as required for nuclear periphery localization and oligomerization. Furthermore, using proteomics, we define the signature protein interactions of the IFI16 pyrin and HIN200 domains and demonstrate the necessity of pyrin for IFI16 interactions with antiviral proteins PML and cGAS. We probe signaling pathways engaged by IFI16, cGAS, and PML using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated knockouts in primary fibroblasts. While IFI16 induces cytokines, only cGAS activates STING/TBK-1/IRF3 and apoptotic responses upon HSV-1 and HCMV infections. cGAS-dependent apoptosis upon DNA stimulation requires both the enzymatic production of cyclic dinucleotides and STING. We show that IFI16, not cGAS or PML, represses HSV-1 gene expression, reducing virus titers. This indicates that regulation of viral gene expression may function as a greater barrier to viral replication than the induction of antiviral cytokines. Altogether, our findings establish coordinated and distinct antiviral functions for IFI16 and cGAS against herpesviruses. How mammalian cells detect and respond to DNA viruses that replicate in the nucleus is poorly understood. Here, we decipher the distinct functions of two viral DNA sensors, IFI16 and cGAS, during active immune signaling upon infection with two herpesviruses, herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV). We show that IFI16

  12. Involvement of DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP Pathways in Human Tissue Kallikrein 1 Protecting Erectile Function in Aged Rats

    PubMed Central

    Tang, Zhe; Rao, Ke; Wang, Tao; Chen, Zhong; Wang, Shaogang; Liu, Jihong; Wang, Daowen

    2017-01-01

    Our previous studies had reported that Human Tissue Kallikrein 1 (hKLK1) preserved erectile function in aged transgenic rats, while the detailed mechanism of hKLK1 protecting erectile function in aged rats through activation of cGMP and cAMP was not mentioned. To explore the latent mechanism, male wild-type Sprague-Dawley rats (WTR) and transgenic rats harboring the hKLK1 gene (TGR) were fed to 4 and 18 months old and divided into four groups: young WTR (yWTR) as the control, aged WTR (aWTR), aged TGR (aTGR) and aged TGRs with HOE140 (aTGRH). Erectile function of all rats was evaluated by cavernous nerve electrostimulation method and measured by the ratio of intracavernous pressure/ mean arterial pressure (ICP/MAP) in rats. Expression levels of cAMP and cGMP were assessed, and related signaling pathways were detected by western blot, immunohistochemistry and RT-PCR. Our experiment results showed erectile function of the aWTR group and aTGRH group was lower compared with those of other two groups. Also, expression levels of cAMP and cGMP were significantly lower than those of other two groups. Moreover, expressions of related signaling pathways including DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP were also downregulated in the corpus cavernosum of rats in aWTR group. Our finding revealed hKLK1 played a protective role in age-related ED. The DDAH/ADMA/NOS/cGMP and COX-2/PTGIS/cAMP pathways that were linked to the mechanism hKLK1 could increase the levels of cGMP and cAMP, which might provide novel therapy targets for age-related ED. PMID:28103290

  13. Anti-allodynic effect of mangiferin in neuropathic rats: Involvement of nitric oxide-cyclic GMP-ATP sensitive K(+) channels pathway and serotoninergic system.

    PubMed

    de Los Monteros-Zuñiga, Antonio Espinosa; Izquierdo, Teresa; Quiñonez-Bastidas, Geovanna Nallely; Rocha-González, Héctor Isaac; Godínez-Chaparro, Beatriz

    The neurobiology of neuropathic pain is caused by injury in the central or peripheral nervous system. Recent evidence points out that mangiferin shows anti-nociceptive effect in inflammatory pain. However, its role in inflammatory and neuropathic pain and the possible mechanisms of action are not yet established. The purpose of this study was to determine the possible anti-allodynic effect of mangiferin in rats with spinal nerve ligation (SNL). Furthermore, we sought to investigate the possible mechanisms of action that contribute to these effects. Mechanical allodynia to stimulation with the von Frey filaments was measured by the up and down method. Intrathecal administration of mangiferin prevented, in a dose-dependent fashion, SNL-induced mechanical allodynia. Mangiferin-induced anti-allodynia was prevented by the intrathecal administration of L-NAME (100μg/rat, non-selective nitric oxide synthase inhibitor), ODQ (10μg/rat, inhibitor of guanylate-cyclase) and glibenclamide (50μg/rat, channel blocker of ATP-sensitive K(+) channels). Moreover, methiothepin (30μg/rat, non-selective 5-HT receptor antagonist), WAY-100635 (6μg/rat, selective 5-HT1A receptor antagonist), SB-224289 (5μg/rat, selective 5-HT1B receptor antagonist), BRL-15572 (4μg/rat, selective 5-HT1D receptor antagonist) and SB-659551 (6μg/rat, selective 5-HT5A receptor antagonist), but not naloxone (50μg/rat, non-selective opioid receptor antagonist), were able to prevent mangiferin-induced anti-allodynic effect. These data suggest that the anti-allodynic effect induced by mangiferin is mediated at least in part by the serotoninergic system, involving the activation of 5-HT1A/1B/1D/5A receptors, as well as the nitric oxide-cyclic GMP-ATP-sensitive K(+) channels pathway, but not by the opioidergic system, in the SNL model of neuropathic pain in rats.

  14. The degenerate EAL-GGDEF domain protein Filp functions as a cyclic di-GMP receptor and specifically interacts with the PilZ-domain protein PXO_02715 to regulate virulence in Xanthomonas oryzae pv. oryzae.

    PubMed

    Yang, Fenghuan; Tian, Fang; Li, Xiaotong; Fan, Susu; Chen, Huamin; Wu, Maosen; Yang, Ching-Hong; He, Chenyang

    2014-06-01

    Degenerate GGDEF and EAL domain proteins represent major types of cyclic diguanylic acid (c-di-GMP) receptors in pathogenic bacteria. Here, we characterized a FimX-like protein (Filp) which possesses both GGDEF and EAL domains in Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice. Both in silico analysis and enzyme assays indicated that the GGDEF and EAL domains of Filp were degenerate and enzymatically inactive. However, Filp bound to c-di-GMP efficiently within the EAL domain, where Q(477), E(653), and F(654) residues were crucial for the binding. Deletion of the filp gene in X. oryzae pv. oryzae resulted in attenuated virulence in rice and reduced type III secretion system (T3SS) gene expression. Complementation analysis with different truncated proteins indicated that REC, PAS, and EAL domains but not the GGDEF domain were required for the full activity of Filp in vivo. In addition, a PilZ-domain protein (PXO_02715) was identified as a Filp interactor by yeast two-hybrid and glutathione-S-transferase pull-down assays. Deletion of the PXO_02715 gene demonstrated changes in bacterial virulence and T3SS gene expression similar to Δfilp. Moreover, both mutants were impaired in their ability to induce hypersensitive response in nonhost plants. Thus, we concluded that Filp was a novel c-di-GMP receptor of X. oryzae pv. oryzae, and its function to regulate bacterial virulence expression might be via the interaction with PXO_02715.

  15. Nitric Oxide–Soluble Guanylyl Cyclase–Cyclic GMP Signaling in the Striatum: New Targets for the Treatment of Parkinson's Disease?

    PubMed Central

    West, Anthony R.; Tseng, Kuei Y.

    2011-01-01

    Striatal nitric oxide (NO)-producing interneurons play an important role in the regulation of corticostriatal synaptic transmission and motor behavior. Striatal NO synthesis is driven by concurrent activation of NMDA and dopamine (DA) D1 receptors. NO diffuses into the dendrites of medium-sized spiny neurons which contain high levels of NO receptors called soluble guanylyl cyclases (sGC). NO-mediated activation of sGC leads to the synthesis of the second messenger cGMP. In the intact striatum, transient elevations in intracellular cGMP primarily act to increase neuronal excitability and to facilitate glutamatergic corticostriatal transmission. NO–cGMP signaling also functionally opposes the inhibitory effects of DA D2 receptor activation on corticostriatal transmission. Not surprisingly, abnormal striatal NO–sGC–cGMP signaling becomes apparent following striatal DA depletion, an alteration thought to contribute to pathophysiological changes observed in basal ganglia circuits in Parkinson's disease (PD). Here, we discuss recent developments in the field which have shed light on the role of NO–sGC–cGMP signaling pathways in basal ganglia dysfunction and motor symptoms associated with PD and l-DOPA-induced dyskinesias. PMID:21747761

  16. PDE5 inhibitors enhance the lethality of pemetrexed through inhibition of multiple chaperone proteins and via the actions of cyclic GMP and nitric oxide

    PubMed Central

    Booth, Laurence; Roberts, Jane L.; Poklepovic, Andrew; Gordon, Sarah; Dent, Paul

    2017-01-01

    Phosphodiesterase 5 (PDE5) inhibitors prevent the breakdown of cGMP that results in prolonged protein kinase G activation and the generation of nitric oxide. PDE5 inhibitors enhanced the anti-NSCLC cell effects of the NSCLC therapeutic pemetrexed. [Pemetrexed + sildenafil] activated an eIF2α – ATF4 – CHOP – Beclin1 pathway causing formation of toxic autophagosomes; activated a protective IRE1 – XBP-1 – chaperone induction pathway; and activated a toxic eIF2α – CHOP – DR4 / DR5 / CD95 induction pathway. [Pemetrexed + sildenafil] reduced the expression of c-FLIP-s, MCL-1 and BCL-XL that was blocked in a cell-type -dependent fashion by either over-expression of HSP90 / GRP78 / HSP70 / HSP27 or by blockade of eIF2α-CHOP signaling. Knock down of PKGI/II abolished the ability of sildenafil to enhance pemetrexed toxicity whereas pan-inhibition of NOS using L-NAME or knock down of [iNOS + eNOS] only partially reduced the lethal drug interaction. Pemetrexed reduced the ATPase activities of HSP90 and HSP70 in an ATM-AMPK-dependent fashion that was enhanced by sildenafil signaling via PKGI/II. The drug combination activated an ATM-AMPK-TSC2 pathway that was associated with reduced mTOR S2448 and ULK-1 S757 phosphorylation and increased ULK-1 S317 and ATG13 S318 phosphorylation. These effects were prevented by chaperone over-expression or by expression of an activated form of mTOR that prevented autophagosome formation and reduced cell killing. In two models of NSCLC, sildenafil enhanced the ability of pemetrexed to suppress tumor growth. Collectively we argue that the combination of [pemetrexed + PDE5 inhibitor] should be explored in a new NSCLC phase I trial. PMID:27903966

  17. Steroid-sensitive gene 1 is a novel cyclic GMP-dependent protein kinase I substrate in vascular smooth muscle cells.

    PubMed

    Wang, Guang-rong; Surks, Howard K; Tang, K Mary; Zhu, Yan; Mendelsohn, Michael E; Blanton, Robert M

    2013-08-23

    NO, via its second messenger cGMP, activates protein kinase GI (PKGI) to induce vascular smooth muscle cell relaxation. The mechanisms by which PKGI kinase activity regulates cardiovascular function remain incompletely understood. Therefore, to identify novel protein kinase G substrates in vascular cells, a λ phage coronary artery smooth muscle cell library was constructed and screened for phosphorylation by PKGI. The screen identified steroid-sensitive gene 1 (SSG1), which harbors several predicted PKGI phosphorylation sites. We observed direct and cGMP-regulated interaction between PKGI and SSG1. In cultured vascular smooth muscle cells, both the NO donor S-nitrosocysteine and atrial natriuretic peptide induced SSG1 phosphorylation, and mutation of SSG1 at each of the two predicted PKGI phosphorylation sites completely abolished its basal phosphorylation by PKGI. We detected high SSG1 expression in cardiovascular tissues. Finally, we found that activation of PKGI with cGMP regulated SSG1 intracellular distribution.

  18. Steroid-sensitive Gene 1 Is a Novel Cyclic GMP-dependent Protein Kinase I Substrate in Vascular Smooth Muscle Cells*

    PubMed Central

    Wang, Guang-rong; Surks, Howard K.; Tang, K. Mary; Zhu, Yan; Mendelsohn, Michael E.; Blanton, Robert M.

    2013-01-01

    NO, via its second messenger cGMP, activates protein kinase GI (PKGI) to induce vascular smooth muscle cell relaxation. The mechanisms by which PKGI kinase activity regulates cardiovascular function remain incompletely understood. Therefore, to identify novel protein kinase G substrates in vascular cells, a λ phage coronary artery smooth muscle cell library was constructed and screened for phosphorylation by PKGI. The screen identified steroid-sensitive gene 1 (SSG1), which harbors several predicted PKGI phosphorylation sites. We observed direct and cGMP-regulated interaction between PKGI and SSG1. In cultured vascular smooth muscle cells, both the NO donor S-nitrosocysteine and atrial natriuretic peptide induced SSG1 phosphorylation, and mutation of SSG1 at each of the two predicted PKGI phosphorylation sites completely abolished its basal phosphorylation by PKGI. We detected high SSG1 expression in cardiovascular tissues. Finally, we found that activation of PKGI with cGMP regulated SSG1 intracellular distribution. PMID:23831687

  19. Kaurenoic acid from Sphagneticola trilobata Inhibits Inflammatory Pain: effect on cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

    PubMed

    Mizokami, Sandra S; Arakawa, Nilton S; Ambrosio, Sergio R; Zarpelon, Ana C; Casagrande, Rubia; Cunha, Thiago M; Ferreira, Sergio H; Cunha, Fernando Q; Verri, Waldiceu A

    2012-05-25

    Kaurenoic acid [ent-kaur-16-en-19-oic acid (1)] is a diterpene present in several plants including Sphagneticola trilobata. The only documented evidence for its antinociceptive effect is that it inhibits the writhing response induced by acetic acid in mice. Therefore, the analgesic effect of 1 in different models of pain and its mechanisms in mice were investigated further. Intraperitoneal and oral treatment with 1 dose-dependently inhibited inflammatory nociception induced by acetic acid. Oral treatment with 1 also inhibited overt nociception-like behavior induced by phenyl-p-benzoquinone, complete Freund's adjuvant (CFA), and both phases of the formalin test. Compound 1 also inhibited acute carrageenin- and PGE(2)-induced and chronic CFA-induced inflammatory mechanical hyperalgesia. Mechanistically, 1 inhibited the production of the hyperalgesic cytokines TNF-α and IL-1β. Furthermore, the analgesic effect of 1 was inhibited by l-NAME, ODQ, KT5823, and glybenclamide treatment, demonstrating that such activity also depends on activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway, respectively. These results demonstrate that 1 exhibits an analgesic effect in a consistent manner and that its mechanisms involve the inhibition of cytokine production and activation of the NO-cyclic GMP-protein kinase G-ATP-sensitive potassium channel signaling pathway.

  20. Protection of Nrf2 against arsenite-induced oxidative damage is regulated by the cyclic guanosine monophosphate-protein kinase G signaling pathway.

    PubMed

    Chen, Chengzhi; Jiang, Xuejun; Gu, Shiyan; Lai, Yanhao; Liu, Yuan; Zhang, Zunzhen

    2016-10-24

    Arsenite has been shown to induce a variety of oxidative damage in mammalian cells. However, the mechanisms underlying cellular responses to its adverse effects remain unknown. We previously showed that the level of Nrf2, a nuclear transcription factor significantly increased in arsenite-treated human bronchial epithelial (HBE) cells suggesting that Nrf2 is involved in responding to arsenite-induced oxidative damage. To explore how Nrf2 can impact arsenite-induced oxidative damage, in this study, we examined Nrf2 activation and its regulation upon cellular arsenite exposure as well as its effects on arsenite-induced oxidative damage in HBE cells. We found that Nrf2 mRNA and protein levels were significantly increased by arsenite in a dose- and time-dependent manner. Furthermore, we showed that over-expression of Nrf2 significantly reduced the level of arsenite-induced oxidative damage in HBE cells including DNA damage, chromosomal breakage, lipid peroxidation and depletion of antioxidants. This indicates a protective role of Nrf2 against arsenite toxicity. This was further supported by the fact that activation of Nrf2 by its agonists, tertiary butylhydroquinone (t-BHQ) and sulforaphane (SFN) resulted in the same protective effects against arsenite toxicity. Moreover, we demonstrated that arsenite-induced activation of Nrf2 was mediated by the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) signaling pathway. This is the first evidence showing that Nrf2 protects against arsenite-induced oxidative damage through the cGMP-PKG pathway. Our study suggests that activation of Nrf2 through the cGMP-PKG signaling pathway in HBE cells may be developed as a new strategy for prevention of arsenite toxicity. © 2016 Wiley Periodicals, Inc. Environ Toxicol, 2016.

  1. Levels of cyclic-AMP and cyclic-GMP in porcine oocyte-cumulus complexes and cumulus-free oocytes derived from small and middle follicles during the first 24-hour period of in vitro maturation.

    PubMed

    Okudaira, Yuichi; Wakai, Takuya; Funahashi, Hiroaki

    2017-02-23

    The objective of this study was to compare the cAMP and cGMP levels in cumulus-oocyte complexes (COCs) derived from the middle follicles (MFs, 3-6 mm in diameter) and small follicles (SFs, 1-3 mm in diameter) of pre-pubertal gilts during the first 24-h period of maturation in vitro (IVM). Both cAMP and cGMP levels in MF- and SF-derived oocytes did not change during this period. Although the cAMP levels increased in the COCs at 10 and 20 h after the start of IVM, the levels of cAMP were significantly higher in MF-derived COCs than in SF-derived COCs at 20 h after the start of IVM. On the other hand, the cGMP levels in COCs decreased to basal levels between 10 and 20 h after the start of the IVM, whereas cGMP levels were lower in SF-derived COCs than in MF-derived COCs during the first 10 h. The number of cumulus cells was larger in the MF-derived COCs than in the SF-derived COCs during the first 20-h period of IVM. The estimated cAMP level per cumulus cell at 10 h after the start of the IVM was higher in SF-derived COCs than in MF-derived COCs, whereas the estimated cGMP level per cumulus cell was no different between MF- and SF-derived COCs. From these results, we conclude that cAMP and cGMP levels in COCs, but not in oocytes, drastically change during the first 20-h period of IVM, and that both cAMP and cGMP levels significantly differ between MF- and SF-derived COCs.

  2. Transcutaneous Electrical Stimulation Increased Nitric Oxide-Cyclic GMP Release Biocaptured Over Skin Surface of Pericardium Meridian and Acupuncture Points in Humans

    PubMed Central

    Ma, Sheng-Xing; Mayer, Emeran; Lee, Paul; Li, Xi-yan; Gao, Ellen Z.

    2015-01-01

    Objectives The purpose of this study was to consecutively capture and quantify nitric oxide (NO) and cGMP, the second messenger of NO, over the skin surface of acupuncture points (acupoints), meridian line without acupoint, and non-meridian control regions of the Pericardium meridian (PC) in humans, and investigate their response to transcutaneous electrical nerve stimulation (TENS). Design, setting, and main outcome measures Adhesive biocapture tubes were attached to the skin surface along PC regions and injected with 2-Phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl solution, an NO-scavenging compound, contacting the skin surface for 20 minutes each during 4 consecutive biocapture intervals. TENS (1.0 mA, 6 Hz, 1.0 msec duration) was applied over acupoints PC 8 and PC 3 during the 2nd biocapture for 20 min. Total nitrite and nitrate (NOx-), the stable metabolic products of NO, and cGMP in biocaptured samples were quantified using chemiluminescence and ELISA. Results NOx- levels in the 1st biocapture over PC regions are almost two fold higher compared to subsequent biocaptures and are higher over PC acupoints versus non-meridian control region. Following TENS, NOx- concentrations over PC regions were significantly increased, and cGMP is predominantly released from the skin surface of PC acupoints. Conclusions TENS induces elevations of NO-cGMP concentrations over local skin region with a high level at acupoints. The enhanced signal molecules improve local circulation, which contributes to beneficial effects of the therapy. PMID:26369251

  3. Phosphodiesterase 9A Controls Nitric-oxide Independent cGMP and Hypertrophic Heart Disease

    PubMed Central

    Lee, Dong I.; Zhu, Guangshuo; Sasaki, Takashi; Cho, Gun-Sik; Hamdani, Nazha; Holewinski, Ronald; Jo, Su-Hyun; Danner, Thomas; Zhang, Manling; Rainer, Peter P.; Bedja, Djahida; Kirk, Jonathan A.; Ranek, Mark J.; Dostmann, Wolfgang R.; Kwon, Chulan; Margulies, Kenneth B.; Van Eyk, Jennifer E.; Paulus, Walter J.; Takimoto, Eiki; Kass, David A.

    2015-01-01

    Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric oxide (NO) and natriuretic peptide (NP) coupled signaling, stimulating phosphorylation changes by protein kinase G (PKG). Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease1,2. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation3. Furthermore, though PDE5A regulates NO-generated cGMP4,5, NO-signaling is often depressed by heart disease6. PDEs controlling NP-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A7,8 is expressed in mammalian heart including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates NP rather than NO-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neuro-hormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of NO-synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phospho-proteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signaling independent of the NO-pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target. PMID:25799991

  4. cGMP and Brown Adipose Tissue.

    PubMed

    Hoffmann, Linda S; Larson, Christopher J; Pfeifer, Alexander

    2016-01-01

    The second messenger cyclic guanosine monophosphate (cGMP) is a key mediator in physiological processes such as vascular tone, and its essential involvement in pathways regulating metabolism has been recognized in recent years. Here, we focus on the fundamental role of cGMP in brown adipose tissue (BAT) differentiation and function. In contrast to white adipose tissue (WAT), which stores energy in the form of lipids, BAT consumes energy stored in lipids to generate heat. This so-called non-shivering thermogenesis takes place in BAT mitochondria, which express the specific uncoupling protein 1 (UCP1). The energy combusting properties of BAT render it a promising target in antiobesity strategies in which BAT could burn the surplus energy that has accumulated in obese and overweight individuals. cGMP is generated by guanylyl cyclases upon activation by nitric oxide or natriuretic peptides. It affects several downstream molecules including cGMP-receptor proteins such as cGMP-dependent protein kinase and is degraded by phosphodiesterases. The cGMP pathway contains several signaling molecules that can increase cGMP signaling, resulting in activation and recruitment of brown adipocytes, and hence can enhance the energy combusting features of BAT. In this review we highlight recent results showing the physiological significance of cGMP signaling in BAT, as well as pharmacological options targeting cGMP signaling that bear a high potential to become BAT-centered therapies for the treatment of obesity.

  5. Time-dependent current decline in cyclic GMP-gated bovine channels caused by point mutations in the pore region expressed in Xenopus oocytes.

    PubMed Central

    Bucossi, G; Eismann, E; Sesti, F; Nizzari, M; Seri, M; Kaupp, U B; Torre, V

    1996-01-01

    1. Amino acids with a charged or a polar residue in the putative pore region, between lysine 346 and glutamate 372 of the alpha-subunit of the cGMP-gated channel from bovine rods were mutated to a different amino acid. The mRNA encoding for the wild-type, i.e. the alpha-subunit, or mutant channels was injected in Xenopus laevis oocytes. 2. When glutamate 363 was mutated to asparagine, serine or alanine, the current activated by a steady cGMP concentration declined in mutant channels. No current decline was observed when glutamate 363 was mutated to aspartate, glutamine or glycine, when theronine 359, 360 and 364 were mutated to alanine or when other charged residues in the pore region were neutralized. 3. The amount of current decline and its time course were significantly voltage dependent. In mutant E363A the current decline developed within about 1.5 s at -100 mV, but in about 6 s at +100 mV. In the same mutant, the current declined to about 55% of its initial level at +100 mV and to about 10% at -100 mV. 4. The current decline in mutants E363A, E363S and E363N was only moderately dependent on the cGMP concentration (from 10 to 1000 microM) and was not caused by a reduced affinity of the mutant channels for cGMP. Analysis of current fluctuations at a single-channel level indicated that current decline was primarily caused by a decrease of the open probability. 5. The wild-type channel was not permeable to dimethylammonium. When glutamate 363 was replaced by a smaller residue such as serine, mutant channels became permeable to dimethylammonium. 6. The current decline observed in mutant channels is reminiscent of desensitization of ligand-gated channels and of inactivation of voltage-gated channels. These results suggest also that gating and permeation through the cGMP-gated channel from bovine rods are intrinsically coupled and that glutamate 363 is part of the molecular structure controlling both the gating and the narrowest region of the pore. PMID:8782105

  6. Plant Cyclic Nucleotide Signalling

    PubMed Central

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc

    2007-01-01

    The presence of the cyclic nucleotides 3′,5′-cyclic adenyl monophosphate (cAMP) and 3′,5′-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed. PMID:19704553

  7. C33(S), a novel PDE9A inhibitor, protects against rat cardiac hypertrophy through upregulating cGMP signaling.

    PubMed

    Wang, Pan-Xia; Li, Zhuo-Ming; Cai, Si-Dong; Li, Jing-Yan; He, Ping; Huang, Yi; Feng, Guo-Shuai; Luo, Hai-Bin; Chen, Shao-Rui; Liu, Pei-Qing

    2017-09-01

    Phosphodiesterase-9A (PDE9A) expression is upregulated during cardiac hypertrophy and heart failure. Accumulating evidence suggests that PDE9A might be a promising therapeutic target for heart diseases. The present study sought to investigate the effects and underlying mechanisms of C33(S), a novel selective PDE9A inhibitor, on cardiac hypertrophy in vitro and in vivo. Treatment of neonatal rat cardiomyocytes (NRCMs) with PE (100 μmol/L) or ISO (1 μmol/L) induced cardiac hypertrophy characterized by significantly increased cell surface areas and increased expression of fetal genes (ANF and BNP). Furthermore, PE or ISO significantly increased the expression of PDE9A in the cells; whereas knockdown of PDE9A significantly alleviated PE-induced hypertrophic responses. Moreover, pretreatment with PDE9A inhibitor C33(S) (50 and 500 nmol/L) or PF-7943 (2 μmol/L) also alleviated the cardiac hypertrophic responses in PE-treated NRCMs. Abdominal aortic constriction (AAC)-induced cardiac hypertrophy and ISO-induced heart failure were established in SD rats. In ISO-treated rats, oral administration of C33(S) (9, 3, and 1 mg·kg(-1)·d(-1), for 3 consecutive weeks) significantly increased fractional shortening (43.55%±3.98%, 54.79%±1.95%, 43.98%±7.96% vs 32.18%±6.28%), ejection fraction (72.97%±4.64%, 84.29%±1.56%, 73.41%±9.37% vs 49.17%±4.20%) and cardiac output (60.01±9.11, 69.40±11.63, 58.08±8.47 mL/min vs 48.97±2.11 mL/min) but decreased the left ventricular internal diameter, suggesting that the transition to heart failure was postponed by C33(S). We further revealed that C33(S) significantly elevated intracellular cGMP levels, phosphorylation of phospholamban (PLB) and expression of SERCA2a in PE-treated NRCMs in vitro and in ISO-induced heart failure model in vivo. Our results demonstrate that C33(S) effectively protects against cardiac hypertrophy and postpones the transition to heart failure, suggesting that it is a promising agent in the treatment of

  8. Cyclic-di-GMP signalling and biofilm-related properties of the Shiga toxin-producing 2011 German outbreak Escherichia coli O104:H4

    PubMed Central

    Richter, Anja M; Povolotsky, Tatyana L; Wieler, Lothar H; Hengge, Regine

    2014-01-01

    In 2011, nearly 4,000 people in Germany were infected by Shiga toxin (Stx)-producing Escherichia coli O104:H4 with > 22% of patients developing haemolytic uraemic syndrome (HUS). Genome sequencing showed the outbreak strain to be related to enteroaggregative E. coli (EAEC), suggesting its high virulence results from EAEC-typical strong adherence and biofilm formation combined to Stx production. Here, we report that the outbreak strain contains a novel diguanylate cyclase (DgcX)—producing the biofilm-promoting second messenger c-di-GMP—that shows higher expression than any other known E. coli diguanylate cyclase. Unlike closely related E. coli, the outbreak strain expresses the c-di-GMP-controlled biofilm regulator CsgD and amyloid curli fibres at 37°C, but is cellulose-negative. Moreover, it constantly generates derivatives with further increased and deregulated production of CsgD and curli. Since curli fibres are strongly proinflammatory, with cellulose counteracting this effect, high c-di-GMP and curli production by the outbreak O104:H4 strain may enhance not only adherence but may also contribute to inflammation, thereby facilitating entry of Stx into the bloodstream and to the kidneys where Stx causes HUS. PMID:25361688

  9. Pharmacological evidence for the participation of NO-cGMP-KATP pathway in the gastric protective effect of curcumin against indomethacin-induced gastric injury in the rat.

    PubMed

    Díaz-Triste, Nadia Estela; González-García, Martha Patricia; Jiménez-Andrade, Juan Miguel; Castañeda-Hernández, Gilberto; Chávez-Piña, Aracely Evangelina

    2014-05-05

    Curcumin, main compound obtained from rizhoma of Curcuma longa, shows antitumoral, antioxidant, anticarcinogenic and gastric protective properties. Recently, it has been demonstrated that curcumin exerts its gastric protective action due to an increase in gastric nitric oxide (NO) levels. However, it is unknown whether these increased NO levels are associated with activation of intracellular signaling pathways. Thus, the purpose of this study was to investigate the role of NO-cGMP-KATP pathway in the gastric protective effect of curcumin during indomethacin-induced gastric injury in the rat. Adult female Wistar rats were gavaged with curcumin (3-300mg/kg, p.o.) or omeprazole (30mg/kg, p.o.) 30min before indomethacin insult (30mg/kg, p.o.). Other groups of rats were administered L-NAME (70mg/kg, i.p.; inhibitor of nitric oxide synthase), ODQ (10mg/kg, i.p.; inhibitor of soluble guanylate cyclase) or glibenclamide (1mg/kg, i.p.; blocker of ATP-sensitive potassium (KATP) channels) 30min before curcumin (30mg/kg, p.o.). 3h after indomethacin administration, rats were sacrificed and gastric injury was evaluated by determining total damaged area. A sample of gastric tissue was harvested and processed to quantify organic nitrite levels. Curcumin significantly protected against indomethacin-induced gastric injury and this effect was comparable to gastroprotective effect by omeprazole. L-NAME, ODQ and glibenclamide significantly prevented the curcumin-mediated gastric protective effect in the indomethacin-induced gastric injury model. Furthermore, curcumin administration induced a significant increase in gastric nitric oxide levels as compared to vehicle administration. Our results show for the first time that curcumin activates NO/cGMP/KATP pathway during its gastro protective action. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. The Cyclic Di-GMP Phosphodiesterase Gene Rv1357c/BCG1419c Affects BCG Pellicle Production and In Vivo Maintenance.

    PubMed

    Flores-Valdez, Mario Alberto; Aceves-Sánchez, Michel de Jesús; Pedroza-Roldán, César; Vega-Domínguez, Perla Jazmín; Prado-Montes de Oca, Ernesto; Bravo-Madrigal, Jorge; Laval, Françoise; Daffé, Mamadou; Koestler, Ben; Waters, Christopher M

    2015-02-01

    Bacteria living in a surface-attached community that contains a heterogeneous population, coated with an extracellular matrix, and showing drug tolerance (biofilms) are often linked to chronic infections. In mycobacteria, the pellicle mode of growth has been equated to an in vitro biofilm and meets several of the criteria mentioned above, while tuberculosis infection presents a chronic (latent) phase of infection. As mycobacteria lack most genes required to control biofilm production by other microorganisms, we deleted or expressed from the hsp60 strong promoter the only known c-di-GMP phosphodiesterase (PDE) gene in Mycobacterium bovis BCG. We found changes in pellicle production, cellular protein profiles, lipid production, resistance to nitrosative stress and maintenance in lungs and spleens of immunocompetent BALB/mice. Our results show that pellicle production and capacity to remain within the host are linked in BCG.

  11. Imidazopyridazine Inhibitors of Plasmodium falciparum Calcium-Dependent Protein Kinase 1 Also Target Cyclic GMP-Dependent Protein Kinase and Heat Shock Protein 90 To Kill the Parasite at Different Stages of Intracellular Development

    PubMed Central

    Moon, Robert W.; Whalley, David; Bowyer, Paul W.; Wallace, Claire; Rochani, Ankit; Nageshan, Rishi K.; Howell, Steven A.; Grainger, Munira; Jones, Hayley M.; Ansell, Keith H.; Chapman, Timothy M.; Taylor, Debra L.; Osborne, Simon A.; Baker, David A.; Tatu, Utpal

    2015-01-01

    Imidazopyridazine compounds are potent, ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1) and of Plasmodium falciparum parasite growth in vitro. Here, we show that these compounds can be divided into two classes depending on the nature of the aromatic linker between the core and the R2 substituent group. Class 1 compounds have a pyrimidine linker and inhibit parasite growth at late schizogony, whereas class 2 compounds have a nonpyrimidine linker and inhibit growth in the trophozoite stage, indicating different modes of action for the two classes. The compounds also inhibited cyclic GMP (cGMP)-dependent protein kinase (PKG), and their potency against this enzyme was greatly reduced by substitution of the enzyme's gatekeeper residue at the ATP binding site. The effectiveness of the class 1 compounds against a parasite line expressing the modified PKG was also substantially reduced, suggesting that these compounds kill the parasite primarily through inhibition of PKG rather than CDPK1. HSP90 was identified as a binding partner of class 2 compounds, and a representative compound bound to the ATP binding site in the N-terminal domain of HSP90. Reducing the size of the gatekeeper residue of CDPK1 enabled inhibition of the enzyme by bumped kinase inhibitors; however, a parasite line expressing the modified enzyme showed no change in sensitivity to these compounds. Taken together, these findings suggest that CDPK1 may not be a suitable target for further inhibitor development and that the primary mechanism through which the imidazopyridazines kill parasites is by inhibition of PKG or HSP90. PMID:26711771

  12. LTQ-XL mass spectrometry proteome analysis expands the Pseudomonas aeruginosa AmpR regulon to include cyclic di-GMP phosphodiesterases and phosphoproteins, and identifies novel open reading frames.

    PubMed

    Kumari, Hansi; Murugapiran, Senthil K; Balasubramanian, Deepak; Schneper, Lisa; Merighi, Massimo; Sarracino, David; Lory, Stephen; Mathee, Kalai

    2014-01-16

    Pseudomonas aeruginosa is well known for its antibiotic resistance and intricate regulatory network, contributing to its success as an opportunistic pathogen. This study is an extension of our transcriptomic analyses (microarray and RNA-Seq) to understand the global changes in PAO1 upon deleting a gene encoding a transcriptional regulator AmpR, in the presence and absence of β-lactam antibiotic. This study was performed under identical conditions to explore the proteome profile of the ampR deletion mutant (PAOΔampR) using LTQ-XL mass spectrometry. The proteomic data identified ~53% of total PAO1 proteins and expanded the master regulatory role of AmpR in determining antibiotic resistance and multiple virulence phenotypes in P. aeruginosa. AmpR proteome analysis identified 853 AmpR-dependent proteins, which include 102 transcriptional regulators and 21 two-component system proteins. AmpR also regulates cyclic di-GMP phosphodiesterases (PA4367, PA4969, PA4781) possibly affecting major virulence systems. Phosphoproteome analysis also suggests a significant role for AmpR in Ser, Thr and Tyr phosphorylation. These novel mechanisms of gene regulation were previously not associated with AmpR. The proteome analysis also identified many unannotated and misannotated ORFs in the P. aeruginosa genome. Thus, our data sheds light on important virulence regulatory pathways that can potentially be exploited to deal with P. aeruginosa infections. The AmpR proteome data not only confirmed the role of AmpR in virulence and resistance to multiple antibiotics, but also expanded the perimeter of AmpR regulon. The data presented here points to the role of AmpR in regulating cyclic di-GMP levels and phosphorylation of Ser, Thr and Tyr, adding another dimension to the regulatory functions of AmpR. We also identify some previously unannotated/misannotated ORFs in the P. aeruginosa genome, indicating the limitations of existing ORF analyses software. This study will contribute towards

  13. Enzymatic Relay Mechanism Stimulates Cyclic GMP Synthesis in Rod Photoresponse: Biochemical and Physiological Study in Guanylyl Cyclase Activating Protein 1 Knockout Mice

    PubMed Central

    Makino, Clint L.; Savchenko, Andrey B.; Dizhoor, Alexander M.

    2012-01-01

    Regulation of cGMP synthesis by retinal membrane guanylyl cyclase isozymes (RetGC1 and RetGC2) in rod and cone photoreceptors by calcium-sensitive guanylyl cyclase activating proteins (GCAP1 and GCAP2) is one of the key molecular mechanisms affecting the response to light and is involved in congenital retinal diseases. The objective of this study was to identify the physiological sequence of events underlying RetGC activation in vivo, by studying the electrophysiological and biochemical properties of mouse rods in a new genetic model lacking GCAP1. The GCAP1−/− retinas expressed normal levels of RetGC isozymes and other phototransduction proteins, with the exception of GCAP2, whose expression was elevated in a compensatory fashion. RetGC activity in GCAP1−/− retinas became more sensitive to Ca2+ and slightly increased. The bright flash response in electroretinogram (ERG) recordings recovered quickly in GCAP1−/−, as well as in RetGC1−/−GCAP1−/−, and RetGC2−/−GCAP1−/− hybrid rods, indicating that GCAP2 activates both RetGC isozymes in vivo. Individual GCAP1−/− rod responses varied in size and shape, likely reflecting variable endogenous GCAP2 levels between different cells, but single-photon response (SPR) amplitude and time-to-peak were typically increased, while recovery kinetics remained faster than in wild type. Recovery from bright flashes in GCAP1−/− was prominently biphasic, because rare, aberrant SPRs producing the slower tail component were magnified. These data provide strong physiological evidence that rod photoresponse recovery is shaped by the sequential recruitment of RetGC isozyme activation by GCAPs according to the different GCAP sensitivities for Ca2+ and specificities toward RetGC isozymes. GCAP1 is the ‘first-response’ sensor protein that stimulates RetGC1 early in the response and thus limits the SPR amplitude, followed by activation of GCAP2 that adds stimulation of both RetGC1 and RetGC2 to speed

  14. Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach.

    PubMed

    Russwurm, Michael; Mullershausen, Florian; Friebe, Andreas; Jäger, Ronald; Russwurm, Corina; Koesling, Doris

    2007-10-01

    The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 microM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays.

  15. Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach

    PubMed Central

    Russwurm, Michael; Mullershausen, Florian; Friebe, Andreas; Jäger, Ronald; Russwurm, Corina; Koesling, Doris

    2007-01-01

    The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 μM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays. PMID:17516914

  16. Transgenic Mice for cGMP Imaging

    PubMed Central

    Thunemann, Martin; Wen, Lai; Hillenbrand, Matthias; Vachaviolos, Angelos; Feil, Susanne; Ott, Thomas; Han, Xiaoxing; Fukumura, Dai; Jain, Rakesh K.; Russwurm, Michael; de Wit, Cor; Feil, Robert

    2014-01-01

    Rationale Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown. Objective To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer–based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues. Methods and Results Mouse lines with smooth muscle–specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase–activatable expression cassette driven by the cytomegalovirus early enhancer/chicken β-actin/β-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were <100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide–induced cGMP transients with peak concentrations of ≈1 to >3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide–stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation. Conclusions These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live

  17. Nitric oxide induces muscular relaxation via cyclic GMP-dependent and -independent mechanisms in the longitudinal muscle of the mouse duodenum.

    PubMed

    Serio, Rosa; Zizzo, Maria Grazia; Mulè, Flavia

    2003-02-01

    The aim of this study was to investigate, in mouse duodenum, the role of nitric oxide (NO) in the relaxation of longitudinal muscle evoked by nerve activation and the coupled action mechanism. Electrical field stimulation (EFS; 0.5 ms, 10-s train duration, supramaximal voltage, at various frequencies) under nonadrenergic noncholinergic conditions evoked muscular relaxation occasionally followed, at the higher stimulus frequencies, by rebound contractions. Inhibition of the synthesis of NO by N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 microM) virtually abolished the evoked relaxation. The relaxation was reduced also by apamin (0.1 microM) and by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a guanylyl cyclase inhibitor. The coadministration of apamin and ODQ produced additive effects on the responses to EFS. Sodium nitroprusside (0.1-100 microM) produced a concentration-dependent reduction of the phasic spontaneous activity and at the highest dose used suppressed phasic activity and induced muscular relaxation. These effects were tetrodotoxin and L-NAME resistant and were antagonized both by apamin and by ODQ. 8-Bromoguanosine 3',5'-cyclic monophosphate (0.1-100 microM) reduced in a concentration-dependent manner the spontaneous mechanical activity and at 100 microM suppressed the phasic activity and induced muscular relaxation, not antagonized by apamin. This study indicates that NO is the primary transmitter released by inhibitory nerves supplying the longitudinal muscle of mouse duodenum and that guanylate cyclase stimulation and opening of Ca(2+)-dependent K(+) channels are independent mechanisms working in parallel to mediate NO action.

  18. Compartmentalized cGMP Responses of Olfactory Sensory Neurons in Caenorhabditis elegans.

    PubMed

    Shidara, Hisashi; Hotta, Kohji; Oka, Kotaro

    2017-04-05

    Cyclic guanosine monophosphate (cGMP) plays a crucial role as a second messenger in the regulation of sensory signal transduction in many organisms. In AWC olfactory sensory neurons of Caenorhabditis elegans, cGMP also has essential and distinctive functions in olfactory sensation and adaptation. According to molecular genetic studies, when nematodes are exposed to odorants, a decrease in cGMP regulates cGMP-gated channels for olfactory sensation. Conversely, for olfactory adaptation, an increase in cGMP activates protein kinase G to modulate cellular physiological functions. Although these opposing cGMP responses in single neurons may occur at the same time, it is unclear how cGMP actually behaves in AWC sensory neurons. A hypothetical explanation for opposing cGMP responses is region-specific behaviors in AWC: for odor sensation, cGMP levels in cilia could decrease, whereas odor adaptation is mediated by increased cGMP levels in soma. Therefore, we visualized intracellular cGMP in AWC with a genetically encoded cGMP indicator, cGi500, and examined spatiotemporal cGMP responses in AWC neurons. The cGMP imaging showed that, after odor exposure, cGMP levels in AWC cilia decreased transiently, whereas levels in dendrites and soma gradually increased. These region-specific responses indicated that the cGMP responses in AWC neurons are explicitly compartmentalized. In addition, we performed Ca(2+) imaging to examine the relationship between cGMP and Ca(2+) These results suggested that AWC sensory neurons are in fact analogous to vertebrate photoreceptor neurons.SIGNIFICANCE STATEMENT Cyclic guanosine monophosphate (cGMP) plays crucial roles in the regulation of sensory signal transduction in many animals. In AWC olfactory sensory neurons of Caenorhabditis elegans, cGMP also has essential and distinctive functions involving olfactory sensation and adaptation. Here, we visualized intracellular cGMP in AWC neurons with a genetically encoded cGMP indicator and examined how

  19. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants

    PubMed Central

    Olivares-González, Lorena; Martínez-Fernández de la Cámara, Cristina; Hervás, David; Marín, María Pilar; Lahoz, Agustin; Millán, José María

    2016-01-01

    Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP) has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE) with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2) for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation) mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities) and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions. PMID:27861632

  20. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

    PubMed

    Olivares-González, Lorena; Martínez-Fernández de la Cámara, Cristina; Hervás, David; Marín, María Pilar; Lahoz, Agustin; Millán, José María; Rodrigo, Regina

    2016-01-01

    Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP) has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE) with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2) for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation) mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities) and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

  1. Direct Activation of cGMP-Dependent Channels of Retinal Rods by the cGMP Phosphodiesterase

    NASA Astrophysics Data System (ADS)

    Bennett, Nelly; Ildefonse, Michele; Crouzy, Serge; Chapron, Yves; Clerc, Armel

    1989-05-01

    The cationic conductances of purified bovine retinal rod membranes were studied by incorporation of vesicles into planar lipid bilayers. When the membranes were stripped of all peripheral proteins [guanine nucleotide-binding protein (G protein) and cGMP phosphodiesterase (3',5'-cyclic-GMP 5'-nucleotidohydrolase), EC 3.1.4.35], sodium and calcium fluxes were almost only observed in the presence of cGMP. Reconstitution experiments in which purified cGMP phosphodiesterase alone or with G protein were reassociated to the vesicles in proportions similar to those found in the native rod provide evidence for a direct interaction between the cGMP-dependent channel protein and the phosphodiesterase. (i) In its inhibited state, phosphodiesterase markedly stimulates the activity of the channels in the presence of cGMP (situation in the dark-adapted rod) but is not capable of activating the channels in the absence of cGMP. (ii) In the absence of cGMP, activation of the phosphodiesterase by G protein with GTP bound (equivalent to photoexcitation) induces the opening of cation channels that have the same conductance for sodium ions as cGMP-activated channels (20-22 pS, with two sublevels of about 7 pS and 13 pS).

  2. cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

    PubMed Central

    Busch, Jennifer L.; Corbin, Jackie D.

    2010-01-01

    To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge. PMID:20716671

  3. Transient hypercapnia reveals an underlying cerebrovascular pathology in a murine model for HIV-1 associated neuroinflammation: role of NO-cGMP signaling and normalization by inhibition of cyclic nucleotide phosphodiesterase-5

    PubMed Central

    2012-01-01

    show that HIV-associated neuroinflammation can cause cerebrovascular pathology through effects on cyclic guanosine monophosphate (cGMP) metabolism and possibly on PDE5 metabolism. PMID:23167821

  4. Transient hypercapnia reveals an underlying cerebrovascular pathology in a murine model for HIV-1 associated neuroinflammation: role of NO-cGMP signaling and normalization by inhibition of cyclic nucleotide phosphodiesterase-5.

    PubMed

    Silva, Jharon; Polesskaya, Oksana; Knight, Walter; Zheng, Johnny Ting; Granger, Megan; Lopez, Tenée; Ontiveros, Fernando; Feng, Changyong; Yan, Chen; Kasischke, Karl A; Dewhurst, Stephen

    2012-11-20

    -associated neuroinflammation can cause cerebrovascular pathology through effects on cyclic guanosine monophosphate (cGMP) metabolism and possibly on PDE5 metabolism.

  5. cGMP Signaling, Phosphodiesterases and Major Depressive Disorder

    PubMed Central

    Reierson, Gillian W; Guo, Shuyu; Mastronardi, Claudio; Licinio, Julio; Wong, Ma-Li

    2011-01-01

    Deficits in neuroplasticity are hypothesized to underlie the pathophysiology of major depressive disorder (MDD): the effectiveness of antidepressants is thought to be related to the normalization of disrupted synaptic transmission and neurogenesis. The cyclic adenosine monophosphate (cAMP) signaling cascade has received considerable attention for its role in neuroplasticity and MDD. However components of a closely related pathway, the cyclic guanosine monophosphate (cGMP) have been studied with much lower intensity, even though this signaling transduction cascade is also expressed in the brain and the activity of this pathway has been implicated in learning and memory processes. Cyclic GMP acts as a second messenger; it amplifies signals received at postsynaptic receptors and activates downstream effector molecules resulting in gene expression changes and neuronal responses. Phosphodiesterase (PDE) enzymes degrade cGMP into 5’GMP and therefore they are involved in the regulation of intracellular levels of cGMP. Here we review a growing body of evidence suggesting that the cGMP signaling cascade warrants further investigation for its involvement in MDD and antidepressant action. PMID:22654729

  6. Cyclic electron flow within PSII protects PSII from its photoinhibition in thylakoid membranes from spinach chloroplasts.

    PubMed

    Miyake, Chikahiro; Okamura, Mitsutaka

    2003-04-01

    Cyclic electron flow within PSII (CEF-PSII) was proven to alleviate the photoinhibition of PSII. We set the conditions where CEF-PSII functioned or did not, by adding nigericin to the reaction mixture for the dissipation of DeltapH across thylakoid membranes, and then the thylakoids were illuminated. When CEF-PSII did not function and the activity of linear electron flow (LEF) was low, light-treated thylakoid membranes largely lost the activity of LEF. The inactivation of LEF was due to the loss of the activity of PSII, but not that of PSI. The inactivation of PSII was suppressed, when CEF-PSII functioned or LEF was enhanced. These results imply that CEF-PSII contributes to the protection of PSII from its photoinhibition with LEF, as an electron sink.

  7. Plant cyclic nucleotide signalling: facts and fiction.

    PubMed

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc; Maathuis, Frans Jm

    2007-11-01

    The presence of the cyclic nucleotides 3',5'-cyclic adenyl monophosphate (cAMP) and 3',5'-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed.

  8. Vortices determine the dynamics of biodiversity in cyclical interactions with protection spillovers

    NASA Astrophysics Data System (ADS)

    Szolnoki, Attila; Perc, Matjaž

    2015-11-01

    If rock beats scissors and scissors beat paper, one might assume that rock beats paper too. But this is not the case for intransitive relationships that make up the famous rock-paper-scissors game. However, the sole presence of paper might prevent rock from beating scissors, simply because paper beats rock. This is the blueprint for the rock-paper-scissors game with protection spillovers, which has recently been introduced as a new paradigm for biodiversity in well-mixed microbial populations. Here we study the game in structured populations, demonstrating that protection spillovers give rise to spatial patterns that are impossible to observe in the classical rock-paper-scissors game. We show that the spatiotemporal dynamics of the system is determined by the density of stable vortices, which may ultimately transform to frozen states, to propagating waves, or to target waves with reversed propagation direction, depending further on the degree and type of randomness in the interactions among the species. If vortices are rare, the fixation to waves and complex oscillatory solutions is likelier. Moreover, annealed randomness in interactions favors the emergence of target waves, while quenched randomness favors collective synchronization. Our results demonstrate that protection spillovers may fundamentally change the dynamics of cyclic dominance in structured populations, and they outline the possibility of programming pattern formation in microbial populations.

  9. Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis

    PubMed Central

    Gausdal, G; Wergeland, A; Skavland, J; Nguyen, E; Pendino, F; Rouhee, N; McCormack, E; Herfindal, L; Kleppe, R; Havemann, U; Schwede, F; Bruserud, Ø; Gjertsen, B T; Lanotte, M; Ségal-Bendirdjian, E; Døskeland, S O

    2013-01-01

    We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients. PMID:23449452

  10. Cyclic AMP can promote APL progression and protect myeloid leukemia cells against anthracycline-induced apoptosis.

    PubMed

    Gausdal, G; Wergeland, A; Skavland, J; Nguyen, E; Pendino, F; Rouhee, N; McCormack, E; Herfindal, L; Kleppe, R; Havemann, U; Schwede, F; Bruserud, O; Gjertsen, B T; Lanotte, M; Ségal-Bendirdjian, E; Døskeland, S O

    2013-02-28

    We show that cyclic AMP (cAMP) elevating agents protect blasts from patients with acute promyelocytic leukemia (APL) against death induced by first-line anti-leukemic anthracyclines like daunorubicin (DNR). The cAMP effect was reproduced in NB4 APL cells, and shown to depend on activation of the generally cytoplasmic cAMP-kinase type I (PKA-I) rather than the perinuclear PKA-II. The protection of both NB4 cells and APL blasts was associated with (inactivating) phosphorylation of PKA site Ser118 of pro-apoptotic Bad and (activating) phosphorylation of PKA site Ser133 of the AML oncogene CREB. Either event would be expected to protect broadly against cell death, and we found cAMP elevation to protect also against 2-deoxyglucose, rotenone, proteasome inhibitor and a BH3-only mimetic. The in vitro findings were mirrored by the findings in NSG mice with orthotopic NB4 cell leukemia. The mice showed more rapid disease progression when given cAMP-increasing agents (prostaglandin E2 analog and theophylline), both with and without DNR chemotherapy. The all-trans retinoic acid (ATRA)-induced terminal APL cell differentiation is a cornerstone in current APL treatment and is enhanced by cAMP. We show also that ATRA-resistant APL cells, believed to be responsible for treatment failure with current ATRA-based treatment protocols, were protected by cAMP against death. This suggests that the beneficial pro-differentiating and non-beneficial pro-survival APL cell effects of cAMP should be weighed against each other. The results suggest also general awareness toward drugs that can affect bone marrow cAMP levels in leukemia patients.

  11. Modulation of basal nitric oxide-dependent cyclic-GMP production by ambient glucose, myo-inositol, and protein kinase C in SH-SY5Y human neuroblastoma cells.

    PubMed Central

    Shindo, H; Thomas, T P; Larkin, D D; Karihaloo, A K; Inada, H; Onaya, T; Stevens, M J; Greene, D A

    1996-01-01

    Defective tissue perfusion and nitric oxide production and altered myo-inositol metabolism and protein kinase C activation have been invoked in the pathogenesis of diabetic complications including neuropathy. The precise cellular compartmentalization and mechanistic interrelationships of these abnormalities remain obscure, and nitric oxide possesses both neurotransmitter and vasodilator activity. Therefore the effects of ambient glucose and myo-inositol on nitric oxide-dependent cGMP production and protein kinase C activity were studied in SH-SY5Y human neuroblastoma cells, a cell culture model for peripheral cholinergic neurons. D-Glucose lowered cellular myo-inositol content, phosphatidylinositol synthesis, and phosphorylation of an endogenous protein kinase C substrate, and specifically reduced nitric oxide-dependent cGMP production a time- and dose-dependent manner with an apparent IC50 of approximately 30 mM. The near maximal decrease in cGMP induced by 50 mM D-glucose was corrected by the addition of protein kinase C agonists or 500 microM myo-inositol to the culture medium, and was reproduced by protein kinase C inhibition or downregulation, or by myo-inositol deficient medium. Sodium nitroprusside increased cGMP in a dose-dependent fashion, with low concentrations (1 microM) counteracting the effects of 50 mM D-glucose or protein kinase C inhibition. The demonstration that elevated D-glucose diminishes basal nitric oxide-dependent cGMP production by myo-inositol depletion and protein kinase C inhibition in peripheral cholinergic neurons provides a potential metabolic basis for impaired nitric oxide production, nerve blood flow, and nerve impulse conduction in diabetes. PMID:8609230

  12. Molecular mechanism of cGMP-mediated smooth muscle relaxation.

    PubMed

    Carvajal, J A; Germain, A M; Huidobro-Toro, J P; Weiner, C P

    2000-09-01

    Contraction and relaxation of smooth muscle is a tightly regulated process involving numerous endogenous substances and their intracellular second messengers. We examine the key role of cyclic guanosine monophosphate (cGMP) in mediating smooth muscle relaxation. We briefly review the current art regarding cGMP generation and degradation, while focusing on the recent identification of the molecular mechanisms underlying cGMP-mediated smooth muscle relaxation. cGMP-induced SM relaxation is mediated mainly by cGMP-dependent protein kinase activation. It involves several molecular events culminating in a reduction in intracellular Ca(2+) concentration and a decrease in the sensitivity of the contractile system to Ca(2+). We propose that the cGMP-induced decrease in Ca(2+) sensitivity is a strategic way to achieve "active relaxation" of the smooth muscle. In summary, we present compelling evidence supporting a key role for cGMP as a mediator of smooth muscle relaxation in physiological and pharmacological settings. Copyright 2000 Wiley-Liss, Inc.

  13. Identification of c-di-GMP-Responsive Riboswitches.

    PubMed

    Peltier, Johann; Soutourina, Olga

    2017-01-01

    Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important signaling molecule for community behavior control, cell morphogenesis, and virulence in bacteria. In addition to protein effectors, this second messenger binds RNA molecules that act as riboswitches to control target gene expression. In this chapter, we describe a method for experimental validation of the functionality of c-di-GMP-responsive riboswitches and the analysis of c-di-GMP control of target gene expression by qRT-PCR and Northern blot. This procedure can be used for the studies of in silico-predicted riboswitch candidates, as well as a targeted experimental approach for exploring the data from next-generation sequencing. The examples on the analysis of type I and type II c-di-GMP-responsive riboswitches in Clostridium difficile are provided to illustrate the application of the method.

  14. Specific labeling and permanent activation of the retinal rod cGMP-activated channel by the photoaffinity analog 8-p-azidophenacylthio-cGMP.

    PubMed Central

    Brown, R L; Gerber, W V; Karpen, J W

    1993-01-01

    Cyclic nucleotide-gated ion channels play a key role in visual excitation and a variety of other signaling pathways. The photoaffinity probe 8-p-azidophenacylthio-cGMP (APT-cGMP) has been developed for structural and functional studies of the cGMP-activated channel of retinal rod outer segments. Using this analog, we have demonstrated both specific labeling of the channel in a partially purified biochemical preparation from bovine rod outer segments and permanent activation of the channel current in excised membrane patches from salamander outer segments. After UV illumination, a 32P-labeled version of APT-cGMP was shown by SDS/PAGE and autoradiography to be covalently attached to the 63-kDa channel subunit. This incorporation was significantly reduced by 8-Br-cGMP but was not reduced by 5'-GMP. In patch-clamp experiments APT-cGMP was a potent activator of the channel; APT-cGMP typically opened half of the channels in a patch at a 10-fold lower concentration than cGMP. Exposure of membrane patches to UV light in the presence of APT-cGMP resulted in a persistent current observed in the absence of bath-applied nucleotide. This current increased with repeated exposure of the patch to both UV light and fresh APT-cGMP, approaching the maximum current originally evoked by saturating (500 microM) cGMP. At this point, addition of 500 microM cGMP caused a negligible increase in current. The persistent current had several other properties expected of current through cGMP-activated channels: it was outwardly rectifying; outward current was blocked > 90% by 2 mM internal Mg2+, whereas inward current was blocked much less efficiently; a low concentration of cGMP caused a larger increase in current atop a half-maximal persistent current than it did originally. We conclude that the persistent current was caused by the covalent tethering of cGMP moieties to channel binding sites, resulting in irreversible channel activation. APT-cGMP should prove useful for further studies of

  15. Role of cGMP in hydrogen sulfide signaling.

    PubMed

    Bibli, Sofia-Iris; Yang, Guangdong; Zhou, Zongmin; Wang, Rui; Topouzis, Stavros; Papapetropoulos, Andreas

    2015-04-30

    The importance of hydrogen sulfide (H2S) in physiology and disease is being increasingly recognized in recent years. Unlike nitric oxide (NO) that signals mainly through soluble guanyl cyclase (sGC)/cGMP, H2S is more promiscuous, affecting multiple pathways. It interacts with ion channels, enzymes, transcription factors and receptors. It was originally reported that H2S does not alter the levels of cyclic nucleotides. More recent publications, however, have shown increases in intracellular cGMP following exposure of cells or tissues to exogenously administered or endogenously produced H2S. Herein, we discuss the evidence for the participation of cGMP in H2S signaling and reconcile the seemingly divergent results presented in the literature on the role of this cyclic nucleotide in the biological actions of H2S. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Mutations of PKA cyclic nucleotide-binding domains reveal novel aspects of cyclic nucleotide selectivity.

    PubMed

    Lorenz, Robin; Moon, Eui-Whan; Kim, Jeong Joo; Schmidt, Sven H; Sankaran, Banumathi; Pavlidis, Ioannis V; Kim, Choel; Herberg, Friedrich W

    2017-07-06

    Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the 3',5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) and the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide selectivity is still not fully understood. Analysis of isolated cyclic nucleotide-binding (CNB) domains of PKA regulatory subunit type Iα (RIα) reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here, we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP specificity. Binding data with the generic purine nucleotide 3',5'-cyclic inosine monophosphate (cIMP) reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding and the molecular basis of cyclic nucleotide specificity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  17. Correlative intravital imaging of cGMP signals and vasodilation in mice

    PubMed Central

    Thunemann, Martin; Schmidt, Kjestine; de Wit, Cor; Han, Xiaoxing; Jain, Rakesh K.; Fukumura, Dai; Feil, Robert

    2014-01-01

    Cyclic guanosine monophosphate (cGMP) is an important signaling molecule and drug target in the cardiovascular system. It is well known that stimulation of the vascular nitric oxide (NO)-cGMP pathway results in vasodilation. However, the spatiotemporal dynamics of cGMP signals themselves and the cGMP concentrations within specific cardiovascular cell types in health, disease, and during pharmacotherapy with cGMP-elevating drugs are largely unknown. To facilitate the analysis of cGMP signaling in vivo, we have generated transgenic mice that express fluorescence resonance energy transfer (FRET)-based cGMP sensor proteins. Here, we describe two models of intravital FRET/cGMP imaging in the vasculature of cGMP sensor mice: (1) epifluorescence-based ratio imaging in resistance-type vessels of the cremaster muscle and (2) ratio imaging by multiphoton microscopy within the walls of subcutaneous blood vessels accessed through a dorsal skinfold chamber. Both methods allow simultaneous monitoring of NO-induced cGMP transients and vasodilation in living mice. Detailed protocols of all steps necessary to perform and evaluate intravital imaging experiments of the vasculature of anesthetized mice including surgery, imaging, and data evaluation are provided. An image segmentation approach is described to estimate FRET/cGMP changes within moving structures such as the vessel wall during vasodilation. The methods presented herein should be useful to visualize cGMP or other biochemical signals that are detectable with FRET-based biosensors, such as cyclic adenosine monophosphate or Ca2+, and to correlate them with respective vascular responses. With further refinement and combination of transgenic mouse models and intravital imaging technologies, we envision an exciting future, in which we are able to “watch” biochemistry, (patho-)physiology, and pharmacotherapy in the context of a living mammalian organism. PMID:25352809

  18. delta-Opioid receptor agonist SNC80 elicits peripheral antinociception via delta(1) and delta(2) receptors and activation of the l-arginine/nitric oxide/cyclic GMP pathway.

    PubMed

    Pacheco, Daniela F; Reis, Gláucia M L; Francischi, Janetti N; Castro, Maria S A; Perez, Andrea C; Duarte, Igor D G

    2005-11-19

    In this study, we characterized the role of delta(1) and delta(2) opioids receptors, as well the involvement of the l-arginine/NO/cGMP pathway in the peripheral antinociception induced by delta-opioid receptor agonist (+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80). The paw pressure test was utilized, in which pain sensitivity is increased by intraplantar injection of prostaglandin E(2) (2 microg). Administration of SNC80 (20, 40 and 80 microg/paw) decreased the hyperalgesia induced by prostaglandin E(2) in a dose-dependent manner. The possibility that the higher dose of SNC80 (80 microg) has a central or systemic effect was excluded, since administration of the drug into the contralateral paw did not elicit antinociception in the right paw. 7-Benzylidenenaltrexone (BNTX), 5, 10 and 20 microg/paw, and 17-(Cyclopropylmethyl)-6,7-didehydro-3,14beta-dihydroxy-4,5alpha-epoxy-6,7-2',3'-benzo[b]furanomorphinan (naltriben), 2.5, 5 and 10 microg/paw, delta(1) and delta(2) opioid receptor antagonist respectively, elicited partial antagonism of the peripheral antinociceptive effect of the SNC80 (80 microg). The BNTX (10 microg/paw)-naltriben (5 microg/paw) combination completely antagonized the peripheral antinociception induced by SNC80 (80 microg). Further, blockers of the l-arginine/NO/cGMP pathway, N(G)-nitro-l-arginine (12, 18 and 24 microg/paw) and methylene blue (125, 250 and 500 microg/paw) were observed reverting the peripheral antinociceptive effect of SNC80. This study provides evidence that the peripheral antinociception induced by SNC80 occurs via delta(1) and delta(2) receptors and may result from l-arginine/NO/cGMP pathway activation.

  19. Autophagy protects end plate chondrocytes from intermittent cyclic mechanical tension induced calcification.

    PubMed

    Xu, Hong-guang; Yu, Yun-fei; Zheng, Quan; Zhang, Wei; Wang, Chuang-dong; Zhao, Xiao-yn; Tong, Wen-xue; Wang, Hong; Liu, Ping; Zhang, Xiao-ling

    2014-09-01

    Calcification of end plate chondrocytes is a major cause of intervertebral disc (IVD) degeneration. However, the underlying molecular mechanism of end plate chondrocyte calcification is still unclear. The aim of this study was to clarify whether autophagy in end plate chondrocytes could protect the calcification of end plate chondrocytes. Previous studies showed that intermittent cyclic mechanical tension (ICMT) contributes to the calcification of end plate chondrocytes in vitro. While autophagy serves as a cell survival mechanism, the relationship of autophagy and induced end plate chondrocyte calcification by mechanical tension in vitro is unknown. Thus, we investigated autophagy, the expression of the autophagy genes, Beclin-1 and LC3, and rat end plate chondrocyte calcification by ICMT. The viability of end plate chondrocytes was examined using the LIVE/DEAD viability/cytotoxicity kit. The reverse transcription-polymerase chain reaction and western blotting were used to detect the expression of Beclin-1; LC3; type I, II and X collagen; aggrecan; and Sox-9 genes. Immunofluorescent and fluorescent microscopy showed decreased autophagy in the 10- and 20-day groups loaded with ICMT. Additionally, Alizarin red and alkaline phosphatase staining detected the palpable calcification of end plate chondrocytes after ICMT treatment. We found that increased autophagy induced by short-term ICMT treatment was accompanied by an insignificant calcification of end plate chondrocytes. To the contrary, the suppressive autophagy inhibited by long-term ICMT was accompanied by a more significant calcification. The process of calcification induced by ICMT was partially resisted by increased autophagy activity induced by rapamycin, implicating that autophagy may prevent end plate chondrocyte calcification. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Effect of interferon on concentrations of cyclic nucleotides in cultured cells.

    PubMed Central

    Tovey, M G; Rochette-Egly, C; Castagna, M

    1979-01-01

    Constant intracellular concentrations of both adenosine 3',5'-cyclic-monophosphate (cyclic AMP) and guanosine 3',5'-cyclic-monophosphate (cyclic GMP) were obtained when leukemia L1210 cells were cultivated under steady-state conditions in the chemostat. In this sensitive and controlled system addition of mouse interferon resulted in a rapid (5-10 min) increase in the intracellular concentration of cyclic GMP, which preceded by several hours an increase in the intracellular concentration of cyclic AMP. In contrast to the effect of interferon, addition of prostaglandin E1 induced a rapid increase in the intracellular concentration of cyclic AMP without markedly affecting the intracellular concentration of cyclic GMP. It is suggested that the rapid effect of interferon on cyclic GMP plays a role in mediating some of the effects of interferon on cells. PMID:226987

  1. Neutron Diffraction Reveals Hydrogen Bonds Critical for cGMP-Selective Activation: Insights for cGMP-Dependent Protein Kinase Agonist Design

    PubMed Central

    2015-01-01

    High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iβ CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). The XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted, explaining its low affinity for cAMP. PMID:25271401

  2. Neutron diffraction reveals hydrogen bonds critical for cGMP-selective activation: Insights for cGMP-dependent protein kinase agonist design

    DOE PAGES

    Huang, Gilbert Y.; Gerlits, Oksana O.; Blakeley, Matthew P.; ...

    2014-10-01

    High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iβ CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). Finally, the XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted,more » explaining its low affinity for cAMP.« less

  3. Evidence for cyclic diguanylate as a vaccine adjuvant with novel immunostimulatory activities.

    PubMed

    Gray, Peter M; Forrest, Gail; Wisniewski, Thomas; Porter, Gene; Freed, Daniel C; DeMartino, Julie A; Zaller, Dennis M; Guo, Zhiqiang; Leone, Joseph; Fu, Tong-Ming; Vora, Kalpit A

    2012-01-01

    Cyclic diguanylate (c-di-GMP), a bacterial signaling molecule, possesses protective immunostimulatory activity in bacterial challenge models. This study explored the potential of c-di-GMP as a vaccine adjuvant comparing it with LPS, CpG oligonucleotides, and a conventional aluminum salt based adjuvant. In this evaluation, c-di-GMP was a more potent activator of both humoral and Th1-like immune responses as evidenced by the robust IgG2a antibody response it induced in mice and the strong IFN-γ, TNF-α and IP-10 responses, it elicited in mice and in vitro in non-human primate peripheral blood mononuclear cells. Further, compared to LPS or CpG, c-di-GMP demonstrated a more pronounced ability to induce germinal center formation, a hallmark of long-term memory, in immunized mice. Together, these data add to the growing body of evidence supporting the utility of c-di-GMP as an adjuvant in vaccination for sustained and robust immune responses and provide a rationale for further evaluation in appropriate models of immunization. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. [Structure and function of c-di-GMP riboswitches].

    PubMed

    Li, Xinfeng; Chen, Fang; Xiao, Jinfeng; He, Jin

    2017-09-25

    Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous nucleotide second messenger present in a wide variety of bacteria. It regulates many important bacterial physiological functions such as biofilm formation, motility, adhesion, virulence and extracellular polysaccharide synthesis. It binds with many different proteins or RNA receptors, one of which is called riboswitch that is usually located at the 5'-untranslational region (5'-UTR) in some mRNA. Riboswitch usually comprises a specific ligand-binding (sensor) domain (named aptamer domain, AD), as well as a variable domain, termed expression platform (EP), to regulate expression of downstream coding sequences. When a specific metabolite concentration exceeds its threshold level, it will bind to its cognate riboswitch receptor to induce a conformational change of 5'-UTR, leading to modulation of downstream gene expression. Two classes of c-di-GMP-binding riboswitches (c-di-GMP-Ⅰ and c-di-GMP-Ⅱ) have been discovered that bind with this second messenger with high affinity to regulate diverse downstream genes, underscoring the importance of this unique RNA receptor in this pathway. Class Ⅰ c-di-GMP riboswitches are present in a wide variety of bacteria, and are most common in the phyla Firmicutes and Proteobacteria, while class Ⅱ c-di-GMP riboswitches typically function as allosteric ribozymes, binding to c-di-GMP to induce folding changes at atypical splicing site junctions to modulate downstream gene expression. This review introduces the discovery, classification, function, and also the affected downstream genes of c-di-GMP riboswitches.

  5. Cyclic adenosine monophosphate-mediated protection against bile acid-induced apoptosis in cultured rat hepatocytes.

    PubMed

    Webster, C R; Anwer, M S

    1998-05-01

    Cyclic adenosine monophosphate (cAMP) has been shown to modulate apoptosis. To evaluate the role of cAMP in bile acid-induced hepatocyte apoptosis, we studied the effect of agents that increase cAMP on the induction of apoptosis by glycochenodeoxycholate (GCDC) in cultured rat hepatocytes. GCDC induced apoptosis in 26.5%+/-1.1% of hepatocytes within 2 hours. Twenty-minute pretreatment of hepatocytes with 100 micromol/L 8-(4-chlorothiophenyl) cAMP (CP-cAMP) resulted in a reduction in the amount of apoptosis to 35.2%+/-3.8% of that seen in hepatocytes treated with GCDC alone. Other agents that increase intracellular cAMP, including dibutyryl cAMP (100 micromol/L), glucagon (200 nmol/L), and a combination of forskolin (20 micromol/L) and 3-isobutyl-1-methylxanthine (20 micromol/L), also inhibited GCDC-induced apoptosis to a similar extent. Pretreatment with the protein kinase A (PKA) inhibitor, KT5720, prevented the protective effect of CP-cAMP and inhibited CP-cAMP-induced activation of PKA activity. Inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin (50 nmol/L), or Ly 294002 (20 micromol/L) also prevented the cytoprotective effect of cAMP. PI3K assays confirmed that wortmannin (50 nmol/L) inhibited PI3K activity, while CP-cAMP had no effect on the activity of this lipid kinase. GCDC increased mitogen-activated protein kinase (MAPK) activity, but had no effect on stress-activated protein kinase (SAPK) activity in hepatocytes. cAMP decreased basal and GCDC-induced MAPK activity and increased SAPK activity. The MAPK kinase inhibitor, PD 98059, inhibited both GCDC-mediated MAPK activation and GCDC-induced apoptosis. 1) agents that increase intracellular cAMP protect against hepatocyte apoptosis induced by hydrophobic bile acids; 2) activation of MAPK by GCDC may be involved in bile acid-induced apoptosis; and 3) cAMP-mediated cytoprotection against bile acid-induced apoptosis appears to involve PKA, MAPK, and PI3K.

  6. Potassium channel stimulation by natriuretic peptides through cGMP-dependent dephosphorylation.

    PubMed

    White, R E; Lee, A B; Shcherbatko, A D; Lincoln, T M; Schonbrunn, A; Armstrong, D L

    1993-01-21

    Natriuretic peptides inhibit the release and action of many hormones through cyclic guanosine monophosphate (cGMP), but the mechanism of cGMP action is unclear. In frog ventricular muscle and guinea-pig hippocampal neurons, cGMP inhibits voltage-activated Ca2+ currents by stimulating phosphodiesterase activity and reducing intracellular cyclic AMP; however, this mechanism is not involved in the action of cGMP on other channels or on Ca2+ channels in other cells. Natriuretic peptide receptors in the rat pituitary also stimulate guanylyl cyclase activity but inhibit secretion by increasing membrane conductance to potassium. In an electrophysiological study on rat pituitary tumour cells, we identified the large-conductance, calcium- and voltage-activated potassium channels (BK) as the primary target of another inhibitory neuropeptide, somatostatin. Here we report that atrial natriuretic peptide also stimulates BK channel activity in GH4C1 cells through protein dephosphorylation. Unlike somatostatin, however, the effect of atrial natriuretic peptide on BK channel activity is preceded by a rapid and potent stimulation of cGMP production and requires cGMP-dependent protein kinase activity. Protein phosphatase activation by cGMP-dependent kinase could explain the inhibitory effects of natriuretic peptides on electrical excitability and the antagonism of cGMP and cAMP in many systems.

  7. Disrupted pulmonary artery cyclic guanosine monophosphate signaling in mice with hyperoxia-induced pulmonary hypertension.

    PubMed

    Lee, Keng Jin; Berkelhamer, Sara K; Kim, Gina A; Taylor, Joann M; O'Shea, Kelly M; Steinhorn, Robin H; Farrow, Kathryn N

    2014-02-01

    Pulmonary hypertension (PH) occurs in 25 to 35% of premature infants with significant bronchopulmonary dysplasia (BPD). Neonatal mice exposed to 14 days of hyperoxia develop BPD-like lung injury and PH. To determinne the impact of hyperoxia on pulmonary artery (PA) cyclic guanosine monophosphate (cGMP) signaling in a murine model of lung injury and PH, neonatal C57BL/6 mice were placed in room air, 75% O2 for 14 days (chronic hyperoxia [CH]) or 75% O2 for 24 hours, followed by 13 days of room air (acute hyperoxia with recovery [AHR]) with or without sildenafil. At 14 days, mean alveolar area, PA medial wall thickness (MWT), right ventricular hypertrophy (RVH), and vessel density were assessed. PA protein was analyzed for cGMP, soluble guanylate cyclase, and PDE5 activity. CH and AHR mice had RVH, but only CH mice had increased alveolar area and MWT and decreased vessel density. In CH and AHR PAs, soluble guanylate cyclase activity was decreased, and PDE5 activity was increased. In CH mice, sildenafil attenuated MWT and RVH but did not improve mean alveolar area or vessel density. In CH and AHR PAs, sildenafil decreased PDE5 activity and increased cGMP. Our results indicate that prolonged hyperoxia leads to lung injury, PH, RVH, and disrupted PA cGMP signaling. Furthermore, 24 hours of hyperoxia causes RVH and disrupted PA cGMP signaling that persists for 13 days. Sildenafil reduced RVH and restored vascular cGMP signaling but did not attenuate lung injury. Thus, hyperoxia can rapidly disrupt PA cGMP signaling in vivo with sustained effects, and concurrent sildenafil therapy can be protective.

  8. Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells.

    PubMed

    Pfannes, Eva K B; Anielski, Alexander; Gerhardt, Matthias; Beta, Carsten

    2013-12-01

    Cyclic GMP (cGMP) is a ubiquitous second messenger in eukaryotic cells. It is assumed to regulate the association of myosin II with the cytoskeleton of motile cells. When cells of the social amoeba Dictyostelium discoideum are exposed to chemoattractants or to increased osmotic stress, intracellular cGMP levels rise, preceding the accumulation of myosin II in the cell cortex. To directly investigate the impact of intracellular cGMP on cytoskeletal dynamics in a living cell, we released cGMP inside the cell by laser-induced photo-cleavage of a caged precursor. With this approach, we could directly show in a live cell experiment that an increase in intracellular cGMP indeed induces myosin II to accumulate in the cortex. Unexpectedly, we observed for the first time that also the amount of filamentous actin in the cell cortex increases upon a rise in the cGMP concentration, independently of cAMP receptor activation and signaling. We discuss our results in the light of recent work on the cGMP signaling pathway and suggest possible links between cGMP signaling and the actin system.

  9. A single, low dose of a cGMP recombinant BCG vaccine elicits protective T cell immunity against the human respiratory syncytial virus infection and prevents lung pathology in mice.

    PubMed

    Céspedes, Pablo F; Rey-Jurado, Emma; Espinoza, Janyra A; Rivera, Claudia A; Canedo-Marroquín, Gisela; Bueno, Susan M; Kalergis, Alexis M

    2017-02-01

    Human respiratory syncytial virus (hRSV) is a major health burden worldwide, causing the majority of hospitalizations in children under two years old due to bronchiolitis and pneumonia. HRSV causes year-to-year outbreaks of disease, which also affects the elderly and immunocompromised adults. Furthermore, both hRSV morbidity and epidemics are explained by a consistently high rate of re-infections that take place throughout the patient life. Although significant efforts have been invested worldwide, currently there are no licensed vaccines to prevent hRSV infection. Here, we describe that a recombinant Bacillus Calmette-Guerin (BCG) vaccine expressing the nucleoprotein (N) of hRSV formulated under current good manufacture practices (cGMP rBCG-N-hRSV) confers protective immunity to the virus in mice. Our results show that a single dose of the GMP rBCG-N-hRSV vaccine retains its capacity to protect mice against a challenge with a disease-causing infection of 1×10(7) plaque-forming units (PFUs) of the hRSV A2 clinical strain 13018-8. Compared to unimmunized infected controls, vaccinated mice displayed reduced weight loss and less infiltration of neutrophils within the airways, as well as reduced viral loads in bronchoalveolar lavages, parameters that are characteristic of hRSV infection in mice. Also, ex vivo re-stimulation of splenic T cells at 28days post-immunization activated a repertoire of T cells secreting IFN-γ and IL-17, which further suggest that the rBCG-N-hRSV vaccine induced a mixed, CD8(+) and CD4(+) T cell response capable of both restraining viral spread and preventing damage of the lungs. All these features support the notion that rBCG-N-hRSV is a promising candidate vaccine to be used in humans to prevent the disease caused by hRSV in the susceptible population.

  10. Cyclic nucleotide responses and radiation-induced mitotic delay in Physarum polycephalum

    SciTech Connect

    Daniel, J.W.; Oleinick, N.L.

    1984-02-01

    The response of the plasmodial levels of cyclic AMP and cyclic GMP in Physarum polycephalum to several putative phosphodiesterase inhibitors and to ionizing radiation has been measured. Isobutylmethylxanthine (2 mM) induces a rapid transient threefold elevation of cyclic AMP alone, with maximum response in about 10 min and return to the base line in about 30 min. Theophylline (2 mM) induces a rapid, sustained twofold elevation of cyclic GMP only. Caffeine (2mM) and Ro-20-1724 (18 ..mu..M) both elicit a rapid transient rise in cyclic AMP, resembling the isobutylmethylxanthine response, and a slow transient elevation of the cyclic GMP level. Of particular interest is the rapid threefold transient elevation of the cyclic AMP, but not of the cyclic GMP, level by ..gamma.. radiation.

  11. Cyclic nucleotide responses and radiation-induced mitotic delay in Physarum polycephalum

    SciTech Connect

    Daniel, J.W.; Oleinick, N.L.

    1984-02-01

    The response of the plasmodial levels of cyclic AMP and cyclic GMP in Physarum polycephalum to several putative phosphodiesterase inhibitors and to ionizing radiation has been measured. Isobutylmethylxanthine (2 mM) induces a rapid transient threefold elevation of cyclic AMP alone, with maximum response in about 10 min and return to the base line in about 30 min. Theophylline (2 mM) induces a rapid, sustained twofold elevation of cyclic GMP only. Caffeine (2mM) and Ro-20-1724 (18 ..mu..M) both elicit a rapid transient rise in cyclic AMP, resembling the isobutylmethylxanthine response, and a slow transient elevation of the cyclic GMP level. Of particular interest is the rapid threefold transient elevation of the cyclic AMP, but not of the cyclic GMP, level by ..gamma.. radiation.

  12. Regulation of cGMP synthesis in cultured podocytes by vasoactive hormones.

    PubMed

    Lewko, B; Gołos, M; Latawiec, E; Angielski, S; Stepinski, J

    2006-12-01

    The podocytes are highly differentiated cells playing a key role in glomerular filtration. Vasoactive factors including angiotensin II (Ang II) and cyclic guanosine 5' monophosphate (cGMP) are synthesized by these cells upon stimulation as well as in the basal state. In this study we have tested whether angiotensin II affects the total synthesis of cGMP in primary culture of rat podocytes. The cells were stimulated with atrial natriuretic peptide (ANP) and/or a nitric oxide (NO) donor, S-nitroso-N-acetyl penicillamine (SNAP), in the absence or presence of Ang II. The cGMP synthesis was determined by radioimmunoassay (RIA). ANP or SNAP alone increased the cGMP synthesis in podocytes although the effects were not additive unless Ang II was present in the medium. Ang II suppressed the ANP-dependent cGMP synthesis whereas SNAP-dependent cGMP production remained unaffected. These effects were prevented by a non-specific antagonist of Ang II receptors (AT), saralasin. Adversely, PD123319, a specific inhibitor of AT2 receptors, augmented inhibition of ANP-dependent and enhanced the NO-dependent cGMP production. Probenecid, an inhibitor of cGMP extrusion from the cells, suppressed the cGMP generation by both ANP and SNAP. We conclude that cGMP synthesis in cultured podocytes is modulated by angiotensin II and that two adversely acting receptors, AT1 and AT2 are involved in this effect. Additionally, production of cGMP might be intrinsically inhibited by cGMP accumulating inside the cells.

  13. Identification of c-di-GMP derivatives resistant to an EAL domain phosphodiesterase

    PubMed Central

    Shanahan, Carly A.; Gaffney, Barbara L.; Jones, Roger A.; Strobel, Scott A.

    2013-01-01

    The bacterial second messenger signaling molecule bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) controls important biological processes such as biofilm formation, virulence response, and motility. This second messenger is sensed by macromolecular targets inside the cell, both protein and RNA, which induce specific phenotypic responses critical for bacterial survival. One class of enzymes responsible for regulating the intracellular concentration of c-di-GMP, and therefore the physiological behavior of the cell, is the EAL domain phosphodiesterases, which degrade the second messenger to its linear form, pGpG. Here, we investigate how base and backbone modifications to c-di-GMP affect the rate of cyclic dinucleotide degradation by an EAL domain protein (CC3396 from Caulobacter crescentus). The doubly substituted thiophosphate analog is highly resistant to hydrolysis by this metabolizing enzyme but can still bind c-di-GMP riboswitch targets. We used these findings to develop a novel ribosyl-phosphate modified derivative of c-di-GMP containing 2′-deoxy and methylphosphonate substitutions that is charge neutral and demonstrate that this analog is also resistant to EAL domain catalyzed degradation. This suggests a general strategy for designing c-di-GMP derivatives with increased enzymatic stability that also possess desirable properties for development as chemical probes of c-di-GMP signaling. PMID:23256840

  14. Near-infrared Light Responsive Synthetic c-di-GMP Module for Optogenetic Applications

    PubMed Central

    2015-01-01

    Enormous potential of cell-based therapeutics is hindered by the lack of effective means to control genetically engineered cells in mammalian tissues. Here, we describe a synthetic module for remote photocontrol of engineered cells that can be adapted for such applications. The module involves photoactivated synthesis of cyclic dimeric GMP (c-di-GMP), a stable small molecule that is not produced by higher eukaryotes and therefore is suitable for orthogonal regulation. The key component of the photocontrol module is an engineered bacteriophytochrome diguanylate cyclase, which synthesizes c-di-GMP from GTP in a light-dependent manner. Bacteriophytochromes are particularly attractive photoreceptors because they respond to light in the near-infrared window of the spectrum, where absorption by mammalian tissues is minimal, and also because their chromophore, biliverdin IXα, is naturally available in mammalian cells. The second component of the photocontrol module, a c-di-GMP phosphodiesterase, maintains near-zero background levels of c-di-GMP in the absence of light, which enhances the photodynamic range of c-di-GMP concentrations. In the E. coli model used in this study, the intracellular c-di-GMP levels could be upregulated by light by >50-fold. Various c-di-GMP-responsive proteins and riboswitches identified in bacteria can be linked downstream of the c-di-GMP-mediated photocontrol module for orthogonal regulation of biological activities in mammals as well as in other organisms lacking c-di-GMP signaling. Here, we linked the photocontrol module to a gene expression output via a c-di-GMP-responsive transcription factor and achieved a 40-fold photoactivation of gene expression. PMID:24926804

  15. An emerging role of cGMP in the treatment of schizophrenia: A review.

    PubMed

    Shim, Seong; Shuman, Michael; Duncan, Erica

    2016-01-01

    Schizophrenia is a progressive psychotic disorder with devastating effects on the broad aspects of human emotion, perception, thought, and psychosocial interactions. Although treatment with antipsychotic drugs, the mainstay in the treatment of schizophrenia, the large number of patients with schizophrenia respond poorly to the pharmacological and, the large number of patients with schizophrenia poorly respond to the pharmacological treatment. Although a variety of novel therapeutics have long been tested, to date, no drugs clinically efficacious for schizophrenia are available. The multiple lines of evidence strongly suggest that the modulation of cyclic guanosine monophosphate (cGMP) is a promising target in promoting the novel therapeutic strategies of schizophrenia beyond the "receptor-dependent" psychopharmacology. cGMP is modulated via regulating its synthesis by N-methyl-d-aspartate receptor (NMDAR) and nitric oxide (NO), which regulate guannylyl cyclase (GC), the enzyme producing cGMP. cGMP is also regulated by phosphodiesterase (PDE), the enzyme hydrolyzing cGMP. In this review, we critically evaluate the therapeutic potential of agents modulating cGMP activity by regulating cGMP synthesis including NMDAR enhancers, NO enhancers, NO inhibitors including minocycline with anti-inflammatory properties and PDE inhibitors in improving the negative, cognitive and positive symptoms of schizophrenia. We also discuss the possible mechanisms by which these agents produce therapeutic effects on schizophrenia including cGMP signaling pathways, oxidative stress, and neuroinflammation.

  16. Structural Basis of Ligand Binding by a C-di-GMP Riboswitch

    SciTech Connect

    Smith, K.; Lipchock, S; Ames, T; Wang, J; Breaker, R; Strobel, S

    2009-01-01

    The second messenger signaling molecule bis-(3{prime}-5{prime})-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP-binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 {angstrom} resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structural data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.

  17. Effect of sildenafil on platelet function and platelet cGMP of patients with erectile dysfunction.

    PubMed

    Akand, M; Gencer, E; Yaman, Ö; Erişgen, G; Tekin, D; Özdiler, E

    2015-12-01

    To investigate the effect of sildenafil on platelet function and cyclic guanosine monophosphate (cGMP) levels in patients with erectile dysfunction, we evaluated the association between erectile function and platelet responses after administration of 100 mg sildenafil. Erectile responses were monitored after 8 daily doses of the drug. Adenosine diphosphate (ADP) and collagen-induced platelet aggregation and simultaneous adenosine triphosphate (ATP) release and cGMP levels were determined before and after sildenafil therapy. Basal levels for platelet aggregation, ATP release and cGMP were compared with age-matched controls. There was no difference among basal levels of platelet responses between patients and controls, except for ADP-induced platelet aggregation (P = 0.04). It was significantly higher in the patient group. Analysis of the responses to sildenafil revealed that for the patients who showed a positive erectile response, there was a significant increase in platelet cGMP (P = 0.028) and a decrease in ADP-induced platelet aggregation (P = 0.04). However, for those who showed a negative or poor erectile response, there was no change in platelet cGMP levels and platelet functions. Sildenafil did not affect collagen-induced platelet responses although cGMP levels of the responders increased. It is concluded that sildenafil increases platelet cGMP in the patients with positive erectile response. Therefore, it has been speculated that platelet cGMP may be used as an index for erectile response.

  18. c-di-GMP induction of Dictyostelium cell death requires the polyketide DIF-1.

    PubMed

    Song, Yu; Luciani, Marie-Françoise; Giusti, Corinne; Golstein, Pierre

    2015-02-15

    Cell death in the model organism Dictyostelium, as studied in monolayers in vitro, can be induced by the polyketide DIF-1 or by the cyclical dinucleotide c-di-GMP. c-di-GMP, a universal bacterial second messenger, can trigger innate immunity in bacterially infected animal cells and is involved in developmental cell death in Dictyostelium. We show here that c-di-GMP was not sufficient to induce cell death in Dictyostelium cell monolayers. Unexpectedly, it also required the DIF-1 polyketide. The latter could be exogenous, as revealed by a telling synergy between c-di-GMP and DIF-1. The required DIF-1 polyketide could also be endogenous, as shown by the inability of c-di-GMP to induce cell death in Dictyostelium HMX44A cells and DH1 cells upon pharmacological or genetic inhibition of DIF-1 biosynthesis. In these cases, c-di-GMP-induced cell death was rescued by complementation with exogenous DIF-1. Taken together, these results demonstrated that c-di-GMP could trigger cell death in Dictyostelium only in the presence of the DIF-1 polyketide or its metabolites. This identified another element of control to this cell death and perhaps also to c-di-GMP effects in other situations and organisms.

  19. Crystal Structure of PKG I:cGMP Complex Reveals a cGMP-Mediated Dimeric Interface that Facilitates cGMP-Induced Activation.

    PubMed

    Kim, Jeong Joo; Lorenz, Robin; Arold, Stefan T; Reger, Albert S; Sankaran, Banumathi; Casteel, Darren E; Herberg, Friedrich W; Kim, Choel

    2016-05-03

    Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R) domain bound with cGMP, which represents the activated state. Although we used a monomeric domain for crystallization, the structure reveals that two R domains form a symmetric dimer where the cGMP bound at high-affinity pockets provide critical dimeric contacts. Small-angle X-ray scattering and mutagenesis support this dimer model, suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cyclic AMP-dependent protein kinase reveals that PKG is drastically different from protein kinase A in its active conformation, suggesting a novel activation mechanism for PKG.

  20. Predicting minimum Al concentrations for protective scale fromation on Ni-base alloys. I - Isothermal oxidation. II - Cyclic oxidation

    NASA Technical Reports Server (NTRS)

    Nesbitt, James A.

    1989-01-01

    Criteria proposed to predict the minimum bulk Al concentration for the formation of protective Al2O3 scales on Ni-based alloys during isothermal oxidation (two criteria proposed by Wagner, 1952 and 1959) and cyclic oxidation (the criteria proposed by Wahl, 1983, and Whittle, 1972/Wahl, 1983) were applied to Ni-Al and Ni-Cr-Al(Zr) alloys, respectively. It is shown that the first Wagner (1952) criterion underpredicted, by a factor of 3, the experimentally observed minimum Al concentration for the formation of an external Al2O3 scale on Ni-Al alloys at 1200 C; the second Wagner criterion predicted a transition from internal oxidation to continuous Al2O3 formation in good agreement with experimentally observed concentrations. It was also found that the two criteria for an Al2O3 scale formation during cyclic oxidation of Ni-Cr-Al(Zr) alloys were inadequate to predict the minimum Al concentration necessary for repeated formation of an Al2O3 scale, regardless of the adherence of the scale.

  1. cGMP reduces the sarcoplasmic reticulum Ca2+ loading in airway smooth muscle cells: a putative mechanism in the regulation of Ca2+ by cGMP.

    PubMed

    Bazán-Perkins, Blanca

    2012-03-01

    Ca(2+) and cGMP have opposite roles in many physiological processes likely due to a complex negative feedback regulation between them. Examples of opposite functions induced by Ca(2+) and cGMP are smooth muscle contraction and relaxation, respectively. A main Ca(2+) storage involved in contraction is sarcoplasmic reticulum (SR); nevertheless, the role of cGMP in the regulation of SR-Ca(2+) has not been completely understood. To evaluate this role, intracellular Ca(2+) concentration ([Ca(2+)]i) was determinated by a ratiometric method in isolated myocytes from bovine trachea incubated with Fura-2/AM. The release of Ca(2+) from SR induced by caffeine was transient, whereas caffeine withdrawal was followed by a [Ca(2+)]i undershoot. Caffeine-induced Ca(2+) transient peak and [Ca(2+)]i undershoot after caffeine were reproducible in the same cell. Dibutyryl cGMP (db-cGMP) blocked the [Ca(2+)]i undershoot and reduced the subsequent caffeine peak (SR-Ca(2+) loading). Both, the opening of SR channels with ryanodine (10 μM) and the blockade of SR-Ca(2+) ATPase with cyclopiazonic acid inhibited the [Ca(2+)]i undershoot as well as the SR-Ca(2+) loading. The addition of db-cGMP to ryanodine (10 μM) incubated cells partially restored the SR-Ca(2+) loading. Cyclic GMP enhanced [Ca(2+)]i undershoot induced by the blockade of ryanodine channels with 50 μM ryanodine. In conclusion, the reduction of SR-Ca(2+) content in airway smooth muscle induced by cGMP can be explained by the combination of SR-Ca(2+) loading and the simultaneous release of SR-Ca(2+). The reduction of SR-Ca(2+) content induced by cGMP might be a putative mechanism limiting releasable Ca(2+) in response to a particular stimulus.

  2. A novel role for a Drosophila homologue of cGMP-specific phosphodiesterase in the active transport of cGMP

    PubMed Central

    Day, Jonathan P.; Houslay, Miles D.; Davies, Shireen-A.

    2005-01-01

    cGMP was first discovered in urine, demonstrating that kidney cells extrude this cyclic nucleotide. Drosophila Malpighian tubules provide a model renal system in which a homologue of mammalian PDE (phosphodiesterase) 6 is expressed. In humans, this cG-PDE (cGMP-specific PDE) is specifically expressed in the retinal system, where it controls visual signal transduction. In order to gain insight into the functional role of DmPDE6 (Drosophila PDE6-like enzyme) in epithelial function, we generated transgenic animals with targeted expression of DmPDE6 to tubule Type I (principal) cells. This revealed localization of DmPDE6 primarily at the apical membranes. As expected, overexpression of DmPDE6 resulted in elevated cG-PDE activity and decreased tubule cGMP content. However, such targeted overexpression of DmPDE6 creates a novel phenotype that manifests itself in inhibition of the active transport and efflux of cGMP by tubules. This effect is specific to DmPDE6 action, as no effect on cGMP transport is observed in tubules from a bovine PDE5 transgenic line which display reduced rates of fluid secretion, an effect not seen in DmPDE6 transgenic animals. Specific ablation of DmPDE6 in tubule principal cells, via expression of a targeted DmPDE6 RNAi (RNA interference) transgene, conferred increased active transport of cGMP, confirming a direct role for DmPDE6 in regulating cGMP transport in tubule principal cells. Pharmacological inhibition of DmPDE6 in wild-type tubules using the cG-PDE inhibitor, zaprinast, similarly results in stimulated cGMP transport. We provide the first demonstration of a novel role for a cG-PDE in modulating cGMP transport and efflux. PMID:16232123

  3. Structural and biochemical characterization of linear dinucleotide analogs bound to the c-di-GMP-I aptamer†,‡

    PubMed Central

    Smith, Kathryn D.; Lipchock, Sarah V.; Strobel, Scott A.

    2011-01-01

    The cyclic dinucleotide c-di-GMP regulates lifestyle transitions in many bacteria, such as the change from a free motile state to a biofilm-forming community. Riboswitches that bind this second messenger are important downstream targets in this bacterial signaling pathway. The breakdown of c-di-GMP in the cell is accomplished enzymatically and results in the linear dinucleotide pGpG. The c-di-GMP-binding riboswitches must be able to discriminate between their cognate cyclic ligand and linear dinucleotides in order to be selective biological switches. It has been reported that the cdi-GMP-I riboswitch binds c-di-GMP five orders of magnitude better than the linear pGpG, but the cause of this large energetic difference in binding is unknown. Here we report binding data and crystal structures of several linear c-di-GMP analogs in complex with the c-di-GMP-I riboswitch. These data reveal the parameters for phosphate recognition and the structural basis of linear dinucleotide binding to the riboswitch. Additionally, the pH dependence of binding shows that exclusion of pGpG is not due to the additional negative charge on the ligand. These data reveal principles that, along with published work, will contribute to the design of c-di-GMP analogs with properties desirable for use as chemical tools and potential therapeutics. PMID:22148472

  4. cGMP-dependent protein kinase enhances Ca2+ current and potentiates the serotonin-induced Ca2+ current increase in snail neurones.

    PubMed

    Paupardin-Tritsch, D; Hammond, C; Gerschenfeld, H M; Nairn, A C; Greengard, P

    Protein phosphorylation catalysed by cyclic AMP-dependent, Ca2+/calmodulin-dependent and Ca2+/diacylglycerol-dependent protein kinases is important both in the modulation of synaptic transmission and in the regulation of neuronal membrane permeability (for reviews see refs 5-7). However, there has previously been no evidence for the involvement of cyclic GMP-dependent protein kinase (cGMP-PK) in the regulation of neuronal function. Serotonin induces an increase of Ca2+ current in a group of identified ventral neurones of the snail Helix aspersa. This effect is probably mediated by cGMP because it is mimicked by the intracellular injection of cGMP or the application of zaprinast, an inhibitor of cGMP-dependent phosphodiesterase. We have now found that the effect of either serotonin or zaprinast on the Ca2+ current is potentiated by the intracellular injection of cGMP-PK. Moreover, the intracellular injection of activated cGMP-PK (cGMP-PK + 1 microM cGMP) greatly enhances the Ca2+ current of the identified ventral neurones seen in the absence of serotonin. These results indicate that cGMP-PK has a physiological role in the control of the membrane permeability of these neurones.

  5. Protection of neuroblastoma Neuro2A cells from hypoxia-induced apoptosis by cyclic phosphatidic acid (cPA).

    PubMed

    Gotoh, Mari; Sano-Maeda, Katsura; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2012-01-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl(2)) to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A) cells with CoCl(2) induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl(2)-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl(2). Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA(1), LPA(2), and LPA(6), which are G-protein coupled receptors that can be activated by cPA. To date, LPA(1) and LPA(2) have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA(1) and LPA(2) on cPA-induced neuroprotective functions, Ki16425, a selective LPA(1) and LPA(3) antagonist, was adopted to know the LPA(1) function and siRNA was used to knockdown the expression of LPA(2). On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl(2)-induced hypoxia damage is mediated via LPA(2).

  6. Development and Validation of a Transcreener Assay for Detection of AMP- and GMP-Producing Enzymes

    PubMed Central

    Kleman-Leyer, Karen M.; Kopp, Andrew L.; Westermeyer, Thane A.; Lowery, Robert G.

    2010-01-01

    Abstract Screening of AMP- and GMP-producing enzymes such as phosphodiesterases (PDEs), ligases, and synthetases would be simplified by the ability to directly detect unmodified nucleoside monophosphates. To address this need, we developed polyclonal and monoclonal antibodies that recognize AMP and GMP with nanomolar sensitivity and high selectivity vs. the corresponding triphosphate and 3′,5′-cyclic monophosphate nucleotides that serve as substrates for many enzymes in these classes. One of these antibodies was used to develop a Transcreener® AMP/GMP assay with a far red fluorescence polarization (FP) readout. This polyclonal antibody exhibited extremely high selectivity, with IC50 ratios of 6,000 for ATP/AMP, 3,810 for cAMP/AMP, and 6,970 for cGMP/GMP. Standard curves mimicking enzymatic conversion of cAMP, cGMP, and ATP to the corresponding monophosphates yielded Z′ values of >0.85 at 10% conversion. The assay reagents were shown to be stable for 24 h at room temperature, both before and after dispensing. The Transcreener AMP/GMP FP assay was used for enzymatic detection of cGMP- and cAMP-dependent PDEs 4A1A, 3A, and 9A2 and ATP-dependent ligases, acetyl CoA synthetase, and ubiquitin-activating enzyme (UBE1). Shifts of >100 mP were observed in the linear part of the progress curves for all enzymes tested, and the PDE isoforms exhibited the expected substrate and inhibitor selectivity. These studies demonstrate that direct immunodetection of AMP and GMP is a flexible, robust enzyme assay method for diverse AMP- and GMP-producing enzymes. Moreover, it eliminates many of the shortcomings of other methods including the need for fluorescently labeled substrates, the low signal:background inherent in substrate depletion assays, and the potential for interference with coupling enzymes. PMID:20158441

  7. Prediction and verification of creep behavior in metallic materials and components for the space shuttle thermal protection system. Volume 2: Phase 2 subsize panel cyclic creep predictions

    NASA Technical Reports Server (NTRS)

    Cramer, B. A.; Davis, J. W.

    1975-01-01

    A method for predicting permanent cyclic creep deflections in stiffened panel structures was developed. The resulting computer program may be applied to either the time-hardening or strain-hardening theories of creep accumulation. Iterative techniques were used to determine structural rotations, creep strains, and stresses as a function of time. Deflections were determined by numerical integration of structural rotations along the panel length. The analytical approach was developed for analyzing thin-gage entry vehicle metallic-thermal-protection system panels subjected to cyclic bending loads at high temperatures, but may be applied to any panel subjected to bending loads. Predicted panel creep deflections were compared with results from cyclic tests of subsize corrugation and rib-stiffened panels. Empirical equations were developed for each material based on correlation with tensile cyclic creep data and both the subsize panels and tensile specimens were fabricated from the same sheet material. For Vol. 1, see N75-21431.

  8. Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor

    PubMed Central

    Hickman, Jason W.; Harwood, Caroline S.

    2008-01-01

    High levels of the intracellular signaling molecule cyclic diguanylate (c-di-GMP) supress motility and activate exopolysaccharide (EPS) production in a variety of bacterial species. In many bacteria part of the effect of c-di-GMP is on gene expression, but the mechanism involved is not known for any species. We have identified the protein FleQ as a c-di-GMP-responsive transcriptional regulator in Pseudomonas aeruginosa. FleQ is known to activate expression of flagella biosynthesis genes. Here we show that it also represses transcription of genes including the pel operon involved in EPS biosynthesis, and that this repression is relieved by c-di-GMP. Our in vivo data indicate that FleQ represses pel transcription and that pel transcription is not repressed when intracellular c-di-GMP levels are high. FleN, a known antiactivator of FleQ also participates in control of pel expression. In in vitro experiments we found that FleQ binds to pel promoter DNA and that this binding is inhibited by c-di-GMP. FleQ binds radiolabeled c-di-GMP in vitro. FleQ does not have amino acid motifs that resemble previously defined c-di-GMP binding domains. Our results show that FleQ is a new type of c-di-GMP binding protein that controls the transcriptional regulation of EPS biosynthesis genes in P. aeruginosa. PMID:18485075

  9. Mechanisms of cGMP-dependent mesangial-cell relaxation: a role for myosin light-chain phosphatase activation.

    PubMed Central

    Torrecillas, G; Díez-Marqués, M L; García-Escribano, C; Bosch, R J; Rodríguez-Puyol, D; Rodríguez-Puyol, M

    2000-01-01

    Although the cGMP-dependent relaxation of contractile cells seems to depend on the ability of the cyclic nucleotide to interfere with intracellular calcium, this does not appear to be the only mechanism involved. The present experiments were designed to analyse alternative mechanisms, trying to test the hypothesis that cGMP could relax rat mesangial cells by activating myosin light-chain phosphatase (MLC-PP), with the subsequent dephosphorylation of myosin light chain (MLC). The effect of a cGMP analogue, dibutyryl cGMP (dbcGMP), on angiotensin II-(AII) and PMA-induced MLC phosphorylation (MLCP) was tested, in the presence of calyculin A (CA), an inhibitor of MLC-PP. MLCP was measured, after cell labelling with (32)P, by immunoprecipitation. dbcGMP prevented the increased MLCP induced by AII or PMA, and this inhibition was blocked by CA. dbcGMP also increased the MLC dephosphorylation observed in cells incubated with AII and in which MLC kinase and protein kinase C activities were blocked. The AII-elicited increased intracellular calcium concentration was only partially inhibited by dbcGMP. These results suggest that the cGMP-induced mesangial-cell relaxation could be due, at least partially, to the stimulation of MLC-PP. PMID:10657260

  10. Multiple roles of the messenger molecule cGMP in testicular function.

    PubMed

    Middendorff, R; Davidoff, M S; Behrends, S; Mewe, M; Miethens, A; Müller, D

    2000-01-01

    The messenger molecule cyclic guanosine monophosphate (cGMP) is produced by different isoforms of the enzyme guanylate cyclase (GC). Natriuretic peptides (ANP and CNP) bind to and activate particulate GCs, whereas NO and CO activate a soluble form of GC. The specific relevance of the cGMP system for reproductive functions has been recently demonstrated by the successful use of sildenafil (Viagra), an inhibitor of cGMP-specific phosphodiesterase type 5, for the treatment of erectile dysfunction. In the testis, cGMP signal transduction pathways are involved in a variety of local functions, based on autocrine or paracrine effects. In particular, cGMP has been suggested to influence motility in spermatozoa, development of testicular germ cells, relaxation of peritubular lamina propria cells, testosterone synthesis in Leydig cells and dilatation of testicular blood vessels. The physiological significance of cGMP accumulation in Scrtoli cells is not yet clear. Taken as a whole, the evidence suggests that cGMP-mediated processes might influence both the potentia coeundi within the penis and the potentia generandi at various levels within the testis.

  11. A calcium-permeable cGMP-activated cation conductance in hippocampal neurons

    NASA Technical Reports Server (NTRS)

    Leinders-Zufall, T.; Rosenboom, H.; Barnstable, C. J.; Shepherd, G. M.; Zufall, F.

    1995-01-01

    Whole-cell patch clamp recordings detected a previously unidentified cGMP-activated membrane conductance in cultured rat hippocampal neurons. This conductance is nonselectively permeable for cations and is completely but reversibly blocked by external Cd2+. The Ca2+ permeability of the hippocampal cGMP-activated conductance was examined in detail, indicating that the underlying ion channels display a high relative permeability for Ca2+. The results indicate that hippocampal neurons contain a cGMP-activated membrane conductance that has some properties similar to the cyclic nucleotide-gated channels previously shown in sensory receptor cells and retinal neurons. In hippocampal neurons this conductance similarly could mediate membrane depolarization and Ca2+ fluxes in response to intracellular cGMP elevation.

  12. Critical Role of Nitric Oxide-cGMP Cascade in the Formation of cAMP-Dependent Long-Term Memory

    ERIC Educational Resources Information Center

    Aonuma, Hitoshi; Mizunami, Makoto; Matsumoto, Yukihisa; Unoki, Sae

    2006-01-01

    Cyclic AMP pathway plays an essential role in formation of long-term memory (LTM). In some species, the nitric oxide (NO)-cyclic GMP pathway has been found to act in parallel and complementary to the cAMP pathway for LTM formation. Here we describe a new role of the NO-cGMP pathway, namely, stimulation of the cAMP pathway to induce LTM. We have…

  13. Critical Role of Nitric Oxide-cGMP Cascade in the Formation of cAMP-Dependent Long-Term Memory

    ERIC Educational Resources Information Center

    Aonuma, Hitoshi; Mizunami, Makoto; Matsumoto, Yukihisa; Unoki, Sae

    2006-01-01

    Cyclic AMP pathway plays an essential role in formation of long-term memory (LTM). In some species, the nitric oxide (NO)-cyclic GMP pathway has been found to act in parallel and complementary to the cAMP pathway for LTM formation. Here we describe a new role of the NO-cGMP pathway, namely, stimulation of the cAMP pathway to induce LTM. We have…

  14. Bixa orellana leaf extract suppresses histamine-induced endothelial hyperpermeability via the PLC-NO-cGMP signaling cascade.

    PubMed

    Yong, Yoke Keong; Chiong, Hoe Siong; Somchit, Muhd Nazrul; Ahmad, Zuraini

    2015-10-14

    Histamine is established as a potent inflammatory mediator and it is known to increased endothelial permeability by promoting gap formation between endothelial cells. Previous studies have shown that aqueous extract of Bixa orellana leaves (AEBO) exhibits antihistamine activity in vivo, yet the mechanism of its action on endothelial barrier function remains unclear. Therefore, the current study aimed to determine the protective effect of AEBO against histamine-induced hyperpermeability in vitro. The endothelial protective effect of AEBO was assess using an in vitro vascular permeability assay kit. Human umbilical vein endothelial cells (HUVEC) were used in the current study. HUVEC were pre-treated with AEBO for 12 h before histamine induction. Vascular permeability was evaluated by the amount of FITC-dextran leakage into the lower chamber. In order to elucidate the mechanism of action of AEBO, phospholipase C (PLC) activity, intracellular calcium level, nitric oxide (NO) concentration, cyclic guanosine monophosphate (cGMP) production and protein kinase C (PKC) activity were determined following histamine challenge. Histamine-induced increased HUVEC permeability was significantly attenuated by pretreatment with AEBO in a time- and concentration-dependent manner. Upregulation of PLC activity caused by histamine in HUVEC was suppressed by pretreatment with AEBO. Pretreatment with AEBO also blocked the production of intracellular calcium induced by histamine in HUVEC. In addition, AEBO suppressed the NO-cGMP signaling cascade when HUVEC were challenged with histamine. Moreover, PKC activity was significantly abolished by pretreatment with AEBO in HUVEC under histamine condition. In conclusion, the present data suggest that AEBO could suppress histamine-induced increased endothelial permeability and the activity may be closely related with the inhibition of the PLC-NO-cGMP signaling pathway and PKC activity.

  15. Synthetic multivalent ligands for cholera & cholera-like toxins: Protected cyclic neoglycopeptides.

    PubMed

    Kumar, Vajinder; Yadav, Narender; Kartha, K P Ravindranathan

    2016-08-05

    Synthesis of a set of novel glycopeptide analogues as potential cholera/cholera-like toxin inhibitors in their protected form is described. They include di-, tri-, tetra- and pentavalent scaffolds. The synthetic steps were achieved using a combination of solvent-free mechanochemical as well as the conventional solution-phase reactions. During the conventional DIC-HOBt-mediated peptide coupling followed for the preparation of certain glycopeptide analogues an interesting in situ Fmoc deprotection was observed which has been demonstrated to hold potential for synthesiszing glycopeptides/neoglycopeptides with extended polyamide chains.

  16. Correlation between airway epithelium-induced relaxation of rat aorta in the co-axial bioassay and cyclic nucleotide levels.

    PubMed

    Hay, D W; Muccitelli, R M; Page, C P; Spina, D

    1992-04-01

    1. In co-axial bioassays, in the presence of indomethacin, addition of histamine (100 microM) or methacholine (100 microM) to guinea-pig trachea produced an epithelium-dependent relaxation of precontracted rat aorta which was associated with an approximately 2 fold elevation in tissue levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP). Removal of the airway epithelium abolished the histamine-induced relaxation of rat aorta and the associated increase in intracellular cyclic GMP. 2. Epithelium-dependent relaxation was not associated with altered adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in rat aorta. Unstimulated intact or denuded guinea-pig trachea also did not affect the levels of cyclic AMP or cyclic GMP in rat aorta. 3. Methylene blue (10 microM) abolished the methacholine-induced, endothelium-derived relaxing factor (EDRF)-mediated rise in intracellular cyclic GMP in rat endothelium-intact aorta alone. In contrast, methylene blue (10 microM) did not affect the methacholine-induced epithelium-dependent rise in intracellular cyclic GMP in rat endothelium-denuded aorta in the co-axial bioassay. 4. Relaxation of the rat aorta without endothelium was associated with increased levels of cyclic GMP (but not cyclic AMP) in response to sodium nitroprusside (5 nM) and of cyclic AMP (but not cyclic GMP) in response to isoprenaline (1 microM). 5. These results provide evidence that the postulated epithelium-derived inhibitory factor (EpDIF) may produce relaxation of vascular tissue via elevation in cyclic GMP levels. Furthermore, some data suggest that EpDIF may act by stimulation of the particulate, rather than the soluble form of guanylate cyclase.

  17. Optogenetic Module for Dichromatic Control of c-di-GMP Signaling.

    PubMed

    Ryu, Min-Hyung; Fomicheva, Anastasia; Moskvin, Oleg V; Gomelsky, Mark

    2017-09-15

    Many aspects of bacterial physiology and behavior, including motility, surface attachment, and the cell cycle, are controlled by cyclic di-GMP (c-di-GMP)-dependent signaling pathways on the scale of seconds to minutes. Interrogation of such processes in real time requires tools for introducing rapid and reversible changes in intracellular c-di-GMP levels. Inducing the expression of genes encoding c-di-GMP-synthetic (diguanylate cyclases) and -degrading (c-di-GMP phosphodiesterase) enzymes by chemicals may not provide adequate temporal control. In contrast, light-controlled diguanylate cyclases and phosphodiesterases can be quickly activated and inactivated. A red/near-infrared-light-regulated diguanylate cyclase, BphS, was engineered previously, yet a complementary light-activated c-di-GMP phosphodiesterase has been lacking. In search of such a phosphodiesterase, we investigated two homologous proteins from Allochromatium vinosum and Magnetococcus marinus, designated BldP, which contain C-terminal EAL-BLUF modules, where EAL is a c-di-GMP phosphodiesterase domain and BLUF is a blue light sensory domain. Characterization of the BldP proteins in Escherichia coli and in vitro showed that they possess light-activated c-di-GMP phosphodiesterase activities. Interestingly, light activation in both enzymes was dependent on oxygen levels. The truncated EAL-BLUF fragment from A. vinosum BldP lacked phosphodiesterase activity, whereas a similar fragment from M. marinus BldP, designated EB1, possessed such activity that was highly (>30-fold) upregulated by light. Following light withdrawal, EB1 reverted to the inactive ground state with a half-life of ∼6 min. Therefore, the blue-light-activated phosphodiesterase EB1 can be used in combination with the red/near-infrared-light-regulated diguanylate cyclase BphS for the bidirectional regulation of c-di-GMP-dependent processes in E. coli as well as other bacterial and nonbacterial cells.IMPORTANCE Regulation of motility

  18. A post-translational, c-di-GMP-dependent mechanism regulating flagellar motility.

    PubMed

    Fang, Xin; Gomelsky, Mark

    2010-06-01

    Elevated levels of the second messenger cyclic dimeric GMP, c-di-GMP, promote transition of bacteria from single motile cells to surface-attached multicellular communities. Here we describe a post-translational mechanism by which c-di-GMP initiates this transition in enteric bacteria. High levels of c-di-GMP induce the counterclockwise bias in Escherichia coli flagellar rotation, which results in smooth swimming. Based on co-immunoprecipitation, two-hybrid and mutational analyses, the E. coli c-di-GMP receptor YcgR binds to the FliG subunit of the flagellum switch complex, and the YcgR-FliG interaction is strengthened by c-di-GMP. The central fragment of FliG binds to YcgR as well as to FliM, suggesting that YcgR-c-di-GMP biases flagellum rotation by altering FliG-FliM interactions. The c-di-GMP-induced smooth swimming promotes trapping of motile bacteria in semi-solid media and attachment of liquid-grown bacteria to solid surfaces, whereas c-di-GMP-dependent mechanisms not involving YcgR further facilitate surface attachment. The YcgR-FliG interaction is conserved in the enteric bacteria, and the N-terminal YcgR/PilZN domain of YcgR is required for this interaction. YcgR joins a growing list of proteins that regulate motility via the FliG subunit of the flagellum switch complex, which suggests that FliG is a common regulatory entryway that operates in parallel with the chemotaxis that utilizes the FliM-entryway.

  19. Cyclic nucleotide phosphodiesterase 3A1 protects the heart against ischemia-reperfusion injury.

    PubMed

    Oikawa, Masayoshi; Wu, Meiping; Lim, Soyeon; Knight, Walter E; Miller, Clint L; Cai, Yujun; Lu, Yan; Blaxall, Burns C; Takeishi, Yasuchika; Abe, Jun-ichi; Yan, Chen

    2013-11-01

    Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5±7.8% vs. 83.9±4.7%) as well as compensatory expansion of left ventricular diameter (4.19±0.19mm vs. 3.10±0.18mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (β-AR) response in TG mice. Importantly, the myocardial infarct size (4.0±1.8% vs. 24.6±3.8%) and apoptotic cell number (1.3±1.0% vs. 5.6±1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates β-AR signaling

  20. Cyclic nucleotide imaging and cardiovascular disease.

    PubMed

    Berisha, Filip; Nikolaev, Viacheslav O

    2017-02-16

    The universal second messengers cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) play central roles in cardiovascular function and disease. They act in discrete, functionally relevant subcellular microdomains which regulate, for example, calcium cycling and excitation-contraction coupling. Such localized cAMP and cGMP signals have been difficult to measure using conventional biochemical techniques. Recent years have witnessed the advent of live cell imaging techniques which allow visualization of these functionally relevant second messengers with unprecedented spatial and temporal resolution at cellular, subcellular and tissue levels. In this review, we discuss these new imaging techniques and give examples how they are used to visualize cAMP and cGMP in physiological and pathological settings to better understand cardiovascular function and disease. Two primary techniques include the use of Förster resonance energy transfer (FRET) based cyclic nucleotide biosensors and nanoscale scanning ion conductance microscopy (SICM). These methods can provide deep mechanistic insights into compartmentalized cAMP and cGMP signaling.

  1. On induction of cell differentiation by cyclic AMP pulses in Dictyostelium discoideum.

    PubMed

    Wurster, B

    1982-01-01

    Repeated pulses of cyclic AMP, applied at intervals of 5 min, efficiently induced differentiation in cells of agip 53, a morphogenetic mutant of Dictyostelium discoideum, strain Ax-2. In contrast, pulses applied at intervals of 2 min did not induce cell differentiation. To analyze this phenomenon the hydrolysis of cyclic AMP between the pulses as well as the effect of the pulses on the intracellular concentrations of cyclic GMP were investigated. Experiments performed in the presence of added cyclic AMP was not the reason of the inefficiency of the pulses applied with a 2-min rhythm. Cyclic AMP pulses applied at intervals of 2 min induced discrete increases of the cyclic GMP concentration. Limited time resolution at the level of cyclic GMP cannot account for the inefficiency of the 2-min pulses.

  2. Cyclic nucleotides of canine antral smooth muscle. Effects of acetylcholine, catecholamines and gastrin.

    PubMed

    Baur, S; Grant, B; Wooton, J

    1981-01-07

    1. The effects of acetylcholine, catecholamines and gastrin on the intracellular content of cyclic AMP and cyclic GMP in antral circular muscle have been determined. 2. Acetylcholine results in a significant but transient increase in intracellular cyclic GMP. 3. Isoproterenol and norepinephrine increase intracellular cyclic AMP. Based on half-maximal effective doses, isoproterenol is 2.7-times more effective than norepinephrine. The increase in intracellular cyclic AMP by both agents is inhibited by propranolol but not phentolamine, indicating that both agents act on the muscle cell by a beta-receptor-coupled mechanism. 4. Gastrin has no demonstrable effect on either cyclic AMP or cyclic GMP. This suggests that while gastrin and acetylcholine can produce a like myoelectric response in the muscle cell, the action of gastrin is mediated by a separate receptor, presumably on the muscle cell, and not by a release of acetylcholine.

  3. Effects of cadmium on the activities of photosystems of Chlorella pyrenoidosa and the protective role of cyclic electron flow.

    PubMed

    Wang, Shuzhi; Zhang, Daoyong; Pan, Xiangliang

    2013-09-01

    Cadmium (Cd) shows high toxicity to aquatic microalgae. Many studies showed that Cd inhibited activities of photosystem II (PSII) but the effects of heavy metals on photosystem I (PSI) and cyclic electron flow (CEF) were still controversial and unclear. The effects of CdCl2 on the activities of PSI, PSII and CEF in Chlorella pyrenoidosa was measured simultaneously in the present study. In presence of 200μM of Cd, ultrastructure of some cells was strongly modified. Cd exposure led to decrease of the activities of photosynthetic oxygen evolution and respiration. PSII was more sensitive to Cd treatment than PSI. Cd treatment showed significant inhibition on the photochemical quantum yield and electron transport rate of PSII. Cd increased the quantum yield of non-light-induced non-photochemical fluorescence quenching, indicating the damage of PSII. The activity of PSI showed tolerance to Cd treatment with concentration less than 100μM in the experiment. Linear electron flow (LEF) made significant contribution to the photochemical quantum yield of PSI of the untreated cells, but decreased with increasing Cd concentration. The contribution of CEF to the yield of PSI increased with increasing Cd concentration. The activation of CEF after exposure to Cd played an essential role for the protection of PSI.

  4. The role of cyclic nucleotide signaling pathways in cancer: targets for prevention and treatment.

    PubMed

    Fajardo, Alexandra M; Piazza, Gary A; Tinsley, Heather N

    2014-02-26

    For more than four decades, the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) have been recognized as important signaling molecules within cells. Under normal physiological conditions, cyclic nucleotides regulate a myriad of biological processes such as cell growth and adhesion, energy homeostasis, neuronal signaling, and muscle relaxation. In addition, altered cyclic nucleotide signaling has been observed in a number of pathophysiological conditions, including cancer. While the distinct molecular alterations responsible for these effects vary depending on the specific cancer type, several studies have demonstrated that activation of cyclic nucleotide signaling through one of three mechanisms-induction of cyclic nucleotide synthesis, inhibition of cyclic nucleotide degradation, or activation of cyclic nucleotide receptors-is sufficient to inhibit proliferation and activate apoptosis in many types of cancer cells. These findings suggest that targeting cyclic nucleotide signaling can provide a strategy for the discovery of novel agents for the prevention and/or treatment of selected cancers.

  5. The Role of Cyclic Nucleotide Signaling Pathways in Cancer: Targets for Prevention and Treatment

    PubMed Central

    Fajardo, Alexandra M.; Piazza, Gary A.; Tinsley, Heather N.

    2014-01-01

    For more than four decades, the cyclic nucleotides cyclic AMP (cAMP) and cyclic GMP (cGMP) have been recognized as important signaling molecules within cells. Under normal physiological conditions, cyclic nucleotides regulate a myriad of biological processes such as cell growth and adhesion, energy homeostasis, neuronal signaling, and muscle relaxation. In addition, altered cyclic nucleotide signaling has been observed in a number of pathophysiological conditions, including cancer. While the distinct molecular alterations responsible for these effects vary depending on the specific cancer type, several studies have demonstrated that activation of cyclic nucleotide signaling through one of three mechanisms—induction of cyclic nucleotide synthesis, inhibition of cyclic nucleotide degradation, or activation of cyclic nucleotide receptors—is sufficient to inhibit proliferation and activate apoptosis in many types of cancer cells. These findings suggest that targeting cyclic nucleotide signaling can provide a strategy for the discovery of novel agents for the prevention and/or treatment of selected cancers. PMID:24577242

  6. The GTP binding protein-dependent activation and deactivation of cGMP phosphodiesterase in rod photoreceptors

    SciTech Connect

    Yamazaki, Akio.

    1989-01-01

    Cyclic GMP (cGMP) has a crucial role in visual transduction. Recent electrophysiological studies clearly indicate the existence of cGMP-activated conductance in photoreceptor plasma membranes. In darkness, Na{sup +}, Ca{sup ++}, and Mg{sup ++} enter rod outer segments (ROS) through cGMP-activated channels while light closes channels by lowering cGMP concentrations through activation of cGMP phosphodiesterase (PDE). Many excellent reviews reference the mechanism of PDE activation in photoreceptors. However, recent progress in understanding the mechanisms regulating cGMP hydrolysis has raised an important question in the PDE-regulation: how does the three-dimensional movement of a subunit of transducin (retinal G protein) relate to the PDE activation Associated with that question, the mechanism of PDE regulation appears to vary at different stages of evolution, for example, frog and bovine photoreceptors. This review examines recent progress of the cGMP hydrolysis mechanism by focusing on the subunit interactions between transducin and PDE. 36 refs., 2 figs.

  7. Prediction and verification of creep behavior in metallic materials and components, for the space shuttle thermal protection system. Volume 1, phase 1: Cyclic materials creep predictions

    NASA Technical Reports Server (NTRS)

    Davis, J. W.; Cramer, B. A.

    1974-01-01

    Cyclic creep response was investigated and design methods applicable to thermal protection system structures were developed. The steady-state (constant temperature and load) and cyclic creep response characteristics of four alloys were studied. Steady-state creep data were gathered through a literature survey to establish reference data bases. These data bases were used to develop empirical equations describing creep as a function of time, temperature, and stress and as a basis of comparison for test data. Steady-state creep tests and tensile cyclic tests were conducted. The following factors were investigated: material thickness and rolling direction; material cyclic creep response under varying loads and temperatures; constant stress and temperature cycles representing flight conditions; changing stresses present in a creeping beam as a result of stress redistribution; and complex stress and temperature profiles representative of space shuttle orbiter trajectories. A computer program was written, applying creep hardening theories and empirical equations for creep, to aid in analysis of test data. Results are considered applicable to a variety of structures which are cyclicly exposed to creep producing thermal environments.

  8. Elevated level of the second messenger c-di-GMP in Comamonas testosteroni enhances biofilm formation and biofilm-based biodegradation of 3-chloroaniline.

    PubMed

    Wu, Yichao; Ding, Yuanzhao; Cohen, Yehuda; Cao, Bin

    2015-02-01

    The bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger that determines bacterial lifestyle between the planktonic and biofilm modes of life. Although the role of c-di-GMP signaling in biofilm development and dispersal has been extensively studied, how c-di-GMP signaling influences environmental bioprocess activities such as biodegradation remains unexplored. To elucidate the impacts of elevating c-di-GMP level on environmental bioprocesses, we constructed a Comamonas testosteroni strain constitutively expressing a c-di-GMP synthase YedQ from Escherichia coli and examined its capability in biofilm formation and biodegradation of 3-chloroaniline (3-CA). The high c-di-GMP strain exhibited an increased binding to Congo red dye, a decreased motility, and an enhanced biofilm formation capability. In planktonic cultures, the strain with an elevated c-di-GMP concentration and the wild type could degrade 3-CA comparably well. However, under batch growth conditions with a high surface to volume ratio, an elevated c-di-GMP concentration in C. testosteroni significantly increased the contribution of biofilms in 3-CA biodegradation. In continuous submerged biofilm reactors, C. testosteroni with an elevated c-di-GMP level exhibited an enhanced 3-CA biodegradation and a decreased cell detachment rate. Taken together, this study provides a novel strategy to enhance biofilm-based biodegradation of toxic xenobiotic compounds through manipulating bacterial c-di-GMP signaling.

  9. Evaluation of a Salmonella Strain Lacking the Secondary Messenger C-di-GMP and RpoS as a Live Oral Vaccine

    PubMed Central

    García, Begoña; Gil, Carmen; García-Ona, Enrique; Burgui, Saioa; Casares, Noelia; Hervás-Stubbs, Sandra; Lasarte, Juan José; Lasa, Iñigo

    2016-01-01

    Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. C-di-GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine. PMID:27537839

  10. CPT-cGMP Is A New Ligand of Epithelial Sodium Channels

    PubMed Central

    Ji, Hong-Long; Nie, Hong-Guang; Chang, Yongchang; Lian, Qizhou; Liu, Shan-Lu

    2016-01-01

    Epithelial sodium channels (ENaC) are localized at the apical membrane of the epithelium, and are responsible for salt and fluid reabsorption. Renal ENaC takes up salt, thereby controlling salt content in serum. Loss-of-function ENaC mutations lead to low blood pressure due to salt-wasting, while gain-of-function mutations cause impaired sodium excretion and subsequent hypertension as well as hypokalemia. ENaC activity is regulated by intracellular and extracellular signals, including hormones, neurotransmitters, protein kinases, and small compounds. Cyclic nucleotides are broadly involved in stimulating protein kinase A and protein kinase G signaling pathways, and, surprisingly, also appear to have a role in regulating ENaC. Increasing evidence suggests that the cGMP analog, CPT-cGMP, activates αβγ-ENaC activity reversibly through an extracellular pathway in a dose-dependent manner. Furthermore, the parachlorophenylthio moiety and ribose 2'-hydroxy group of CPT-cGMP are essential for facilitating the opening of ENaC channels by this compound. Serving as an extracellular ligand, CPT-cGMP eliminates sodium self-inhibition, which is a novel mechanism for stimulating salt reabsorption in parallel to the traditional NO/cGMP/PKG signal pathway. In conclusion, ENaC may be a druggable target for CPT-cGMP, leading to treatments for kidney malfunctions in salt reabsorption. PMID:27019621

  11. Compartmentalization of extracellular cGMP determines absorptive or secretory responses in the rat jejunum

    PubMed Central

    Jin, Xiao-Hong; Siragy, Helmy M.; Guerrant, Richard L.; Carey, Robert M.

    1999-01-01

    We examined potential mechanisms by which angiotensin subtype-2 (AT2) receptor stimulation induces net fluid absorption and serosal guanosine cyclic 3′,5′-monophosphate (cGMP) formation in the rat jejunum. l-arginine (l-ARG) given intravenously or interstitially enhanced net fluid absorption and cGMP formation, which were completely blocked by the nitric oxide (NO) synthase inhibitor, N-nitro-l-arginine methylester (l-NAME), but not by the specific AT2 receptor antagonist, PD-123319 (PD). Dietary sodium restriction also increased jejunal interstitial fluid cGMP and fluid absorption. Both could be blocked by PD or l-NAME, suggesting that the effects of sodium restriction occur via ANG II at the AT2 receptor. l-ARG–stimulated fluid absorption was blocked by the soluble guanylyl cyclase inhibitor 1-H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (ODQ). Cyclic GMP–specific phosphodiesterase in the interstitial space decreased extracellular cGMP content and prevented the absorptive effects of l-ARG. Angiotensin II (ANG II) caused an increase in net Na+ and Cl– ion absorption and 22Na+ unidirectional efflux (absorption) from the jejunal loop. In contrast, intraluminal heat-stable enterotoxin of Escherichia coli (STa) increased loop cGMP and fluid secretion that were not blocked by either l-NAME or ODQ. These findings suggest that ANG II acts at the serosal side via AT2 receptors to stimulate cGMP production via soluble guanylyl cyclase activation and absorption through the generation of NO, but that mucosal STa activation of particulate guanylyl cyclase causes secretion independently of NO, thus demonstrating the opposite effects of cGMP in the mucosal and serosal compartments of the jejunum. PMID:9916128

  12. Activation of cGMP-Dependent Protein Kinase Stimulates Cardiac ATP-Sensitive Potassium Channels via a ROS/Calmodulin/CaMKII Signaling Cascade

    PubMed Central

    Chai, Yongping; Zhang, Dai-Min; Lin, Yu-Fung

    2011-01-01

    Background Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (KATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue. Methods and Findings Single-channel recordings of cardiac KATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type KATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H2O2 scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H2O2 also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of KATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions. Conclusion The present study provides novel evidence that PKG exerts dual regulation of cardiac KATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H2O2 in

  13. Cyclic nucleotide phosphodiesterases (PDEs): coincidence detectors acting to spatially and temporally integrate cyclic nucleotide and non-cyclic nucleotide signals.

    PubMed

    Maurice, Donald H; Wilson, Lindsay S; Rampersad, Sarah N; Hubert, Fabien; Truong, Tammy; Kaczmarek, Milosz; Brzezinska, Paulina; Freitag, Silja I; Umana, M Bibiana; Wudwud, Alie

    2014-04-01

    The cyclic nucleotide second messengers cAMP and cGMP each affect virtually all cellular processes. Although these hydrophilic small molecules readily diffuse throughout cells, it is remarkable that their ability to activate their multiple intracellular effectors is spatially and temporally selective. Studies have identified a critical role for compartmentation of the enzymes which hydrolyse and metabolically inactivate these second messengers, the PDEs (cyclic nucleotide phosphodiesterases), in this specificity. In the present article, we describe several examples from our work in which compartmentation of selected cAMP- or cGMP-hydrolysing PDEs co-ordinate selective activation of cyclic nucleotide effectors, and, as a result, selectively affect cellular functions. It is our belief that therapeutic strategies aimed at targeting PDEs within these compartments will allow greater selectivity than those directed at inhibiting these enzymes throughout the cells.

  14. Amyloid-β Peptide Is Needed for cGMP-Induced Long-Term Potentiation and Memory.

    PubMed

    Palmeri, Agostino; Ricciarelli, Roberta; Gulisano, Walter; Rivera, Daniela; Rebosio, Claudia; Calcagno, Elisa; Tropea, Maria Rosaria; Conti, Silvia; Das, Utpal; Roy, Subhojit; Pronzato, Maria Adelaide; Arancio, Ottavio; Fedele, Ernesto; Puzzo, Daniela

    2017-07-19

    High levels of amyloid-β peptide (Aβ) have been related to Alzheimer's disease pathogenesis. However, in the healthy brain, low physiologically relevant concentrations of Aβ are necessary for long-term potentiation (LTP) and memory. Because cGMP plays a key role in these processes, here we investigated whether the cyclic nucleotide cGMP influences Aβ levels and function during LTP and memory. We demonstrate that the increase of cGMP levels by the phosphodiesterase-5 inhibitors sildenafil and vardenafil induces a parallel release of Aβ due to a change in the approximation of amyloid precursor protein (APP) and the β-site APP cleaving enzyme 1. Moreover, electrophysiological and behavioral studies performed on animals of both sexes showed that blocking Aβ function, by using anti-murine Aβ antibodies or APP knock-out mice, prevents the cGMP-dependent enhancement of LTP and memory. Our data suggest that cGMP positively regulates Aβ levels in the healthy brain which, in turn, boosts synaptic plasticity and memory.SIGNIFICANCE STATEMENT Amyloid-β (Aβ) is a key pathogenetic factor in Alzheimer's disease. However, low concentrations of endogenous Aβ, mimicking levels of the peptide in the healthy brain, enhance hippocampal long-term potentiation (LTP) and memory. Because the second messenger cGMP exerts a central role in LTP mechanisms, here we studied whether cGMP affects Aβ levels and function during LTP. We show that cGMP enhances Aβ production by increasing the APP/BACE-1 convergence in endolysosomal compartments. Moreover, the cGMP-induced enhancement of LTP and memory was disrupted by blockade of Aβ, suggesting that the physiological effect of the cyclic nucleotide on LTP and memory is dependent upon Aβ. Copyright © 2017 the authors 0270-6474/17/376926-12$15.00/0.

  15. cGMP Accumulation Causes Photoreceptor Degeneration in CNG Channel Deficiency: Evidence of cGMP Cytotoxicity Independently of Enhanced CNG Channel Function

    PubMed Central

    Xu, Jianhua; Morris, Lynsie; Thapa, Arjun; Ma, Hongwei; Michalakis, Stylianos; Biel, Martin; Baehr, Wolfgang; Peshenko, Igor V.; Dizhoor, Alexander M.

    2013-01-01

    Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca2+ influx in rod and cone photoreceptors. cGMP, the native ligand of the photoreceptor CNG channels, has been associated with cytotoxicity when its levels rise above normal due to defects in photoreceptor phosphodiesterase (PDE6) or regulation of retinal guanylyl cyclase (retGC). We found a massive accumulation of cGMP in CNGA3-deficient retina and investigated whether cGMP accumulation plays a role in cone degeneration in CNG channel deficiency. The time course study showed that the retinal cGMP level in Cnga3−/−;Nrl−/− mice with CNGA3 deficiency on a cone-dominant background was sharply increased at postnatal day 8 (P8), peaked around P10–P15, remained high through P30–P60, and returned to near control level at P90. This elevation pattern correlated with photoreceptor apoptotic death, which peaked around P15–P20. In Cnga3−/−;Gucy2e−/− mice lacking retGC1, cone density and expression levels of cone-specific proteins were significantly increased compared with Cnga3−/−, consistent with a role of cGMP accumulation as the major contributor to cone death caused by CNG channel deficiency. The activity and expression levels of cGMP-dependent protein kinase G (PKG) were significantly increased in Cnga3−/−;Nrl−/− retina compared with Nrl−/−, suggesting an involvement of PKG regulation in cell death. Our results indicate that cGMP accumulation in photoreceptors can itself exert cytotoxic effect in cones, independently of CNG channel activity and Ca2+ influx. PMID:24027293

  16. Supramolecular polymer formation by cyclic dinucleotides and intercalators affects dinucleotide enzymatic processing.

    PubMed

    Nakayama, Shizuka; Zhou, Jie; Zheng, Yue; Szmacinski, Henryk; Sintim, Herman O

    2016-03-01

    Cyclic dinucleotides form supramolecular aggregates with intercalators, and this property could be utilized in nanotechnology and medicine. Atomic force microscopy and electrophoretic mobility shift assays were used to show that cyclic diguanylic acid (c-di-GMP) forms G-wires in the presence of intercalators. The average fluorescence lifetime of thiazole orange, when bound to c-di-GMP was greater than when bound to DNA G-quadruplexes or dsDNA. The stability of c-di-GMP supramolecular polymers is dependent on both the nature of the cation present and the intercalator. C-di-GMP or cyclic diadenylic acid/intercalator complexes are more resistant to cleavage by YybT, a phosphodiesterase, than the uncomplexed nucleotides. Cleavage of bacterial cyclic dinucleotides could be slowed down via complexation with small molecules and that this could be utilized for diverse applications in nanotechnology and medicine.

  17. Supramolecular polymer formation by cyclic dinucleotides and intercalators affects dinucleotide enzymatic processing

    PubMed Central

    Nakayama, Shizuka; Zhou, Jie; Zheng, Yue; Szmacinski, Henryk; Sintim, Herman O

    2016-01-01

    Background: Cyclic dinucleotides form supramolecular aggregates with intercalators, and this property could be utilized in nanotechnology and medicine. Methods & results: Atomic force microscopy and electrophoretic mobility shift assays were used to show that cyclic diguanylic acid (c-di-GMP) forms G-wires in the presence of intercalators. The average fluorescence lifetime of thiazole orange, when bound to c-di-GMP was greater than when bound to DNA G-quadruplexes or dsDNA. The stability of c-di-GMP supramolecular polymers is dependent on both the nature of the cation present and the intercalator. C-di-GMP or cyclic diadenylic acid/intercalator complexes are more resistant to cleavage by YybT, a phosphodiesterase, than the uncomplexed nucleotides. Conclusion: Cleavage of bacterial cyclic dinucleotides could be slowed down via complexation with small molecules and that this could be utilized for diverse applications in nanotechnology and medicine. PMID:28031943

  18. Hindlimb-suspension and spaceflight both alter cGMP levels in rat choroid plexus.

    PubMed

    Carcenac, C; Herbute, S; Masseguin, C; Mani-Ponset, L; Maurel, D; Briggs, R; Guell, A; Gabrion, J B

    1999-10-01

    Effects of actual and simulated weightlessness on choroidal guanylate cyclase activity were evaluated by assaying the production of cyclic guanosine monophosphate (cGMP), a second messenger involved in mechanisms regulating the secretion of cerebrospinal fluid (CSF) in choroid plexus. Cyclic cGMP was measured, using radio-immunoassay, in choroidal extracts of hindlimb-suspended rats (HLS rats), adapted to an anti-orthostatic restraint for 30 min., or for 3, 9 or 14 days and after a 17-day spaceflight (Life and Microgravity SpaceLab experiment; LMS). Basal cGMP levels were slightly but significantly decreased in the first 30 min. of the HLS experiment, whereas they were significantly increased in rats adapted to longer anti-orthostatic restraints. LMS flight rats demonstrated a similar increase in the choroidal cGMP baseline. After natriuretic peptide stimulation, i.e. using ANP (atrial natriuretic peptide) or BNP (brain natriuretic peptide), choroidal cGMP contents were typically increased (by 1.5-2 times; p<0.05) in control rats (LMS and HLS experiments), but not significantly elevated in suspended rats, except for those adapted to HLS for 14 days. In these animals the ANP-dependent cGMP production was significantly increased (by about 3 times; p<0.005). The ANP- or BNP-dependent responses were similarly abolished in LMS flight rats, which were dissected 4-6 hours after return to Earth's gravity. The role of corticosteroids was also investigated during the LMS experiment. Results on choroidal functions revealed a lack of significant change of cGMP levels between adrenalectomized and sham-operated rats. For the first time, it is reported that both basal and ANP- stimulated cGMP levels are dramatically changed over the first 14 days of suspension, i.e. with experiments known to simulate some effects of weightlessness. Basal choroidal cGMP levels are also increased after 17 days in space, suggesting that space adaptation also impacts choroidal guanylate cyclase

  19. In vivo cGMP levels in frog photoreceptor cells as a function of light exposure.

    PubMed

    Barbehenn, E K; Klotz, K L; Noelker, D M; Nelson, R; Chader, G J; Passonneau, J V

    1986-11-01

    By employing a combination of highly sensitive radioimmunoassays and histochemical techniques, an in vivo time course of cGMP levels has been determined in the outer segment, photoreceptor cell and outer plexiform layers of frog retina. Frogs (Rana pipiens) were dark-adapted overnight and either frozen rapidly (approximately 3 sec) in liquid nitrogen or exposed to periods of light varying between 0.1 sec and 2 hr before freezing. Frozen retinal sections were cut, freeze-dried, and samples of individual layers dissected out and analysed for cGMP. In the outer plexiform layer, there was a 42% drop in cGMP concentration after 2 sec of light (250 ft candles) followed by a 34% rise after 2 min; a steep concentration gradient formed around the layer after the 2 min exposure. In both the outer-segment layer and photoreceptor-cell layer (which includes outer segments, inner segments and outer nuclear layers), cGMP levels declined from a dark value of 56 mumol kg-1 (dry) to 9 mumol kg-1 (dry) as a result of increasing exposure to several types of light source: levels appear to be primarily a function of total ft candle min. Cyclic GMP concentrations at the longest exposures (2 min with a fiber optic light source or 2 hr with fluorescent room light) reached identical minimum levels. In the outer segments, a 15% decrease in cGMP was observed after 0.1 sec of light exposure. Although the freezing time is too long to be able to say whether the 15% decrease in cGMP at the 0.1 sec exposure is involved in transduction, the low identical levels reached gradually after longer exposures appear to indicate that a light-induced biochemical adjustment in cGMP metabolism occurs over a relatively long time period separate from the msec time course of the transduction process.

  20. Alterations in adipocytokines and cGMP homeostasis in morbid obesity patients reverse after bariatric surgery.

    PubMed

    Felipo, Vicente; Urios, Amparo; García-Torres, Maria L; El Mlili, Nisrin; del Olmo, Juan A; Civera, Miguel; Ortega, Joaquin; Ferrandez, Antonio; Martínez-Valls, Jose; Cassinello, Norberto; Montoliu, Carmina

    2013-02-01

    Obesity-associated nonalcoholic fatty liver disease (NAFLD), covering from simple steatosis to nonalcoholic steatohepatitis (NASH), is a common cause of chronic liver disease. Aberrant production of adipocytokines seems to play a main role in most obesity-associated disorders. Changes in adipocytokines in obesity could be mediated by alterations in cyclic GMP (cGMP) homeostasis. The aims of this work were: (1) to study the role of altered cGMP homeostasis in altered adipocytokines in morbid obesity, (2) to assess whether these alterations are different in simple steatosis or NASH, and (3) to assess whether these changes reverse in obese patients after bariatric surgery. In 47 patients with morbid obesity and 45 control subjects, the levels in blood of adipocytokines, cGMP, nitric oxide (NO) metabolites, and atrial natriuretic peptide (ANP) were studied. Whether weight loss after a bariatric surgery reverses the changes in these parameters was evaluated. NO metabolites and leptin increase (and adiponectin decreases) similarly in patients with steatosis or NASH, suggesting that these changes are due to morbid obesity and not to liver disease. Inflammation and cGMP homeostasis are affected both by morbid obesity and by liver disease. The increases in interleukin 6 (IL-6), interleukin 18 (IL-18), plasma cGMP, ANP, and the decrease in cGMP in lymphocytes are stronger in patients with NASH than with steatosis. All these changes reverse completely after bariatric surgery and weight loss, except IL-18. Altered cGMP homeostasis seems to contribute more than inflammation to changes in leptin and adiponectin in morbid obesity. Copyright © 2012 The Obesity Society.

  1. Biophysical techniques for detection of cAMP and cGMP in living cells.

    PubMed

    Sprenger, Julia U; Nikolaev, Viacheslav O

    2013-04-12

    Cyclic nucleotides cAMP and cGMP are ubiquitous second messengers which regulate myriads of functions in virtually all eukaryotic cells. Their intracellular effects are often mediated via discrete subcellular signaling microdomains. In this review, we will discuss state-of-the-art techniques to measure cAMP and cGMP in biological samples with a particular focus on live cell imaging approaches, which allow their detection with high temporal and spatial resolution in living cells and tissues. Finally, we will describe how these techniques can be applied to the analysis of second messenger dynamics in subcellular signaling microdomains.

  2. Deposition and cyclic oxidation behavior of a protective (Mo,W)(Si,Ge) [sub 2] coating on Nb-base alloys

    SciTech Connect

    Mueller, A.; Wang, G. . Dept. of Materials Science and Engineering); Rapp, R.A. )

    1992-05-01

    A multicomponent diffusion coating has been developed to protect Nb-base alloys from high-temperature environmental attach. A solid solution of molybdenum and tungsten disilicide (Mo, W)Si[sub 2], constituted the primary coating layer which supported a slow-growing protective silica scale in service. Germanium additions were made during the coating process to improve the cyclic oxidation resistance by increasing the thermal expansion coefficient of the vitreous silica film formed and to avoid pesting by decreasing the viscosity of the protective film. In this paper, the development of the halide-activated pack cementation coating process to produce this (Mo,W)(Si,Ge)[sub 2] coating on Nb-base alloys is described. The results of cyclic oxidation for coupons coated under different conditions in air at 1370[degrees]C are presented. Many coupons have successfully passed 200 1 h cyclic oxidation tests at 1370[degrees]C with weight-gain values in the range of 1.2 to 1.6 mg/cm[sup 2].

  3. Isolated regulatory domains of cGMP-dependent protein kinase Ialpha and Ibeta retain dimerization and native cGMP-binding properties and undergo isoform-specific conformational changes.

    PubMed

    Richie-Jannetta, Robyn; Busch, Jennifer L; Higgins, Kristin A; Corbin, Jackie D; Francis, Sharron H

    2006-03-17

    Molecular mechanisms that provide for cGMP activation of cGMP-dependent protein kinase (PKG) are unknown. PKGs are dimeric; each monomer contains a regulatory (R) and catalytic (C) domain. In this study, isolated recombinant R domains of PKGIalpha-(Delta349-670) and PKGIbeta-(Delta364-685) containing the dimerization and autoinhibitory subdomains and two allosteric cGMP-binding sites were expressed in Sf9 cells. Both R domains were dimers with elongated conformations (Stokes radii of 44 and 51 A, respectively, and frictional coefficients of 1.6 and 1.8, respectively). Exchange dissociation kinetics and K(D) values for cGMP were similar for each holoenzyme and its isolated R domain, indicating that under these conditions the C domain does not appreciably alter cGMP-binding functions of the R domain. As determined by gel filtration chromatography, cGMP binding caused elongation of the PKGIalpha-isolated R domain and contraction of the PKGIbeta-isolated R domain. Cyclic GMP-bound forms of the isoforms have similar physical dimensions that may reflect a common conformation of active isoforms. Elongation of the PKGIbeta holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGIalpha R domain may partially account for the elongation of wild type PKGIalpha associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation.

  4. Advances in targeting cyclic nucleotide phosphodiesterases

    PubMed Central

    Maurice, Donald H.; Ke, Hengming; Ahmad, Faiyaz; Wang, Yousheng; Chung, Jay; Manganiello, Vincent C.

    2014-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants. PMID:24687066

  5. cGMP regulates hydrogen peroxide accumulation in calcium-dependent salt resistance pathway in Arabidopsis thaliana roots.

    PubMed

    Li, Jisheng; Wang, Xiaomin; Zhang, Yanli; Jia, Honglei; Bi, Yurong

    2011-10-01

    3',5'-cyclic guanosine monophosphate (cGMP) is an important second messenger in plants. In the present study, roles of cGMP in salt resistance in Arabidopsis roots were investigated. Arabidopsis roots were sensitive to 100 mM NaCl treatment, displaying a great increase in electrolyte leakage and Na(+)/K(+) ratio and a decrease in gene expression of the plasma membrane (PM) H(+)-ATPase. However, application of exogenous 8Br-cGMP (an analog of cGMP), H(2)O(2) or CaCl(2) alleviated the NaCl-induced injury by maintaining a lower Na(+)/K(+) ratio and increasing the PM H(+)-ATPase gene expression. In addition, the inhibition of root elongation and seed germination under salt stress was removed by 8Br-cGMP. Further study indicated that 8Br-cGMP-induced higher NADPH levels for PM NADPH oxidase to generate H(2)O(2) by regulating glucose-6-phosphate dehydrogenase (G6PDH) activity. The effect of 8Br-cGMP and H(2)O(2) on ionic homeostasis was abolished when Ca(2+) was eliminated by glycol-bis-(2-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, a Ca(2+) chelator) in Arabidopsis roots under salt stress. Taken together, cGMP could regulate H(2)O(2) accumulation in salt stress, and Ca(2+) was necessary in the cGMP-mediated signaling pathway. H(2)O(2), as the downstream component of cGMP signaling pathway, stimulated PM H(+)-ATPase gene expression. Thus, ion homeostasis was modulated for salt tolerance.

  6. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    SciTech Connect

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Ohman, Dennis E.; Howell, P. Lynne

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.

  7. Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa

    DOE PAGES

    Whitney, John C.; Robinson, Howard; Whitfield, Gregory B.; ...

    2015-05-15

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZmore » domain fold with a dimerization mode not previously observed for this family of proteins. Moreover, calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. Our results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa.« less

  8. Dimeric c-di-GMP Is Required for Post-translational Regulation of Alginate Production in Pseudomonas aeruginosa*

    PubMed Central

    Whitney, John C.; Whitfield, Gregory B.; Marmont, Lindsey S.; Yip, Patrick; Neculai, A. Mirela; Lobsanov, Yuri D.; Robinson, Howard; Ohman, Dennis E.; Howell, P. Lynne

    2015-01-01

    Pseudomonas aeruginosa is an opportunistic human pathogen that secretes the exopolysaccharide alginate during infection of the respiratory tract of individuals afflicted with cystic fibrosis and chronic obstructive pulmonary disease. Among the proteins required for alginate production, Alg44 has been identified as an inner membrane protein whose bis-(3′,5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) binding activity post-translationally regulates alginate secretion. In this study, we report the 1.8 Å crystal structure of the cytoplasmic region of Alg44 in complex with dimeric self-intercalated c-di-GMP and characterize its dinucleotide-binding site using mutational analysis. The structure shows that the c-di-GMP binding region of Alg44 adopts a PilZ domain fold with a dimerization mode not previously observed for this family of proteins. Calorimetric binding analysis of residues in the c-di-GMP binding site demonstrate that mutation of Arg-17 and Arg-95 alters the binding stoichiometry between c-di-GMP and Alg44 from 2:1 to 1:1. Introduction of these mutant alleles on the P. aeruginosa chromosome show that the residues required for binding of dimeric c-di-GMP in vitro are also required for efficient alginate production in vivo. These results suggest that the dimeric form of c-di-GMP represents the biologically active signaling molecule needed for the secretion of an important virulence factor produced by P. aeruginosa. PMID:25817996

  9. Cyclic electron flow may provide some protection against PSII photoinhibition in rice (Oryza sativa L.) leaves under heat stress.

    PubMed

    Essemine, Jemaa; Xiao, Yi; Qu, Mingnan; Mi, Hualing; Zhu, Xin-Guang

    2017-04-01

    Previously we have shown that a quick down-regulation in PSI activity compares to that of PSII following short-term heat stress for two rice groups including C4023 and Q4149, studied herein. These accessions were identified to have different natural capacities in driving cyclic electron flow (CEF) around PSI; i.e., low CEF (lcef) and high CEF (hcef) for C4023 and Q4149, respectively. The aim of this study was to investigate whether these two lines have different mechanisms of protecting photosystem II from photodamage under heat stress. We observed a stepwise alteration in the shape of Chl a fluorescence induction (OJIP) with increasing temperature treatment. The effect of 44°C treatment on the damping in Chl a fluorescence was more pronounced in C4023 than in Q4149. Likewise, we noted a disruption in the I-step, a decline in the Fv due to a strong damping in the Fm, and a slight increase in the F0. Normalized data demonstrated that the I-step seems more susceptible to 44°C in C4023 than in Q4149. We also measured the redox states of plastocyanin (PC) and P700 by monitoring the transmission changes at 820nm (I820), and observed a disturbance in the oxidation/reduction kinetics of PC and P700. The decline in the amplitude of their oxidation was shown to be about 29% and 13% for C4023 and Q4149, respectively. The electropotential component (Δφ) of ms-DLE appeared more sensitive to temperature stress than the chemical component (ΔpH), and the impact of heat was more evident and drastic in C4023 than in Q4149. Under heat stress, we noticed a concomitant decline in the primary photochemistry of PSII as well as in both the membrane energization process and the lumen protonation for both accessions, and it is evident that heat affects these parameters more in C4023 than in Q4149. All these data suggest that higher CET can confer higher photoprotection to PSII in rice lines, which can be a desirable trait during rice breeding, especially in the context of a "warming

  10. Genetic Modulation of c-di-GMP Turnover Affects Multiple Virulence Traits and Bacterial Virulence in Rice Pathogen Dickeya zeae

    PubMed Central

    Chen, Yufan; Lv, Mingfa; Liao, Lisheng; Gu, Yanfang; Liang, Zhibin; Shi, Zurong; Liu, Shiyin; Zhou, Jianuan; Zhang, Lianhui

    2016-01-01

    The frequent outbreaks of rice foot rot disease caused by Dickeya zeae have become a significant concern in rice planting regions and countries, but the regulatory mechanisms that govern the virulence of this important pathogen remain vague. Given that the second messenger cyclic di-GMP (c-di-GMP) is associated with modulation of various virulence-related traits in various microorganisms, here we set to investigate the role of the genes encoding c-di-GMP metabolism in the regulation of the bacterial physiology and virulence by construction all in-frame deletion mutants targeting the annotated c-di-GMP turnover genes in D. zeae strain EC1. Phenotype analyses identified individual mutants showing altered production of exoenzymes and phytotoxins, biofilm formation and bacterial motilities. The results provide useful clues and a valuable toolkit for further characterization and dissection of the regulatory complex that modulates the pathogenesis and persistence of this important bacterial pathogen. PMID:27855163

  11. The prokaryote messenger c-di-GMP triggers stalk cell differentiation in Dictyostelium.

    PubMed

    Chen, Zhi-hui; Schaap, Pauline

    2012-08-30

    Cyclic di-(3′:5′)-guanosine monophosphate (c-di-GMP) is a major prokaryote signalling intermediate that is synthesized by diguanylate cyclases and triggers sessility and biofilm formation. We detected the first eukaryote diguanylate cyclases in all major groups of Dictyostelia. On food depletion, Dictyostelium discoideum amoebas collect into aggregates, which first transform into migrating slugs and then into sessile fruiting structures. These structures consist of a spherical spore mass that is supported by a column of stalk cells and a basal disk. A polyketide, DIF-1, which induces stalk-like cells in vitro, was isolated earlier. However, its role in vivo proved recently to be restricted to basal disk formation. Here we show that the Dictyostelium diguanylate cyclase, DgcA, produces c-di-GMP as the morphogen responsible for stalk cell differentiation. Dictyostelium discoideum DgcA synthesized c-di-GMP in a GTP-dependent manner and was expressed at the slug tip, which is the site of stalk cell differentiation. Disruption of the DgcA gene blocked the transition from slug migration to fructification and the expression of stalk genes. Fructification and stalk formation were restored by exposing DgcA-null slugs to wild-type secretion products or to c-di-GMP. Moreover, c-di-GMP, but not cyclic di-(3′:5′)-adenosine monophosphate, induced stalk gene expression in dilute cell monolayers. Apart from identifying the long-elusive stalk-inducing morphogen, our work also identifies a role for c-di-GMP in eukaryotes.

  12. The role of a parasite-specific allosteric site in the distinctive activation behavior of Eimeria tenella cGMP-dependent protein kinase.

    PubMed

    Salowe, Scott P; Wiltsie, Judyann; Liberator, Paul A; Donald, Robert G K

    2002-04-02

    A cGMP-dependent protein kinase (PKG) was recently identified as an anticoccidial target for the apicomplexan parasite Eimeria tenella [Gurnett, A., Liberator, P. A., Dulski, P., Salowe, S., Donald, R. G. K., Anderson, J., Wiltsie, J., Diaz, C., Harris, G., Chang, B., Darkin-Rattray, S. J., Nare, B., Crumley, T., Blum, P., Misura, A., Tamas, T., Sardana, M., Yuan, J., Biftu, T., and Schmatz, D. (2002) J. Biol. Chem. (in press)]. Unlike the PKGs of higher organisms that have two cGMP binding sites in their regulatory domain, the PKG from Eimeria tenella (Et-PKG) contains three putative cGMP binding sites and has distinctive activation properties, including a very large stimulation by cGMP ( approximately 1000-fold) with significant cooperativity (Hill coefficient of 1.7). During our investigation of Et-PKG activation, we found that 8-substituted cGMP analogues are weak partial activators. For example, 8-NBD-cGMP provides a maximal stimulation of activity of only 20-fold with little evident cooperativity, although cGMP can synergize with the analogue to provide full activation. The results suggest that partial activation is a consequence of restricted binding of 8-NBD-cGMP to a subset of cGMP sites in the enzyme. Site-directed mutagenesis of conserved arginine and glutamate residues in the parasite-specific third cGMP site confirms that this site is an important functional participant in the allosteric regulation of the kinase and that it exhibits very high selectivity against 8-NBD-cGMP. Since the results are consistent with full activation of Et-PKG requiring cyclic nucleotide binding in all three allosteric sites, one role for the additional cGMP site may be to establish a stricter regulatory mechanism for the kinase activity than is present in the PKGs of higher organisms containing only two allosteric sites.

  13. PDE2 at the crossway between cAMP and cGMP signalling in the heart.

    PubMed

    Weber, Silvio; Zeller, Miriam; Guan, Kaomei; Wunder, Frank; Wagner, Michael; El-Armouche, Ali

    2017-10-01

    The cyclic nucleotides cAMP and cGMP are central second messengers in cardiac cells and critical regulators of cardiac physiology as well as pathophysiology. Consequently, subcellular compartmentalization allows for spatiotemporal control of cAMP/cGMP metabolism and subsequent regulation of their respective effector kinases PKA or PKG is most important for cardiac function in health and disease. While acute cAMP-mediated signalling is a mandatory prerequisite for the physiological fight-or-flight response, sustained activation of this pathway may lead to the progression of heart failure. In contrast, acute as well as sustained cGMP-mediated signalling can foster beneficial features, e.g. anti-hypertrophic and vasodilatory effects. These two signalling pathways seem to be intuitively counteracting and there is increasing evidence for a functionally relevant crosstalk between cAMP and cGMP signalling pathways on the level of cyclic nucleotide hydrolysing phosphodiesterases (PDEs). Among this diverse group of enzymes, PDE2 may fulfill a unique integrator role. Equipped with dual substrate specificity for cAMP as well as for cGMP, it is the only cAMP hydrolysing PDE, which is allosterically activated by cGMP. Recent studies have revealed strongly remodelled cAMP/cGMP microdomains and subcellular concentration profiles in different cardiac pathologies, leading to a putatively enhanced involvement of PDE2 in cAMP/cGMP breakdown and crosstalk compared to the other cardiac PDEs. This review sums up the current knowledge about molecular properties and regulation of PDE2 and explains the complex signalling network encompassing PDE2 in order to better understand the functional role of PDE2 in distinct cell types in cardiac health and disease. Moreover, this review gives an outlook in which way PDE2 may serve as a therapeutic target to treat cardiac disease. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Formation and dimerization of the phosphodiesterase active site of the Pseudomonas aeruginosa MorA, a bi-functional c-di-GMP regulator.

    PubMed

    Phippen, Curtis William; Mikolajek, Halina; Schlaefli, Henry George; Keevil, Charles William; Webb, Jeremy Stephen; Tews, Ivo

    2014-12-20

    Diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively synthesise and hydrolyse the secondary messenger cyclic dimeric GMP (c-di-GMP), and both activities are often found in a single protein. Intracellular c-di-GMP levels in turn regulate bacterial motility, virulence and biofilm formation. We report the first structure of a tandem DGC-PDE fragment, in which the catalytic domains are shown to be active. Two phosphodiesterase states are distinguished by active site formation. The structures, in the presence or absence of c-di-GMP, suggest that dimerisation and binding pocket formation are linked, with dimerisation being required for catalytic activity. An understanding of PDE activation is important, as biofilm dispersal via c-di-GMP hydrolysis has therapeutic effects on chronic infections. Copyright © 2014. Published by Elsevier B.V.

  15. Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent.

    PubMed

    Carvalho-Costa, P G; Branco, L G S; Leite-Panissi, C R A

    2014-12-01

    Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3',5'-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress.

  16. Temperature affects c-di-GMP signaling and biofilm formation in Vibrio cholerae

    PubMed Central

    Townsley, Loni; Yildiz, Fitnat H.

    2016-01-01

    SUMMARY Biofilm formation is crucial to the environmental survival and transmission of Vibrio cholerae, the facultative human pathogen responsible for the disease cholera. During its infectious cycle V. cholerae experiences fluctuations in temperature within the aquatic environment and during the transition between human host and aquatic reservoirs. In this study, we report that biofilm formation is induced at low temperatures through increased levels of the signaling molecule, cyclic diguanylate (c-di-GMP). Strains harboring in-frame deletions of all V. cholerae genes that are predicted to encode diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) were screened for their involvement in low-temperature-induced biofilm formation and Vibrio polysaccharide (VPS) gene expression. Of the 52 mutants tested, deletions of six DGCs and three PDEs were found to affect these phenotypes at low temperatures. Unlike wild type, a strain lacking all six DGCs did not exhibit a low-temperature-dependent increase in c-di-GMP, indicating that these DGCs are required for temperature modulation of c-di-GMP levels. We also show that temperature modulates c-di-GMP levels in a similar fashion in the Gram-negative pathogen Pseudomonas aeruginosa but not in the Gram-positive pathogen Listeria monocytogenes. This study uncovers the role of temperature in environmental regulation of biofilm formation and c-di-GMP signaling. PMID:25684220

  17. Engineering a novel c-di-GMP-binding protein for biofilm dispersal.

    PubMed

    Ma, Qun; Yang, Zhonghua; Pu, Mingming; Peti, Wolfgang; Wood, Thomas K

    2011-03-01

    Bacteria prefer to grow attached to themselves or an interface, and it is important for an array of applications to make biofilms disperse. Here we report simultaneously the discovery and protein engineering of BdcA (formerly YjgI) for biofilm dispersal using the universal signal 3,5-cyclic diguanylic acid (c-di-GMP). The bdcA deletion reduced biofilm dispersal, and production of BdcA increased biofilm dispersal to wild-type level. Since BdcA increases motility and extracellular DNA production while decreasing exopolysaccharide, cell length and aggregation, we reasoned that BdcA decreases the concentration of c-di-GMP, the intracellular messenger that controls cell motility through flagellar rotation and biofilm formation through synthesis of curli and cellulose. Consistently, c-di-GMP levels increase upon deleting bdcA, and purified BdcA binds c-di-GMP but does not act as a phosphodiesterase. Additionally, BdcR (formerly YjgJ) is a negative regulator of bdcA. To increase biofilm dispersal, we used protein engineering to evolve BdcA for greater c-di-GMP binding and found that the single amino acid change E50Q causes nearly complete removal of biofilms via dispersal without affecting initial biofilm formation. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.

  18. Tetrameric c-di-GMP Mediates Effective Transcription Factor Dimerization to Control Streptomyces Development

    PubMed Central

    Tschowri, Natalia; Schumacher, Maria A.; Schlimpert, Susan; Chinnam, Naga babu; Findlay, Kim C.; Brennan, Richard G.; Buttner, Mark J.

    2014-01-01

    Summary The cyclic dinucleotide c-di-GMP is a signaling molecule with diverse functions in cellular physiology. Here, we report that c-di-GMP can assemble into a tetramer that mediates the effective dimerization of a transcription factor, BldD, which controls the progression of multicellular differentiation in sporulating actinomycete bacteria. BldD represses expression of sporulation genes during vegetative growth in a manner that depends on c-di-GMP-mediated dimerization. Structural and biochemical analyses show that tetrameric c-di-GMP links two subunits of BldD through their C-terminal domains, which are otherwise separated by ∼10 Å and thus cannot effect dimerization directly. Binding of the c-di-GMP tetramer by BldD is selective and requires a bipartite RXD-X8-RXXD signature. The findings indicate a unique mechanism of protein dimerization and the ability of nucleotide signaling molecules to assume alternative oligomeric states to effect different functions. PMID:25171413

  19. Bacterial c-di-GMP affects hematopoietic stem/progenitors and their niches through STING.

    PubMed

    Kobayashi, Hiroshi; Kobayashi, Chiharu I; Nakamura-Ishizu, Ayako; Karigane, Daiki; Haeno, Hiroshi; Yamamoto, Kimiyo N; Sato, Taku; Ohteki, Toshiaki; Hayakawa, Yoshihiro; Barber, Glen N; Kurokawa, Mineo; Suda, Toshio; Takubo, Keiyo

    2015-04-07

    Upon systemic bacterial infection, hematopoietic stem and progenitor cells (HSPCs) migrate to the periphery in order to supply a sufficient number of immune cells. Although pathogen-associated molecular patterns reportedly mediate HSPC activation, how HSPCs detect pathogen invasion in vivo remains elusive. Bacteria use the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) for a variety of activities. Here, we report that c-di-GMP comprehensively regulated both HSPCs and their niche cells through an innate immune sensor, STING, thereby inducing entry into the cell cycle and mobilization of HSPCs while decreasing the number and repopulation capacity of long-term hematopoietic stem cells. Furthermore, we show that type I interferon acted as a downstream target of c-di-GMP to inhibit HSPC expansion in the spleen, while transforming growth factor-β was required for c-di-GMP-dependent splenic HSPC expansion. Our results define machinery underlying the dynamic regulation of HSPCs and their niches during bacterial infection through c-di-GMP/STING signaling.

  20. Acute stress-induced antinociception is cGMP-dependent but heme oxygenase-independent

    PubMed Central

    Carvalho-Costa, P.G.; Branco, L.G.S.; Leite-Panissi, C.R.A.

    2014-01-01

    Endogenous carbon monoxide (CO), which is produced by the enzyme heme oxygenase (HO), participates as a neuromodulator in physiological processes such as thermoregulation and nociception by stimulating the formation of 3′,5′-cyclic guanosine monophosphate (cGMP). In particular, the acute physical restraint-induced fever of rats can be blocked by inhibiting the enzyme HO. A previous study reported that the HO-CO-cGMP pathway plays a key phasic antinociceptive role in modulating noninflammatory acute pain. Thus, this study evaluated the involvement of the HO-CO-cGMP pathway in antinociception induced by acute stress in male Wistar rats (250-300 g; n=8/group) using the analgesia index (AI) in the tail flick test. The results showed that antinociception induced by acute stress was not dependent on the HO-CO-cGMP pathway, as neither treatment with the HO inhibitor ZnDBPG nor heme-lysinate altered the AI. However, antinociception was dependent on cGMP activity because pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ) blocked the increase in the AI induced by acute stress. PMID:25387672

  1. Temperature affects c-di-GMP signalling and biofilm formation in Vibrio cholerae.

    PubMed

    Townsley, Loni; Yildiz, Fitnat H

    2015-11-01

    Biofilm formation is crucial to the environmental survival and transmission of Vibrio cholerae, the facultative human pathogen responsible for the disease cholera. During its infectious cycle, V. cholerae experiences fluctuations in temperature within the aquatic environment and during the transition between human host and aquatic reservoirs. In this study, we report that biofilm formation is induced at low temperatures through increased levels of the signalling molecule, cyclic diguanylate (c-di-GMP). Strains harbouring in frame deletions of all V. cholerae genes that are predicted to encode diguanylate cyclases (DGCs) or phosphodiesterases (PDEs) were screened for their involvement in low-temperature-induced biofilm formation and Vibrio polysaccharide gene expression. Of the 52 mutants tested, deletions of six DGCs and three PDEs were found to affect these phenotypes at low temperatures. Unlike wild type, a strain lacking all six DGCs did not exhibit a low-temperature-dependent increase in c-di-GMP, indicating that these DGCs are required for temperature modulation of c-di-GMP levels. We also show that temperature modulates c-di-GMP levels in a similar fashion in the Gram-negative pathogen Pseudomonas aeruginosa but not in the Gram-positive pathogen Listeria monocytogenes. This study uncovers the role of temperature in environmental regulation of biofilm formation and c-di-GMP signalling. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  2. The c-di-GMP phosphodiesterase BifA regulates biofilm development in Pseudomonas putida.

    PubMed

    Jiménez-Fernández, Alicia; López-Sánchez, Aroa; Calero, Patricia; Govantes, Fernando

    2015-02-01

    We previously showed the isolation of biofilmpersistent Pseudomonas putida mutants that fail to undergo biofilm dispersal upon entry in stationary phase. Two such mutants were found to bear insertions in PP0914, encoding a GGDEF/EAL domain protein with high similarity to Pseudomon asaeruginosa BifA. Here we show the phenotypic characterization of a ΔbifA mutant in P. putida KT2442.This mutant displayed increased biofilm and pellicle formation, cell aggregation in liquid medium and decreased starvation-induced biofilm dispersal relative to the wild type. Unlike its P. aeruginosa counterpart, P. putida BifA did not affect swarming motility. The hyperadherent phenotype of the ΔbifA mutant correlates with a general increase in cyclic diguanylate (c-di-GMP) levels, Congo Red-binding exopolyaccharide production and transcription of the adhesin-encoding lapA gene. Integrity of the EAL motif and a modified GGDEF motif (altered to GGDQF)were crucial for BifA activity, and c-di-GMP depletion by overexpression of a heterologous c-di-GMP phosphodiesterase in the ΔbifA mutant restored wild-type biofilm dispersal and lapA expression.Our results indicate that BifA is a phosphodiesterase involved in the regulation of the c-di-GMP pool and required for the generation of the low c-di-GMP signal that triggers starvation-induced biofilm dispersal.

  3. White - cGMP Interaction Promotes Fast Locomotor Recovery from Anoxia in Adult Drosophila

    PubMed Central

    2017-01-01

    Increasing evidence indicates that the white (w) gene in Drosophila possesses extra-retinal functions in addition to its classical role in eye pigmentation. We have previously shown that w+ promotes fast and consistent locomotor recovery from anoxia, but how w+ modulates locomotor recovery is largely unknown. Here we show that in the absence of w+, several PDE mutants, especially cyclic guanosine monophosphate (cGMP)-specific PDE mutants, display wildtype-like fast locomotor recovery from anoxia, and that during the night time, locomotor recovery was light-sensitive in white-eyed mutant w1118, and light-insensitive in PDE mutants under w1118 background. Data indicate the involvement of cGMP in the modulation of recovery timing and presumably, light-evoked cGMP fluctuation is associated with light sensitivity of locomotor recovery. This was further supported by the observations that w-RNAi-induced delay of locomotor recovery was completely eliminated by upregulation of cGMP through multiple approaches, including PDE mutation, simultaneous overexpression of an atypical soluble guanylyl cyclase Gyc88E, or sildenafil feeding. Lastly, prolonged sildenafil feeding promoted fast locomotor recovery from anoxia in w1118. Taken together, these data suggest that a White-cGMP interaction modulates the timing of locomotor recovery from anoxia. PMID:28060942

  4. Activation of the cGMP/nitric oxide signal transduction system by nicotine in the retina.

    PubMed

    Blute, Todd A; Strang, Christianne; Keyser, Kent T; Eldred, William D

    2003-01-01

    Acetylcholine is one of the primary excitatory neurotransmitters/neuromodulators in the retina, but little is known about the downstream signaling pathways it can activate. The present study immunocytochemically examines the potential sources of acetylcholine and the location of the nicotinic cholinergic receptors in the turtle retina. It also examines how activation of these receptors can influence the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signal-transduction pathways. Photoreceptors, amacrine cells, and potentially ganglion cells contain choline acetyltransferase-like immunoreactivity (LI). Nicotinic acetylcholine receptors are immunocytochemically localized on photoreceptors, horizontal, bipolar, and ganglion cells. Nitric oxide imaging indicates that stimulation with nicotine increases NO production primarily in photoreceptors, horizontal, Muller, bipolar, and ganglion cells. In turn, very select populations of amacrine cells respond to this NO with increased levels of cGMP-LI. Selective inhibitors reveal that nitric oxide synthase is involved in most, but not all, of these increases in cGMP-LI. These results show that acetylcholine can activate the NO/cGMP signal-transduction pathways in both the inner and outer retina. This indicates that both of the major excitatory retinal transmitters, glutamate and acetylcholine, can stimulate NO production that increases levels of cGMP-LI in overlapping populations of retinal cells.

  5. cGMP modulates responses to queen mandibular pheromone in worker honey bees

    PubMed Central

    Fussnecker, Brendon L.; McKenzie, Alexander M.; Grozinger, Christina M.

    2013-01-01

    Responses to social cues, such as pheromones, can be modified by genotype, physiology, or environmental context. Honey bee queens produce a pheromone (queen mandibular pheromone; QMP) which regulates aspects of worker bee behavior and physiology. Forager bees are less responsive to QMP than young bees engaged in brood care, suggesting that physiological changes associated with behavioral maturation modulate response to this pheromone. Since 3′, 5′-cyclic guanosine monophosphate (cGMP) is a major regulator of behavioral maturation in workers, we examined its role in modulating worker responses to QMP. Treatment with a cGMP analog resulted in significant reductions in both behavioral and physiological responses to QMP in young caged workers. Treatment significantly reduced attraction to QMP and inhibited the QMP-mediated increase in vitellogenin RNA levels in the fat bodies of worker bees. Genome-wide analysis of brain gene expression patterns demonstrated that cGMP has a larger effect on expression levels than QMP, and that QMP has specific effects in the presence of cGMP, suggesting that some responses to QMP may be dependent on an individual bees' physiological state. Our data suggest that cGMP-mediated processes play a role in modulating responses to QMP in honey bees at the behavioral, physiological, and molecular levels. PMID:21626397

  6. Diacylglycerol analogs inhibit the rod cGMP-gated channel by a phosphorylation-independent mechanism.

    PubMed Central

    Gordon, S. E.; Downing-Park, J.; Tam, B.; Zimmerman, A. L.

    1995-01-01

    The electrical response to light in retinal rods is mediated by cyclic nucleotide-gated, nonselective cation channels in the outer segment plasma membrane. Although cGMP appears to be the primary light-regulated second messenger, cellular levels of other substances, including Ca2+ and phosphatidylinositol-4,5-bisphosphate, are also sensitive to the level of illumination. We now show that diacylglycerol (DAG) analogs reversibly suppress the cGMP-activated conductance in excised patches from frog rod outer segments. This suppression did not require nucleoside triphosphates, indicating that a phosphorylation reaction was not involved. DAG was more effective at low than at high [cGMP]: with 50 microM 8-Br-cGMP, the DAG analog 1,2-dioctanoyl-sn-glycerol (1,2-DiC8) reduced the current with an IC50 of approximately 22 microM (Hill coefficient, 0.8), whereas with 1.2 microM 8-Br-cGMP, only approximately 1 microM 1,2-DiC8 was required to halve the current. DAG reduced the apparent affinity of the channels for cGMP: 4 microM 1,2-DiC8 produced a threefold increase in the K1/2 for channel activation by 8-Br-cGMP, as well as a threefold reduction in the maximum current, without changing the apparent stoichiometry or cooperativity of cGMP binding. Inhibition by 1,2-DiC8 was not relieved by supersaturating concentrations of 8-Br-cGMP, suggesting that DAG did not act by competitive inhibition of cGMP binding. Furthermore, DAG did not seem to significantly reduce single-channel conductance. A DAG analog similar to 1,2-DiC8--1,3-dioctanoyl-sn-glycerol (1,3-DiC8)--suppressed the current with the same potency as 1,2-DiC8, whereas an ethylene glycol of identical chain length (DiC8-EG) was much less effective. Our results suggest that DAG allosterically interferes with channel opening, and raise the question of whether DAG is involved in visual transduction. PMID:8527654

  7. BolA Is Required for the Accurate Regulation of c-di-GMP, a Central Player in Biofilm Formation

    PubMed Central

    Dressaire, Clémentine; Barahona, Susana; Galego, Lisete; Kaever, Volkhard; Jenal, Urs

    2017-01-01

    ABSTRACT The bacterial second messenger cyclic dimeric GMP (c-di-GMP) is a nearly ubiquitous intracellular signaling molecule involved in the transition from the motile to the sessile/biofilm state in bacteria. C-di-GMP regulates various cellular processes, including biofilm formation, motility, and virulence. BolA is a transcription factor that promotes survival in different stresses and is also involved in biofilm formation. Both BolA and c-di-GMP participate in the regulation of motility mechanisms leading to similar phenotypes. Here, we establish the importance of the balance between these two factors for accurate regulation of the transition between the planktonic and sessile lifestyles. This balance is achieved by negative-feedback regulation of BolA and c-di-GMP. BolA not only contributes directly to the motility of bacteria but also regulates the expression of diguanylate cyclases and phosphodiesterases. This expression modulation influences the synthesis and degradation of c-di-GMP, while this signaling metabolite has a negative influence in bolA mRNA transcription. Finally, we present evidence of the dominant role of BolA in biofilm, showing that, even in the presence of elevated c-di-GMP levels, biofilm formation is reduced in the absence of BolA. C-di-GMP is one of the most important bacterial second messengers involved in several cellular processes, including virulence, cell cycle regulation, biofilm formation, and flagellar synthesis. In this study, we unravelled a direct connection between the bolA morphogene and the c-di-GMP signaling molecule. We show the important cross-talk that occurs between these two molecular regulators during the transition between the motile/planktonic and adhesive/sessile lifestyles in Escherichia coli. This work provides important clues that can be helpful in the development of new strategies, and the results can be applied to other organisms with relevance for human health. PMID:28928205

  8. A Minimal Threshold of c-di-GMP Is Essential for Fruiting Body Formation and Sporulation in Myxococcus xanthus

    PubMed Central

    Skotnicka, Dorota; Trampari, Eleftheria; Liang, Jennifer; Kaever, Volkhard; Malone, Jacob G.; Singer, Mitchell; Søgaard-Andersen, Lotte

    2016-01-01

    Generally, the second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-di-GMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be—at least partially—functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus. PMID:27214040

  9. Multifunctional cyclic D,L-α-peptide architectures stimulate non-insulin dependent glucose uptake in skeletal muscle cells and protect them against oxidative stress.

    PubMed

    Shapira, Renana; Rudnick, Safra; Daniel, Bareket; Viskind, Olga; Aisha, Vered; Richman, Michal; Ayasolla, Kamesh R; Perelman, Alex; Chill, Jordan H; Gruzman, Arie; Rahimipour, Shai

    2013-09-12

    Oxidative stress directly correlates with the early onset of vascular complications and the progression of peripheral insulin resistance in diabetes. Accordingly, exogenous antioxidants augment insulin sensitivity in type 2 diabetic patients and ameliorate its clinical signs. Herein, we explored the unique structural and functional properties of the abiotic cyclic D,L-α-peptide architecture as a new scaffold for developing multifunctional agents to catalytically decompose ROS and stimulate glucose uptake. We showed that His-rich cyclic D,L-α-peptide 1 is very stable under high H2O2 concentrations, effectively self-assembles to peptide nanotubes, and increases the uptake of glucose by increasing the translocation of GLUT1 and GLUT4. It also penetrates cells and protects them against oxidative stress induced under hyperglycemic conditions at a much lower concentration than α-lipoic acid (ALA). In vivo studies are now required to probe the mode of action and efficacy of these abiotic cyclic D,L-α-peptides as a novel class of antihyperglycemic compounds.

  10. Cyclic diguanylate regulation of Bacillus cereus group biofilm formation.

    PubMed

    Fagerlund, Annette; Smith, Veronika; Røhr, Åsmund K; Lindbäck, Toril; Parmer, Marthe P; Andersson, K Kristoffer; Reubsaet, Leon; Økstad, Ole Andreas

    2016-08-01

    Biofilm formation can be considered a bacterial virulence mechanism. In a range of Gram-negatives, increased levels of the second messenger cyclic diguanylate (c-di-GMP) promotes biofilm formation and reduces motility. Other bacterial processes known to be regulated by c-di-GMP include cell division, differentiation and virulence. Among Gram-positive bacteria, where the function of c-di-GMP signalling is less well characterized, c-di-GMP was reported to regulate swarming motility in Bacillus subtilis while having very limited or no effect on biofilm formation. In contrast, we show that in the Bacillus cereus group c-di-GMP signalling is linked to biofilm formation, and to several other phenotypes important to the lifestyle of these bacteria. The Bacillus thuringiensis 407 genome encodes eleven predicted proteins containing domains (GGDEF/EAL) related to c-di-GMP synthesis or breakdown, ten of which are conserved through the majority of clades of the B. cereus group, including Bacillus anthracis. Several of the genes were shown to affect biofilm formation, motility, enterotoxin synthesis and/or sporulation. Among these, cdgF appeared to encode a master diguanylate cyclase essential for biofilm formation in an oxygenated environment. Only two cdg genes (cdgA, cdgJ) had orthologs in B. subtilis, highlighting differences in c-di-GMP signalling between B. subtilis and B. cereus group bacteria.

  11. May Cyclic Nucleotides Be a Source for Abiotic RNA Synthesis?

    NASA Astrophysics Data System (ADS)

    Costanzo, Giovanna; Pino, Samanta; Botta, Giorgia; Saladino, Raffaele; di Mauro, Ernesto

    2011-12-01

    Nucleic bases are obtained by heating formamide in the presence of various catalysts. Formamide chemistry also allows the formation of acyclonucleosides and the phosphorylation of nucleosides in every possible position, also affording 2',3' and 3',5' cyclic forms. We have reported that 3',5' cyclic GMP and 3',5' cyclic AMP polymerize in abiotic conditions yielding short oligonucleotides. The characterization of this reaction is being pursued, several of its parameters have been determined and experimental caveats are reported. The yield of non-enzymatic polymerization of cyclic purine nucleotides is very low. Polymerization is strongly enhanced by the presence of base-complementary RNA sequences.

  12. Thermodynamics of Activation Gating in Olfactory-Type Cyclic Nucleotide-Gated (CNGA2) Channels

    PubMed Central

    Nache, Vasilica; Kusch, Jana; Biskup, Christoph; Schulz, Eckhard; Zimmer, Thomas; Hagen, Volker; Benndorf, Klaus

    2008-01-01

    Olfactory-type cyclic nucleotide-gated (CNG) ion channels open by the binding of cyclic nucleotides to a binding domain in the C-terminus. Employing the Eyring rate theory, we performed a thermodynamic analysis of the activation gating in homotetrameric CNGA2 channels. Lowering the temperature shifted the concentration-response relationship to lower concentrations, resulting in a decrease of both the enthalpy ΔH and entropy ΔS upon channel opening, suggesting that the order of an open CNGA2 channel plus its environment is higher than that of the closed channel. Activation time courses induced by cGMP concentration jumps were used to study thermodynamics of the transition state. The activation enthalpies ΔH‡ were positive at all cGMP concentrations. In contrast, the activation entropy ΔS‡ was positive at low cGMP concentrations and became then negative at increasing cGMP concentrations. The enthalpic and entropic parts of the activation energies approximately balance each other at all cGMP concentrations, leaving the free enthalpy of activation in the range between 19 and 21 kcal/mol. We conclude that channel activation proceeds through different pathways at different cGMP concentrations. Compared to the unliganded channel, low cGMP concentrations generate a transitional state of lower order whereas high cGMP concentrations generate a transitional state of higher order. PMID:18567637

  13. A cyclic nucleotide sensitive promoter reporter system suitable for bacteria and plant cells.

    PubMed

    Wheeler, Janet I; Freihat, Lubna; Irving, Helen R

    2013-11-09

    Cyclic AMP (cAMP) and cyclic GMP (cGMP) have roles in relaying external signals and modifying gene expression within cells in all phyla. Currently there are no reporter systems suitable for bacteria and plant cells that measure alterations in downstream gene expression following changes in intracellular levels of cyclic nucleotides. As the plant protein OLIGOPEPTIDE TRANSPORTER X (OPTX) is upregulated by cGMP, we fused the OPTX promoter to a luciferase reporter gene (OPTX:LUC) to develop a plant cell reporter of cGMP-induced gene expression. We prepared a second construct augmented with three mammalian cGMP response elements (OPTXcGMPRE:LUC) and a third construct containing five gibberellic acid response elements (OPTXGARE:LUC). All three constructs were tested in bacteria and isolated plant protoplasts. Membrane permeable cGMP enhanced luciferase activity of OPTX:LUC and OPTXGARE:LUC in protoplasts. Treatment with the plant hormone gibberellic acid which acts via cGMP also generated downstream luciferase activity. However, membrane permeable cAMP induced similar responses to cGMP in protoplasts. Significantly increased luciferase activity occurred in bacteria transformed with either OPTXcGMPRE:LUC or OPTXGARE:LUC in response to membrane permeable cAMP and cGMP. Bacteria co-transformed with OPTXcGMPRE:LUC or OPTXGARE:LUC and the soluble cytoplasmic domain of phytosulfokine receptor1 (PSKR1; a novel guanylate cyclase) had enhanced luciferase activity following induction of PSKR1 expression. We have developed promoter reporter systems based on the plant OPTX promoter that can be employed in bacteria and isolated plant cells. We have shown that it can be used in bacteria to screen recombinant proteins for guanylate cyclase activity as increases in intracellular cGMP levels result in altered gene transcription and luciferase activity.

  14. Development of ion-plated aluminide diffusion coatings for thermal cyclic oxidation and hot corrosion protection of a nickel-based superalloy and a stainless steel

    NASA Astrophysics Data System (ADS)

    Elsawy, Abdel Raouf

    This project was carried out at the University of Toronto and Cametoid Ltd of Whitby, Ontario. Ohno continuous casting; a novel net shape casting technique, was used to generate, Al-Y, Al-Ce, Al-La, and Al-Si-Y, in form of 1.6 to 1.7 mm diameter alloy wires. These alloy wires exhibited suitable properties for use as feed materials to an Ion Vapor Deposition facility. The deposition parameters were optimized to provide coatings with a compact and cohesive columnar structure with reduced porosity and diffusion barriers that were essential to ensure the success of the diffusion process in the subsequent stage. Solid-state diffusion heat treatment processes were developed in order to form the stable aluminide phases, AlNi and FeAl, on IN738 and S310 substrates, respectively. Experiments simulating the coating service conditions and environments encountered during the prospective aerospace and fuel cell applications were conducted to evaluate the performance of each aluminide coating developed during this study. Thermal cyclic oxidation and molten sulfate corrosion studies were performed on coated IN738 pins at 1050°C and 900°C, respectively, simulating the service environment of turbine engine blades and other hot section components. Molten carbonate corrosion behavior was investigated for coated S310 coupons that were immersed in, or covered with a thin film of molten carbonate, at 650°C, in air plus 30%CO2, to simulate the operating conditions of the cathode-side separator plates of molten carbonate fuel cells. The behavior of the reactive elements, yttrium, cerium, lanthanum, and silicon in enhancing the adhesion of the protective aluminum oxide scale was determined by weight variation experiments, structural examination and compositional analysis. The influence of the base material elements, nickel, chromium, and iron, on the formation of protective oxides was investigated. All coatings were found to provide significant improvement for thermal cyclic oxidation

  15. C-di-GMP regulates Pseudomonas aeruginosa stress response to tellurite during both planktonic and biofilm modes of growth

    PubMed Central

    Chua, Song Lin; Sivakumar, Krishnakumar; Rybtke, Morten; Yuan, Mingjun; Andersen, Jens Bo; Nielsen, Thomas E.; Givskov, Michael; Tolker-Nielsen, Tim; Cao, Bin; Kjelleberg, Staffan; Yang, Liang

    2015-01-01

    Stress response plays an important role on microbial adaptation under hostile environmental conditions. It is generally unclear how the signaling transduction pathway mediates a stress response in planktonic and biofilm modes of microbial communities simultaneously. Here, we showed that metalloid tellurite (TeO32–) exposure induced the intracellular content of the secondary messenger cyclic di-GMP (c-di-GMP) of Pseudomonas aeruginosa. Two diguanylate cyclases (DGCs), SadC and SiaD, were responsible for the increased intracellular content of c-di-GMP. Enhanced c-di-GMP levels by TeO32– further increased P. aeruginosa biofilm formation and resistance to TeO32–. P. aeruginosa ΔsadCΔsiaD and PAO1/plac-yhjH mutants with low intracellular c-di-GMP content were more sensitive to TeO32– exposure and had low relative fitness compared to the wild-type PAO1 planktonic and biofilm cultures exposed to TeO32–. Our study provided evidence that c-di-GMP level can play an important role in mediating stress response in microbial communities during both planktonic and biofilm modes of growth. PMID:25992876

  16. Activation of phosphodiesterase 5 and inhibition of guanylate cyclase by cGMP-dependent protein kinase in smooth muscle.

    PubMed Central

    Murthy, K S

    2001-01-01

    The regulation of cGMP-specific phosphodiesterase (PDE) 5 and soluble guanylate cyclase (GC) by cGMP- and cAMP-dependent protein kinases (PKG and PKA respectively) was examined in gastric smooth muscle. The NO donor, sodium nitroprusside (SNP), stimulated PDE5 phosphorylation and activity, which was blocked by the selective PKG inhibitor, KT5823, resulting in an elevation of cGMP levels. Activation of PKA either directly by Sp-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole 3',5'-cyclic monophosphothioate, or via isoproterenol- and forskolin-dependent increase in cAMP, also caused an increase in PDE5 phosphorylation and activity, but only in the presence of cGMP; consistent with the dependence of PDE5 phosphorylation and activity on cGMP binding to allosteric sites in the regulatory domain of PDE5. The selective PKA inhibitors, myristoylated protein kinase inhibitor and H-89, blocked the increase in PDE5 phosphorylation and activity induced by PKA. SNP also stimulated soluble GC phosphorylation and activity. KT5823 abolished phosphorylation and augmented soluble GC activity, implying feedback inhibition of soluble GC by PKG-dependent phosphorylation. Phosphorylation by PKG was direct and could be induced in vitro. Activation of PKA had no effect on soluble GC. Thus cGMP levels are regulated by PKG- and PKA-dependent activation of PDE5 and PKG-specific inhibition of soluble GC. PMID:11696008

  17. Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins.

    PubMed

    Römling, Ute; Liang, Zhao-Xun; Dow, J Maxwell

    2017-03-01

    Cyclic di-GMP was the first cyclic dinucleotide second messenger described, presaging the discovery of additional cyclic dinucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, and they include both enzymatically active and inactive members, with a subset involved in synthesis and degradation of other cyclic dinucleotides. Here, we summarize current knowledge of sequence and structural variations that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL, and HD-GYP domain proteins.

  18. CSF Concentrations of cAMP and cGMP Are Lower in Patients with Creutzfeldt-Jakob Disease but Not Parkinson's Disease and Amyotrophic Lateral Sclerosis

    PubMed Central

    Oeckl, Patrick; Steinacker, Petra; Lehnert, Stefan; Jesse, Sarah; Kretzschmar, Hans A.; Ludolph, Albert C.; Otto, Markus; Ferger, Boris

    2012-01-01

    Background The cyclic nucleotides cyclic adenosine-3′,5′-monophosphate (cAMP) and cyclic guanosine-3′,5′-monophosphate (cGMP) are important second messengers and are potential biomarkers for Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and Creutzfeldt-Jakob disease (CJD). Methodology/Principal Findings Here, we investigated by liquid chromatography/tandem mass spectrometry (LC-MS/MS) the cerebrospinal fluid (CSF) concentrations of cAMP and cGMP of 82 patients and evaluated their diagnostic potency as biomarkers. For comparison with a well-accepted biomarker, we measured tau concentrations in CSF of CJD and control patients. CJD patients (n = 15) had lower cAMP (−70%) and cGMP (−55%) concentrations in CSF compared with controls (n = 11). There was no difference in PD, PD dementia (PDD) and ALS cases. Receiver operating characteristic (ROC) curve analyses confirmed cAMP and cGMP as valuable diagnostic markers for CJD indicated by the area under the curve (AUC) of 0.86 (cAMP) and 0.85 (cGMP). We calculated a sensitivity of 100% and specificity of 64% for cAMP and a sensitivity of 67% and specificity of 100% for cGMP. The combination of both nucleotides increased the sensitivity to 80% and specificity to 91% for the term cAMPxcGMP (AUC 0.92) and to 93% and 100% for the ratio tau/cAMP (AUC 0.99). Conclusions/Significance We conclude that the CSF determination of cAMP and cGMP may easily be included in the diagnosis of CJD and could be helpful in monitoring disease progression as well as in therapy control. PMID:22396786

  19. Ecklonia cava Polyphenol Has a Protective Effect against Ethanol-Induced Liver Injury in a Cyclic AMP-Dependent Manner

    PubMed Central

    Yamashita, Haruka; Goto, Mayu; Matsui-Yuasa, Isao; Kojima-Yuasa, Akiko

    2015-01-01

    Previously, we showed that Ecklonia cava polyphenol (ECP) treatment suppressed ethanol-induced increases in hepatocyte death by scavenging intracellular reactive oxygen species (ROS) and maintaining intracellular glutathione levels. Here, we examined the effects of ECP on the activities of alcohol-metabolizing enzymes and their regulating mechanisms in ethanol-treated hepatocytes. Isolated hepatocytes were incubated with or without 100 mM ethanol. ECP was dissolved in dimethylsulfoxide. ECP was added to cultured cells that had been incubated with or without ethanol. The cells were incubated for 0–24 h. In cultured hepatocytes, the ECP treatment with ethanol inhibited cytochrome P450 2E1 (CYP2E1) expression and activity, which is related to the production of ROS when large quantities of ethanol are oxidized. On the other hand, ECP treatment with ethanol increased the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase. These changes in activities of CYP2E1 and ADH were suppressed by treatment with H89, an inhibitor of protein kinase A. ECP treatment with ethanol enhanced cyclic AMP concentrations compared with those of control cells. ECP may be a candidate for preventing ethanol-induced liver injury via regulating alcohol metabolic enzymes in a cyclic AMP-dependent manner. PMID:26096275

  20. Ecklonia cava Polyphenol Has a Protective Effect against Ethanol-Induced Liver Injury in a Cyclic AMP-Dependent Manner.

    PubMed

    Yamashita, Haruka; Goto, Mayu; Matsui-Yuasa, Isao; Kojima-Yuasa, Akiko

    2015-06-18

    Previously, we showed that Ecklonia cava polyphenol (ECP) treatment suppressed ethanol-induced increases in hepatocyte death by scavenging intracellular reactive oxygen species (ROS) and maintaining intracellular glutathione levels. Here, we examined the effects of ECP on the activities of alcohol-metabolizing enzymes and their regulating mechanisms in ethanol-treated hepatocytes. Isolated hepatocytes were incubated with or without 100 mM ethanol. ECP was dissolved in dimethylsulfoxide. ECP was added to cultured cells that had been incubated with or without ethanol. The cells were incubated for 0-24 h. In cultured hepatocytes, the ECP treatment with ethanol inhibited cytochrome P450 2E1 (CYP2E1) expression and activity, which is related to the production of ROS when large quantities of ethanol are oxidized. On the other hand, ECP treatment with ethanol increased the activity of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase. These changes in activities of CYP2E1 and ADH were suppressed by treatment with H89, an inhibitor of protein kinase A. ECP treatment with ethanol enhanced cyclic AMP concentrations compared with those of control cells. ECP may be a candidate for preventing ethanol-induced liver injury via regulating alcohol metabolic enzymes in a cyclic AMP-dependent manner.

  1. Receptors and cGMP signalling mechanism for E. coli enterotoxin in opossum kidney

    SciTech Connect

    Forte, L.R.; Krause, W.J.; Freeman, R.H. Harry S. Truman Memorial Veterans Medical Center, Columbia, MO )

    1988-11-01

    Receptors for the heat-stable enterotoxin produced by Escherichia coli were found in the kidney and intestine of the North American opossum and in cultured renal cell lines. The enterotoxin markedly increased guanosine 3{prime},5{prime}-cyclic monophosphate (cGMP) production in slices of kidney cortex and medulla, in suspensions of intestinal mucosa, and in the opossum kidney (OK) and rat kangaroo kidney (PtK-2) cell lines. In contrast, atrial natriuretic factor elicited much smaller increases in cGMP levels of kidney, intestine, or cultured kidney cell lines. The enterotoxin receptors in OK cells had a molecular mass of approximately 120 kDa when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of receptors crosslinked with {sup 125}I-enterotoxin. The occurrence of receptors for the E. coli peptide in OK implies that these receptors may be involved in the regulation of renal tubular function in the opossum. E. coli enterotoxin caused a much larger increase in urine cGMP excretion than did atrial natriuretic factor when these peptides were injected intravenously into opossums. However, atrial natriuretic factor elicited a marked diuresis, natriuresis, and increased urinary excretion of calcium, phosphate, potassium, and magnesium. In contrast, the enterotoxin did not acutely influence OK fluid and electrolyte excretion. Thus the substantial increase in cGMP synthesis produced by the bacterial peptide in OK cortex and medulla in vitro and the increased renal excretion of cGMP in vivo were not associated with changes in electrolyte or water excretion. Whether cGMP represents a second messenger molecule in the kidney is an interesting question that was raised but not answered in this series of experiments.

  2. Basal cGMP regulates the resting pacemaker potential frequency of cultured mouse colonic interstitial cells of Cajal.

    PubMed

    Shahi, Pawan Kumar; Choi, Seok; Jeong, Yu Jin; Park, Chan Guk; So, Insuk; Jun, Jae Yeoul

    2014-07-01

    Cyclic guanosine 3',5'-monophosphate (cGMP) inhibited the generation of pacemaker activity in interstitial cells of Cajal (ICCs) from the small intestine. However, cGMP role on pacemaker activity in colonic ICCs has not been reported yet. Thus, we investigated the role of cGMP in pacemaker activity regulation by colonic ICCs. We performed a whole-cell patch-clamp and Ca(2+) imaging in cultured ICCs from mouse colon. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) increased the pacemaker potential frequency, whereas zaprinast (an inhibitor of phosphodiesterase) and cell-permeable 8-bromo-cGMP decreased the pacemaker potential frequency. KT-5823 (an inhibitor of protein kinase G [PKG]) did not affect the pacemaker potential. L-N(G)-nitroarginine methyl ester (L-NAME, an inhibitor of nitric oxide [NO] synthase) increased the pacemaker potential frequency, whereas (±)-S-nitroso-N-acetylpenicillamine (SNAP, a NO donor) decreased the pacemaker potential frequency. Glibenclamide (an ATP-sensitive K(+) channel blocker) did not block the effects of cell-permeable 8-bromo-cGMP and SNAP. Recordings of spontaneous intracellular Ca(2+) ([Ca(2+)]i) oscillations revealed that ODQ and L-NAME increased [Ca(2+)]i oscillations. In contrast, zaprinast, 8-bromo cGMP, and SNAP decreased the [Ca(2+)]i oscillations. Basal cGMP levels regulate the resting pacemaker potential frequency by the alteration on Ca(2+) release via a PKG-independent pathway. Additionally, the endogenous release of NO seems to be responsible maintaining basal cGMP levels in colonic ICCs.

  3. New Functions and Subcellular Localization Patterns of c-di-GMP Components (GGDEF Domain Proteins) in B. subtilis.

    PubMed

    Bedrunka, Patricia; Graumann, Peter L

    2017-01-01

    The universal and pleiotropic cyclic dinucleotide second messenger c-di-GMP is most prominently known to inversely regulate planktonic and sessile lifestyles of Gram-negative species. In the Gram-positive model organism Bacillus subtilis, intracellular c-di-GMP levels are modulated by a concise set of three diguanylate cylases (DgcK, DgcP, DgcW) and one phosphodiesterase (PdeH). Two recent studies have reported the negative influence of the c-di-GMP receptor DgrA (PilZ domain protein) on swarming motility indicating a conserved role of this second messenger across the bacterial domain. However, it has been suggested that the degenerated GGDEF protein YdaK and the inactive EAL domain protein YkuI may also function as c-di-GMP receptors regulating potentially other processes than motility. Here we describe a novel c-di-GMP dependent signaling network in B. subtilis regulating the production of an unknown exopolysaccharide (EPS) that leads to strongly altered colony morphologies upon overproduction. The network consists of the c-di-GMP receptor YdaK and the c-di-GMP synthetase DgcK. Both proteins establish a spatially close signal-effector cluster at the membrane. The cytoplasmic DgcP synthetase can complement for DgcK only upon overproduction, while the third c-di-GMP synthetase, DgcW, of B. subtilis is not part of the signaling pathway. Removal of the regulatory EAL domain from DgcW reveals a distinct function in biofilm formation. Therefore, our study is compatible with the "local pool signaling" hypothesis, but shows that in case of the yda operon, this can easily be overcome by overproduction of non-cognate DGCs, indicating that global pools can also confer signals to regulatory circuits in a Gram-positive bacterium.

  4. Regulation of Hippocampal cGMP Levels as a Candidate to Treat Cognitive Deficits in Huntington’s Disease

    PubMed Central

    Saavedra, Ana; Giralt, Albert; Arumí, Helena; Alberch, Jordi; Pérez-Navarro, Esther

    2013-01-01

    Huntington’s disease (HD) patients and mouse models show learning and memory impairment associated with hippocampal dysfunction. The neuronal nitric oxide synthase/3',5'-cyclic guanosine monophosphate (nNOS/cGMP) pathway is implicated in synaptic plasticity, and in learning and memory processes. Here, we examined the nNOS/cGMP pathway in the hippocampus of HD mice to determine whether it can be a good therapeutic target for cognitive improvement in HD. We analyzed hippocampal nNOS and phosphodiesterase (PDE) 5 and 9 levels in R6/1 mice, and cGMP levels in the hippocampus of R6/1, R6/2 and HdhQ7/Q111 mice, and of HD patients. We also investigated whether sildenafil, a PDE5 inhibitor, could improve cognitive deficits in R6/1 mice. We found that hippocampal cGMP levels were 3-fold lower in 12-week-old R6/1 mice, when they show deficits in object recognition memory and in passive avoidance learning. Consistent with hippocampal cGMP levels, nNOS levels were down-regulated, while there were no changes in the levels of PDE5 and PDE9 in R6/1 mice. A single intraperitoneal injection of sildenafil (3 mg/Kg) immediately after training increased cGMP levels, and improved memory in R6/1 mice, as assessed by using the novel object recognition and the passive avoidance test. Importantly, cGMP levels were also reduced in R6/2 mouse and human HD hippocampus. Therefore, the regulation of hippocampal cGMP levels can be a suitable treatment for cognitive impairment in HD. PMID:24040016

  5. 3'-5' cyclic-guanosine monophosphate increase in rat brain hippocampus after gamma-hydroxybutyrate administration. Prevention by valproate and naloxone

    SciTech Connect

    Vayer, P.; Gobaille, S.; Mandel, P.; Maitre, M.

    1987-08-03

    An increase (123%) of cyclic GMP (cGMP) was observed in the h