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Sample records for epac-selective cyclic amp

  1. Cyclic AMP in rat pancreatic islets

    International Nuclear Information System (INIS)

    Grill, V.; Borglund, E.; Cerasi, E.; Uppsala Univ.

    1977-01-01

    The incorporation of [ 3 H]adenine into cyclic AMP was studied in rat pancreatic islets under varying conditions of labeling. Prolonging the exposure to [ 3 H]adenine progressively augmented the islet cyclic [ 3 H]AMP level. Islets labeled for different periods of time and subsequently incubated (without adenine) in the presence of D-glucose or cholera toxin showed stimulations of intra-islet cyclic [ 3 H]AMP that were proportionate to the levels of radioactive nucleotide present under non-stimulatory conditions. Labeling the islets in a high glucose concentration (27.7 mM) did not modify the nucleotide responses to glucose or cholera toxin. The specific activity of cyclic [ 3 H]AMP, determined by simultaneous assay of cyclic [ 3 H]AMP and total cyclic AMP, was not influenced by glucose or cholera toxin. Glucose had no effect on the specific activity of labeled ATP

  2. 3' : 5'-Cyclic AMP-dependent 3'

    NARCIS (Netherlands)

    Mato, José M.; Krens, Frans A.; Haastert, Peter J.M. van; Konijn, Theo M.

    1977-01-01

    Suspensions of 3':5'-cyclic AMP (cAMP)-sensitive cells of Dictyostelium discoideum responded to a cAMP pulse with increased 3':5'-cyclic GMP (cGMP) levels. Under the assay conditions used (2 × 10^8 cells per ml in 10 mM phosphate buffer, pH 6.0) cAMP (5 × 10-8 M final concentration) increased cGMP

  3. 21 CFR 862.1230 - Cyclic AMP test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cyclic AMP test system. 862.1230 Section 862.1230....1230 Cyclic AMP test system. (a) Identification. A cyclic AMP test system is a device intended to measure the level of adenosine 3′, 5′-monophosphate (cyclic AMP) in plasma, urine, and other body fluids...

  4. Caffeine, cyclic AMP and postreplication repair of mammalian DNA

    International Nuclear Information System (INIS)

    Ehmann, U.K.

    1976-01-01

    The methylxanthines, caffeine and theophylline, inhibit postreplication repair of DNA in mammalian cells. Because they also inhibit cyclic AMP phosphodiesterase, it was thought that there might be some connection between concentrations of cyclic AMP and postreplication repair. This possibility was tested by performing DNA sedimentation experiments with a cyclic AMP-resistant mouse lymphoma cell mutant and its wild-type counterpart. The results show that there is no connection between cellular cyclic AMP concentrations and the rate of postreplication repair. Therefore, it is more likely that caffeine and theophylline inhibit postreplication repair by some other means, such as by binding to DNA

  5. Microgravity changes in heart structure and cyclic-AMP metabolism

    Science.gov (United States)

    Philpott, D. E.; Fine, A.; Kato, K.; Egnor, R.; Cheng, L.

    1985-01-01

    The effects of microgravity on cardiac ultrastructure and cyclic AMP metabolism in tissues of rats flown on Spacelab 3 are reported. Light and electron microscope studies of cell structure, measurements of low and high Km phosphodiesterase activity, cyclic AMP-dependent protein kinase activity, and regulatory subunit compartmentation show significant deviations in flight animals when compared to ground controls. The results indicate that some changes have occurred in cellular responses associated with catecholamine receptor interactions and intracellular signal processing.

  6. [Prognostic significance of the cyclic AMP concentration in acute leukemias].

    Science.gov (United States)

    Paietta, E; Mittermayer, K; Schwarzmeier, J D

    1979-01-01

    In patients with acute leukemia (myeloblastic, lymphoblastic, undifferentiated) proliferation kinetics and cyclic adenosine-3', 5'-monophosphate (cAMP) concentration of the leukemic cells were studied for their significance in the prediction of responsiveness to cytostatic therapy. Patients with good clinical response had significantly faster turnover and lower cAMP-levels than those who failed to respond to treatment.

  7. Discovery of a cAMP Deaminase That Quenches Cyclic AMP-Dependent Regulation

    Science.gov (United States)

    Goble, Alissa M.; Feng, Youjun; Raushel, Frank M.; Cronan, John E.

    2013-01-01

    An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of the universal second messenger, cyclic-3’, 5’-adenosine monophosphate (cAMP). The enzyme, which we have named CadD, is encoded by the human pathogenic bacterium Leptospira interrogans. Although CadD is annotated as an adenosine deaminase, the protein specifically deaminates cAMP to cyclic-3’, 5’-inosine monophosphate (cIMP) with a kcat/Km of 2.7 ± 0.4 × 105 M−1 s−1 and has no activity on adenosine, adenine, or 5’-adenosine monophosphate (AMP). This is the first identification of a deaminase specific for cAMP. Expression of CadD in Escherichia coli mimics the loss of adenylate cyclase in that it blocks growth on carbon sources that require the cAMP-CRP transcriptional activator complex for expression of the cognate genes. The cIMP reaction product cannot replace cAMP as the ligand for CRP binding to DNA in vitro and cIMP is a very poor competitor of cAMP activation of CRP for DNA binding. Transcriptional analyses indicate that CadD expression represses expression of several cAMP-CRP dependent genes. CadD adds a new activity to the cAMP metabolic network and may be a useful tool in intracellular study of cAMP-dependent processes. PMID:24074367

  8. Cyclic AMP system in muscle tissue during prolonged hypokinesia

    Science.gov (United States)

    Antipenko, Y. A.; Bubeyev, Y. A.; Korovkin, B. F.; Mikhaleva, N. P.

    1980-01-01

    Components of the cyclic Adenosine-cyclic-35-monophosphate (AMP) system in the muscle tissue of white rats were studied during 70-75 days of hypokinesia, created by placing the animals in small booths which restricted their movements, and during the readaptation period. In the initial period, cyclic AMP levels and the activities of phosphodiesterase and adenylate cyclase in muscle tissue were increased. The values for these indices were roughly equal for controls and experimental animals during the adaptation period, but on the 70th day of the experiment cAMP levels dropped, phosphodiesterase activity increased, and the stimulative effect of epinephrine on the activity of adenylate cyclase decreased. The indices under study normalized during the readaptation period.

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

    OpenAIRE

    Škrnjug, Ivana; Guzmán, Carlos Alberto; Ruecker, Christine

    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....

  10. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

    DEFF Research Database (Denmark)

    Søgaard-Andersen, L; Martinussen, J; Møllegaard, N E

    1990-01-01

    We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) Cyt...

  11. Radioprotection of mouse intestine by inhibitors of cyclic amp phosphodiesterase

    International Nuclear Information System (INIS)

    Lehnert, S.

    1979-01-01

    The survival of colony-forming units of the jejunal crypt was used to assay the radioprotective capacity of various inhibitors of cyclic AMP phosphodiesterase. DL-152, RO-20-1724 and the methyl xanthines, caffeine, theophylline, and methyl isbutyl xanthine (MIX) were all found to have some radioprotective effect. The degree of radioprotecton depended on the route of administration of the drug and on the timing of administration with respect to irradiation. Optimum survival of crypt stem cells was found following intraperitoneal administration of DL-152 (60 min before irradiation) or MIX (30 min before irradiaton), and following intravenous administration of caffeine (60 to 120 min before irradiaton) or theophylline (60 min before irradiation). When these protocols were used, crypt stem cell survival could be enhanced by a factor of from 6 to 7. All the compounds investigated produced some elevation of cyclic AMP content of the whole jejunum; this was found to be simultaneous with or to precede the period of maximum radioprotection. Cyclic AMP was localized with immunofluorescent staining; following injection of DL-152 it was found to be elevated in all parts of the jejunum but to the greatest extent in the lower part of the crypt. Survival curves for crypt stem cells from MIX and DL-152 treated mice were found to have almost the same exponential slope as the saline-injected control, suggesting that the mechanism of protection does not depend on induction of hypoxia

  12. Somite chrondrogenesis: alterations in cyclic AMP levels and proteoglycan synthesis

    International Nuclear Information System (INIS)

    Vasan, Nagaswamistri; Lamb, K.M.; Heick, A.E.

    1985-01-01

    Cyclic AMP (cAMP) levels have been shown to have a positive influence on chondrogenesis in limb buds and pelvic cartilage. In the present study the level of cAMP was measured during somite chondrogenesis in vitro and found to decrease from 1.38 pmol/μg DNA on day 0 to 0.9 pmol/μg DNA on day 6. Inclusion of notochord with somites caused a marked recution, with levels decreasing from 1.41 pmol/μg DNA on day 0 to 0.36 pmol/μg DNA on day 6. Concurrently, the incorporation of radioactive sulfate into sulfated glycosaminoglycans increased from day 3 to day 6 by 38% in somite and 77% in somite-notochord explants. The aggregation of proteoglycans was analyzed by gel chromatography and found to increase with a corresponding decrease in cAMP levels. The result indicate that a decrease in cAMP levels may be necessary for chondrogenic expression in somites. (author)

  13. Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

    Directory of Open Access Journals (Sweden)

    Ivana Škrnjug

    Full Text Available 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.

  14. Cyclic GMP-AMP displays mucosal adjuvant activity in mice.

    Science.gov (United States)

    Škrnjug, Ivana; Guzmán, Carlos Alberto; Rueckert, Christine; Ruecker, Christine

    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.

  15. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa

    1990-01-01

    Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated

  16. Down-regulation of Cell Surface Cyclic AMP Receptors and Desensitization of Cyclic AMP-stimulated Adenylate Cyclase by Cyclic AMP in Dictyostelium discoideum. Kinetics and Concentration Dependence

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1987-01-01

    cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylate cyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of

  17. "cAMP sponge": a buffer for cyclic adenosine 3', 5'-monophosphate.

    Directory of Open Access Journals (Sweden)

    Konstantinos Lefkimmiatis

    Full Text Available BACKGROUND: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP. METHODS/PRINCIPAL FINDINGS: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIbeta of protein kinase A (PKA. Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named "cAMP sponge" was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. CONCLUSIONS: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.

  18. Insulin alters the target size of the peripheral cyclic AMP phosphodiesterase but not the integral cyclic GMP-stimulated cyclic AMP phosphodiesterase in liver plasma membranes

    International Nuclear Information System (INIS)

    Wallace, A.V.; Martin, B.R.; Houslay, M.D.

    1990-01-01

    Radiation inactivation of the two high affinity cyclic AMP phosphodiesterases (PDE) found in liver plasma membranes afforded an estimation of their molecular target sizes in situ. The activity of the peripheral plasma membrane PDE decayed as a single exponential with a target size corresponding to a monomer of circa 54 kDa. The integral, cyclic GMP-stimulated PDE decayed as a dimer of circa 125 kDa. Preincubation of plasma membranes with insulin (10nM), prior to irradiation, caused the target size of only the peripheral plasma membrane PDE to increase. We suggest that insulin addition causes the peripheral plasma membrane PDE to alter its coupling to an integral plasma membrane protein with a target size of circa 90 kDa

  19. Radioimmunoassays for cyclic AMP cross-react with phosphodiesterase inhibitors and buffer components

    NARCIS (Netherlands)

    Sinha, B; Semmler, J; Haen, E; Moeller, J; Endres, S

    We addressed the issue of cross-reactivity of several commonly used phosphodiesterase inhibitors with radioimmunoassays for cyclic AMP, after we had observed a considerably high cross-reactivity with a noncommercial antibody. Theophylline, pentoxifylline, penthydroxifylline (BL 194), albifylline

  20. Binding of the cyclic AMP receptor protein of Escherichia coli to RNA polymerase.

    Science.gov (United States)

    Pinkney, M; Hoggett, J G

    1988-03-15

    Fluorescence polarization studies were used to study the interaction of a fluorescein-labelled conjugate of the Escherichia coli cyclic AMP receptor protein (F-CRP) and RNA polymerase. Under conditions of physiological ionic strength, F-CRP binds to RNA polymerase holoenzyme in a cyclic AMP-dependent manner; the dissociation constant was about 3 microM in the presence of cyclic AMP and about 100 microM in its absence. Binding to core RNA polymerase under the same conditions was weak (Kdiss. approx. 80-100 microM) and independent of cyclic AMP. Competition experiments established that native CRP and F-CRP compete for the same binding site on RNA polymerase holoenzyme and that the native protein binds about 3 times more strongly than does F-CRP. Analytical ultracentrifuge studies showed that CRP binds predominantly to the monomeric rather than the dimeric form of RNA polymerase.

  1. Aspirin effects on lymphocyte cyclic AMP levels in normal human subjects.

    Science.gov (United States)

    Snider, D E; Parker, C W

    1976-01-01

    In purified lymphocytes from the peripheral blood of healthy human subjects who had ingested therapeutic doses of aspirin, there was a significant decrease in resting cyclic AMP levels as well as a partial inhibition of the rise in cyclic AMP with isoproterenol or prostaglandin E1. These changes were seen as early as 30 min after aspirin ingestion and did not appear to result from aspirin effects on lymphocyte recovery, purity, viability, or relative number of thymus- or bone marrow-derived lymphocytes. In contrast, the direct addition of aspirin to suspensions of purified peripheral lymphocytes did not significantly alter their cyclic AMP levels. However, an effect of aspirin could be obtained in vitro if aspirin was added to unprocessed whole blood during the dextran sedimentation phase of the cell purification. Thus the effect of aspirin on lymphocyte cyclic AMP metabolism, may be indirect, through other cells present in the peripheral blood. PMID:182720

  2. The Cyclic AMP-Vfr Signaling Pathway in Pseudomonas aeruginosa Is Inhibited by Cyclic Di-GMP

    DEFF Research Database (Denmark)

    Almblad, Henrik; Harrison, Joe J; Rybtke, Morten

    2015-01-01

    infection give rise to rugose small colony variants (RSCVs), which are hyper-biofilm-forming mutants that commonly possess mutations that increase production of the biofilm-promoting secondary messenger cyclic di-GMP (c-di-GMP). We show that RSCVs display a decreased production of acute virulence factors...... as a direct result of elevated c-di-GMP content. Overproduction of c-di-GMP causes a decrease in the transcription of virulence factor genes that are regulated by the global virulence regulator Vfr. The low level of Vfr-dependent transcription is caused by a low level of its coactivator, cyclic AMP (c......AMP), which is decreased in response to a high level of c-di-GMP. Mutations that cause reversion of the RSCV phenotype concomitantly reactivate Vfr-cAMP signaling. Attempts to uncover the mechanism underlying the observed c-di-GMP-mediated lowering of cAMP content provided evidence that it is not caused...

  3. The interplay between cyclic AMP and insulin during obesity development

    DEFF Research Database (Denmark)

    Borkowski, Kamil

    Insulin and cAMP signalling are related to two opposite metabolic responses. Insulin secretion is elicited in response to food availability and trigger catabolic processes like lipogenesis and glycogen synthesis with a purpose of energy storage. On the other hand cAMP signalling is associated...

  4. Relation of intracellular cyclic AMP to the shape of mammalian cell survival curves

    International Nuclear Information System (INIS)

    Lehnert, S.

    1975-01-01

    Results of experiments with V79 cells growing in tissue culture indicate that the reproductive survival of cells following irradiation is influenced by the level of intracellular 3', 5'-cyclic adenosine monophosphate (cyclic AMP) at the time of irradiation. Cells containing high levels of cyclic AMP induced by treatments with drugs show a characteristic survival curve in which the extent of the shoulder is increased so that the survival after low doses is enhanced. The exponential slope or D 0 , however, is decreased so that at high doses the survival of cells containing high levels of cyclic AMP may be less than that of controls. Naturally occurring changes in radiosensitivity such as those observed as cells pass through the division cycle, may also be related to parallel changes in cyclic AMP concentration occurring during the cycle. Injection of mice with compounds producing elevated cyclic AMP prior to whole-body irradiation increases survival at seven days post-irradiation. The shape of the survival curve for intestinal stem cells in these mice differs from that of the control in having an increased extrapolation number; no change in D 0 is observed in this in vivo situation. (author)

  5. Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

    Science.gov (United States)

    Melmed, RN; Karanian, PJ; Berlin, RD

    1981-01-01

    We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J

  6. Is the level of cyclic AMP in the pyloric caeca of the starfish Asterias rubens related to the reproductive cycle?

    NARCIS (Netherlands)

    Voogt, P.A.; Rheenen, J.W.A. van

    1984-01-01

    1. 1. Cyclic AMP levels were significantly higher (P = 0.01) in female than in male animals. The average difference found was 0.50 pmole/mg dry weight. 2. 2. Cyclic AMP levels were negatively correlated to the pyloric caeca-index. In females this correlation was −0.54 (P < 0.05). 3. 3. Cyclic

  7. Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain

    Science.gov (United States)

    Cohen, C. R.; Mills, I.; Du, W.; Kamal, K.; Sumpio, B. E.

    1997-01-01

    The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

  8. Cyclic AMP Pathway Activation and Extracellular Zinc Induce Rapid Intracellular Zinc Mobilization in Candida albicans

    Science.gov (United States)

    Kjellerup, Lasse; Winther, Anne-Marie L.; Wilson, Duncan; Fuglsang, Anja T.

    2018-01-01

    Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this “zinc flux” could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen. PMID:29619016

  9. The Interplay between Cyclic AMP, MAPK, and NF-κB Pathways in Response to Proinflammatory Signals in Microglia

    Directory of Open Access Journals (Sweden)

    Mousumi Ghosh

    2015-01-01

    Full Text Available Cyclic AMP is an important intracellular regulator of microglial cell homeostasis and its negative perturbation through proinflammatory signaling results in microglial cell activation. Though cytokines, TNF-α and IL-1β, decrease intracellular cyclic AMP, the mechanism by which this occurs is poorly understood. The current study examined which signaling pathways are responsible for decreasing cyclic AMP in microglia following TNF-α stimulation and sought to identify the role cyclic AMP plays in regulating these pathways. In EOC2 microglia, TNF-α produced a dramatic reduction in cyclic AMP and increased cyclic AMP-dependent PDE activity that could be antagonized by Rolipram, myristoylated-PKI, PD98059, or JSH-23, implicating a role for PDE4, PKA, MEK, and NF-κB in this regulation. Following TNF-α there were significant increases in iNOS and COX-2 immunoreactivity, phosphorylated ERK1/2 and NF-κB-p65, IκB degradation, and NF-κB p65 nuclear translocation, which were reduced in the presence of high levels of cyclic AMP, indicating that reductions in cyclic AMP during cytokine stimulation are important for removing its inhibitory action on NF-κB activation and subsequent proinflammatory gene expression. Further elucidation of the signaling crosstalk involved in decreasing cyclic AMP in response to inflammatory signals may provide novel therapeutic targets for modulating microglial cell activation during neurological injury and disease.

  10. Cyclic di-AMP regulation of osmotic homeostasis is essential in Group B Streptococcus.

    Directory of Open Access Journals (Sweden)

    Laura Devaux

    2018-04-01

    Full Text Available Cyclic nucleotides are universally used as secondary messengers to control cellular physiology. Among these signalling molecules, cyclic di-adenosine monophosphate (c-di-AMP is a specific bacterial second messenger recognized by host cells during infections and its synthesis is assumed to be necessary for bacterial growth by controlling a conserved and essential cellular function. In this study, we sought to identify the main c-di-AMP dependent pathway in Streptococcus agalactiae, the etiological agent of neonatal septicaemia and meningitis. By conditionally inactivating dacA, the only diadenyate cyclase gene, we confirm that c-di-AMP synthesis is essential in standard growth conditions. However, c-di-AMP synthesis becomes rapidly dispensable due to the accumulation of compensatory mutations. We identified several mutations restoring the viability of a ΔdacA mutant, in particular a loss-of-function mutation in the osmoprotectant transporter BusAB. Identification of c-di-AMP binding proteins revealed a conserved set of potassium and osmolyte transporters, as well as the BusR transcriptional factor. We showed that BusR negatively regulates busAB transcription by direct binding to the busAB promoter. Loss of BusR repression leads to a toxic busAB expression in absence of c-di-AMP if osmoprotectants, such as glycine betaine, are present in the medium. In contrast, deletion of the gdpP c-di-AMP phosphodiesterase leads to hyperosmotic susceptibility, a phenotype dependent on a functional BusR. Taken together, we demonstrate that c-di-AMP is essential for osmotic homeostasis and that the predominant mechanism is dependent on the c-di-AMP binding transcriptional factor BusR. The regulation of osmotic homeostasis is likely the conserved and essential function of c-di-AMP, but each species has evolved specific c-di-AMP mechanisms of osmoregulation to adapt to its environment.

  11. Mechanism for iron control of the Vibrio fischeri luminescence system: involvement of cyclic AMP and cyclic AMP receptor protein and modulation of DNA level.

    Science.gov (United States)

    Dunlap, P V

    1992-07-01

    Iron controls luminescence in Vibrio fischeri by an indirect but undefined mechanism. To gain insight into that mechanism, the involvement of cyclic AMP (cAMP) and cAMP receptor protein (CRP) and of modulation of DNA levels in iron control of luminescence were examined in V. fischeri and in Escherichia coli containing the cloned V. fischeri lux genes on plasmids. For V. fischeri and E. coli adenylate cyclase (cya) and CRP (crp) mutants containing intact lux genes (luxR luxICDABEG), presence of the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid) (EDDHA) increased expression of the luminescence system like in the parent strains only in the cya mutants in the presence of added cAMP. In the E. coli strains containing a plasmid with a Mu dl(lacZ) fusion in luxR, levels of beta-galactosidase activity (expression from the luxR promoter) and luciferase activity (expression from the lux operon promoter) were both 2-3-fold higher in the presence of EDDHA in the parent strain, and for the mutants this response to EDDHA was observed only in the cya mutant in the presence of added cAMP. Therefore, cAMP and CRP are required for the iron restriction effect on luminescence, and their involvement in iron control apparently is distinct from the known differential control of transcription from the luxR and luxICDABEG promoters by cAMP-CRP. Furthermore, plasmid and chromosomal DNA levels were higher in E. coli and V. fischeri in the presence of EDDHA. The higher DNA levels correlated with an increase in expression of chromosomally encoded beta-galactosidase in E. coli and with a higher level of autoinducer in cultures of V. fischeri. These results implicate cAMP-CRP and modulation of DNA levels in the mechanism of iron control of the V. fischeri luminescence system.

  12. Transcriptional regulation of the tyrosine hydroxylase gene by glucocorticoid and cyclic AMP

    International Nuclear Information System (INIS)

    Lewis, E.J.; Harrington, C.A.; Chikaraishi, D.M.

    1987-01-01

    Glucocorticoid and cyclic AMP increase tyrosine hydroxylase (TH) activity and mRNA levels in pheochromocytoma cultures. The transcriptional activity of the TH gene, as measured by nuclear run-on assay, is also increased when cultures are treated with the synthetic glucocorticoid dexamethasone or agents that increase intracellular cyclic AMP, such as forskolin and 8-BrcAMP. Both inducers effect transcriptional changes within 10 min after treatment and are maximal after 30 min for forskolin and after 60 min for dexamethasone. The 5' flanking sequences of the TH gene were fused to the bacterial gene chloramphenicol acetyltransferase (CAT), and the hybrid gene was transfected into pheochromocytoma cultures and GH 4 pituitary cells. In both cell lines, a region of the TH gene containing bases -272 to +27 conferred induction of CAT by cyclic AMP, but not by glucocorticoid. The same results were found when a region of the TH gene containing -773 to + 27 was used. Thus, the sequences required for induction of TH by cyclic AMP are contained within 272 bases of 5' flanking sequence, but sequences sufficient for glucocorticoid regulation are not contained with 773 bases

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

    OpenAIRE

    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 mo...

  14. Earl Sutherland (1915-1974) [corrected] and the discovery of cyclic AMP.

    Science.gov (United States)

    Blumenthal, Stanley A

    2012-01-01

    In 1945, Earl Sutherland (1915-1974) [corrected] and associates began studies of the mechanism of hormone-induced glycogen breakdown in the liver. In 1956, their efforts culminated in the identification of cyclic AMP, an ancient molecule generated in many cell types in response to hormonal and other extracellular signals. Cyclic AMP, the original "second messenger," transmits such signals through pathways that regulate a diversity of cellular functions and capabilities: metabolic processes such as lipolysis and glycogenolysis; hormone secretion; the permeability of ion channels; gene expression; cell proliferation and survival. Indeed, it can be argued that the discovery of cyclic AMP initiated the study of intracellular signaling pathways, a major focus of contemporary biomedical inquiry. This review presents relevant details of Sutherland's career; summarizes key contributions of his mentors, Carl and Gerti Cori, to the knowledge of glycogen metabolism (contributions that were the foundation for his own research); describes the experiments that led to his identification, isolation, and characterization of cyclic AMP; assesses the significance of his work; and considers some aspects of the impact of cyclic nucleotide research on clinical medicine.

  15. Effect of drugs on lipid methylation, receptor-adenylate cyclase coupling and cyclic AMP secretion in Dictyostelium discoideum

    NARCIS (Netherlands)

    Van Waarde, Aren; Van Haastert, P.J.M.

    1986-01-01

    Intercellular communication in Dictyostelium discoldeum takes place by means of cyclic AMP-induced cyclic AMP-synthesis and secretion. Since phospholipid methylation has been suggested to play a role in receptor-adenylate cyclase coupling, we examined the effects of transmethylation inhibitors on

  16. The cyclic AMP cascade is altered in the fragile X nervous system.

    Directory of Open Access Journals (Sweden)

    Daniel J Kelley

    2007-09-01

    Full Text Available Fragile X syndrome (FX, the most common heritable cause of mental retardation and autism, is a developmental disorder characterized by physical, cognitive, and behavioral deficits. FX results from a trinucleotide expansion mutation in the fmr1 gene that reduces levels of fragile X mental retardation protein (FMRP. Although research efforts have focused on FMRP's impact on mGluR signaling, how the loss of FMRP leads to the individual symptoms of FX is not known. Previous studies on human FX blood cells revealed alterations in the cyclic adenosine 3', 5'-monophosphate (cAMP cascade. We tested the hypothesis that cAMP signaling is altered in the FX nervous system using three different model systems. Induced levels of cAMP in platelets and in brains of fmr1 knockout mice are substantially reduced. Cyclic AMP induction is also significantly reduced in human FX neural cells. Furthermore, cAMP production is decreased in the heads of FX Drosophila and this defect can be rescued by reintroduction of the dfmr gene. Our results indicate that a robust defect in cAMP production in FX is conserved across species and suggest that cAMP metabolism may serve as a useful biomarker in the human disease population. Reduced cAMP induction has implications for the underlying causes of FX and autism spectrum disorders. Pharmacological agents known to modulate the cAMP cascade may be therapeutic in FX patients and can be tested in these models, thus supplementing current efforts centered on mGluR signaling.

  17. The effect of in vitro procedures on cyclic AMP accumulation in human leucocytes

    DEFF Research Database (Denmark)

    Johansen, Torben; Friis, U G; Rasmussen, A K

    1994-01-01

    The aim of this study was to investigate the effect of various methodological procedures or protocols on cyclic AMP formation in human leucocytes. The data showed that: (1) ATP content and lactate production was unaffected by hypotonic lysis during leucocyte isolation; (2) there was a linear rela...

  18. Modification of radiation-induced division delay by caffeine analogues and dibutyryl cyclic AMP

    International Nuclear Information System (INIS)

    Kimler, B.F.; Leeper, D.B.; Snyder, M.H.; Rowley, R.; SChneiderman, M.H.

    1982-01-01

    The mitotic selection procedure for cell cycle analysis was utilized to investigate the concentration-dependent modification of x-radiation-induced division delay in Chinese hamster ovary (CHO) cells by methyl xanthines (caffeine, theophylline, and theobromine) and by dibutyryl cyclic AMP. The methyl xanthines (concentrations from 0.5 to 1000 μg/ml) all reduced radiation-induced division delay with the effect being linear between approximately 100 and 1000 μg/ml. After doses of 100-300 rad, delay was reduced by 75, 94 or 83 per cent at 1000 μg/ml for each drug, respectively. However, the addition of dibutyryl cyclic AMP had an opposite effect: radiation-induced delay was increased by the concentration range of 0.3 to 300 μg/ml. These results indicate that in mammalian cells the control of cell cycle progression and the modification of radiation-induced division delay are not simply related to intracellular levels of cyclic AMP. Rather, there appear to be at least two competing mechanisms which are differentially affected by caffeine analogues or by direct addition of dibutyryl cyclic AMP. The direct effect of caffeine and the methyl xanthines on membrane calcium permeability is considered. (author)

  19. Modification of radiation-induced division delay by caffeine analogues and dibutyryl cyclic AMP

    Energy Technology Data Exchange (ETDEWEB)

    Kimler, B.F.; Leeper, D.B.; Snyder, M.H.; Rowley, R.; Schneiderman, M.H. (Thomas Jefferson Univ., Philadelphia, PA (USA). Hospital)

    1982-01-01

    The mitotic selection procedure for cell cycle analysis was utilized to investigate the concentration-dependent modification of x-radiation-induced division delay in Chinese hamster ovary (CHO) cells by methyl xanthines (caffeine, theophylline, and theobromine) and by dibutyryl cyclic AMP. The methyl xanthines (concentrations from 0.5 to 1000 ..mu..g/ml) all reduced radiation-induced division delay with the effect being linear between approximately 100 and 1000 ..mu..g/ml. After doses of 100-300 rad, delay was reduced by 75, 94 or 83 per cent at 1000 ..mu..g/ml for each drug, respectively. However, the addition of dibutyryl cyclic AMP had an opposite effect: radiation-induced delay was increased by the concentration range of 0.3 to 300 ..mu..g/ml. These results indicate that in mammalian cells the control of cell cycle progression and the modification of radiation-induced division delay are not simply related to intracellular levels of cyclic AMP. Rather, there appear to be at least two competing mechanisms which are differentially affected by caffeine analogues or by direct addition of dibutyryl cyclic AMP. The direct effect of caffeine and the methyl xanthines on membrane calcium permeability is considered.

  20. Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells

    Science.gov (United States)

    Anwar, Zubeya; Albert, Jennifer L; Gubby, Sharon E; Boyle, John P; Roberts, Jonathon A; Webb, Tania E; Boarder, Michael R

    1999-01-01

    In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors.None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPγS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin.Comparison of the concentration response curves to ATPγS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides.Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1,3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N6-cyclopentyladenosine nor CGS 21680 were agonists.These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors. PMID:10510459

  1. Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment

    Directory of Open Access Journals (Sweden)

    Shravan Babu Girada

    2017-10-01

    Conclusions: The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex.

  2. Cellular regulation of basal and FSH-stimulated cyclic AMP production in irradiated rat testes

    International Nuclear Information System (INIS)

    Kangasniemi, M.; Kaipia, A.; Toppari, J.; Mali, P.; Huhtaniemi, I.; Parvinen, M.

    1990-01-01

    Basal and follicle-stimulating hormone (FSH)-stimulated cyclic AMP (cAMP) productions by seminiferous tubular segments from irradiated adult rats were investigated at defined stages of the epithelial cycle when specific spermatogenic cells were low in number. Seven days post-irradiation, depletion of spermatogonia did not influence the basal cAMP production, but FSH response increased in stages II-VIII. Seventeen days post-irradiation when spermatocytes were low in number, there was a small increase in basal cAMP level in stages VII-VIII and FSH-stimulated cAMP production increased in stages VII-XII and XIII-I. At 38 days when pachytene spermatocytes and round spermatids (steps 1-6) were low in number, a decreased basal cAMP production was measured in stages II-VI and IX-XII. FSH-stimulated cAMP output increased in stages VII-XII but decreased in stages II-VI. At 52 days when all spermatids were low in number, basal cAMP levels decreased in all stages of the cycle, whereas FSH response was elevated only in stages VII-XII. All spermatogenic cell types seem to have an effect on cAMP production by the seminiferous tubule in a stage-specific fashion. Germ cells appear to regulate Sertoli cell FSH response in a paracrine way, and a part of cAMP may originate from spermatids stimulated by an unknown FSH-dependent Sertoli cell factor. The FSH-dependent functions may control such phenomena as spermatogonial proliferation, final maturation of spermatids, and onset of meiosis

  3. Role of coronary endothelium in cyclic AMP formation by the heart

    International Nuclear Information System (INIS)

    Kroll, K.; Schrader, J.

    1986-01-01

    In order to quantify the activation of adenylate cyclase of the coronary endothelium in vivo, endothelial adenine nucleotides of isolated guinea pig hearts were selectively pre-labeled by intracoronary infusion of tritiated (H3)-adenosine, and the coronary efflux of H3-cAMP was measured. The adenosine receptor agonist, NECA (12 μM), increased total cAMP release 4 fold, and raised H3-cAMP release 22 fold. Several classes of coronary vasodilators (adenosine, L-PIA, D-PIA, the beta 2-adrenergic agonist procaterol, and PGE1) caused dose-dependent increases in endothelial-derived H3-cAMP release. These increases were accompanied by decreases in vascular resistance, at agonist doses without positive intropic effects. Hypoxic perfusion also raised H3-cAMP release, and this was antagonized by theophylline. It is concluded: (1) cyclic AMP formation by coronary endothelium can dominate total cAMP production by the heart; (2) coronary endothelial adenylate cyclase-coupled receptors for adenosine (A2), catecholamines (beta2) and prostaglandins are activated in parallel with coronary vasodilation; (3) endothelial adenylate cyclase can be activated by endogenous adenosine

  4. CHARACTERIZATION OF P2-PURINOCEPTOR MEDIATED CYCLIC-AMP FORMATION IN MOUSE C2C12 MYOTUBES

    NARCIS (Netherlands)

    HENNING, RH; DUIN, M; DENHERTOG, A; NELEMANS, A

    1 The formation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2Cl2 myotubes. 2 ATP (100 muM) and ATPgammaS (100 muM) caused a sustained increase in cyclic AMP content of the cells,

  5. Cyclic GMP-AMP Synthase is Activated by Double-stranded DNA-Induced Oligomerization

    OpenAIRE

    Li, Xin; Shu, Chang; Yi, Guanghui; Chaton, Catherine T.; Shelton, Catherine L.; Diao, Jiasheng; Zuo, Xiaobing; Kao, C Cheng; Herr, Andrew B.; Li, Pingwei

    2013-01-01

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide 2′,5′ cGAMP that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and ...

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

    OpenAIRE

    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...

  7. Regulatory action of catalpol from radix rehmanniae on β-adrenoceptor-cyclic AMP system

    International Nuclear Information System (INIS)

    Liu Qingfeng; Sun Qixiang; Hu Yaer; Xia Zongqin

    2004-01-01

    Objective: It has been proved in previous works from this laboratory that the Yin tonic effect of water extract of Radix Rehmanniae is closely related to its down-regulation effect on β adrenoceptor-cyclic AMP system. The purpose of this work is to investigate whether catalpol from Radix Rehmanniae is the effective active component of this plant drug. Methods: Catalpol was extracted from fresh Radix Rehmanniae by organic solvents and purified by low pressure silica gel column and recrystallization. Experimental mice were divided into 4 groups. Three groups of hyperthyroid mouse models were prepared with subcutaneous injection of T 3 and two of them were additionally fed oral catalpol at low and high dose respectively. One group receiving neither T 3 nor catalpol served as controls. Plasma cAMP levels in these mice were measured with RIA 10 min after subcutaneous injection of a single dose of isoproterenol. Results: After injection of catalpol, the plasma cAMP content rose markedly to above 700 pmol/ml in control mice while that of the hyperthyroid mice rose to a even higher levels. Simultaneous additional oral administration of catalpol with purity >97% for 6 days significantly lowered the cAMP level in hyperthyroid mice. With catalpol dose of 1.56 mg/mouse/d and 6.0 mg/mouse/d, the plasma cAMP levels were 978 ± 283 pmol/ml and 1070 ± 394 pmol/ml respectively, the values in the group without catalpol being 1408 ± 568 pmol/ml and 1491 ± 486 pmol/ml, both P<0.05. Conclusion: Catalpol can repeat the regulatory effect of water extract of Radix Rehmanniae on β adrenoceptor-cyclic AMP system, that is, down regulate the response of hyperthyroid mice to isoproterenol. This result suggests that catalpol is an effective active component of the Yin tonic effect of Radix Rehmanniae. (authors)

  8. Cyclic AMP-receptor proteins in heart muscle of rats flown on Cosmos 1887

    Science.gov (United States)

    Mednieks, Maija I.; Popova, Irina A.; Grindeland, Richard E.

    1991-01-01

    The cellular compartmentalization of the cyclic AMP-receptor proteins in heart ventricular tissue obtained from rats flown on the Cosmos 1887 is determined. Photoaffinity labeling of soluble and particular cell fractions with a (32P)-8-azido analog of cyclic AMP is followed by electrophoretic separation of the proteins and by autoradiographic identification of the labeled isoforms of cAPK R subunits. It is shown that RII in the particulate subcellular fraction was significantly decreased in heart cells from rats in the flight group when compared to controls. Protein banding patterns in both the cytoplasmic fraction and in a fraction enriched in chromatin-bound proteins exhibited some variability in tissues of individual animals, but showed no changes that could be directly attributed to flight conditions. No significant change was apparent in the distribution of RI or RII cyclic AMP binding in the soluble fractions. It is inferred that the cardiac cell integrity or its protein content is not compromised under flight conditions.

  9. Prostaglandin A1 metabolism and inhibition of cyclic AMP extrusion by avian erythrocytes

    International Nuclear Information System (INIS)

    Heasley, L.E.; Brunton, L.L.

    1985-01-01

    Prostaglandins (PG) inhibit active cyclic AMP export from pigeon red cells, PGA1 and PGA2 most potently. To probe the mechanism of this action of PGA1, the authors have studied the interaction of [ 3 H]PGA1 with suspensions of pigeon red cells. The interaction of PGA1 with pigeon red cells is a multistep process of uptake, metabolism, and secretion. [ 3 H] PGA1 rapidly enters red cells and is promptly metabolized to a compound(s) that remains in the aqueous layer after ethylacetate extraction. The glutathione-depleting agent, diamide, inhibits formation of the PGA1 metabolite. The red cells secrete the polar metabolite of PGA1 by a saturable mechanism that lowered temperatures inhibit. Because uptake and metabolism progress with much greater rates than metabolite secretion, red cells transiently concentrate the polar compound intracellularly. Onset and reversal of inhibition of cyclic AMP export by PGA1 coincide with accumulation and secretion of PGA1 metabolite, suggesting that the polar metabolite acts at an intracellular site to inhibit cyclic AMP efflux

  10. Proliferation kinetics and cyclic AMP as prognostic factors in adult acute leukemia.

    Science.gov (United States)

    Paietta, E; Mittermayer, K; Schwarzmeier, J

    1980-07-01

    In 41 adult patients with acute leukemia (myeloblastic, lymphoblastic, and undifferentiated), proliferation kinetics (as determined by double-label autoradiography) and cyclic adenosine 3',5'-monophosphate (cAMP) concentration were studied for their significance in the prediction of responsiveness to cytostatic therapy. Patients with good clinical response had significantly shorter turnover times and higher labeling indices in the bone marrow than did those who failed to respond to treatment. Cases for which cell kinetics did not correlate with clinical response were explained by variance in the distribution of leukemic blasts between the proliferative cell cycle and the resting pool. Good clinical response was also found to be associated with low levels of cAMP in leukemic cells prior to therapy, whereas high cAMP contents predicted failure. Low cAMP concentrations, however, did not necessarily correlate with short turnover times and vice versa. This might be due to fluctuations of the cAMP concentrations during the cell cycle.

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

    Science.gov (United States)

    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.

  12. Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in Vibrio vulnificus.

    Science.gov (United States)

    Kim, Choon-Mee; Kim, Seong-Jung; Shin, Sung-Heui

    2012-04-01

    The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.

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

    Directory of Open Access Journals (Sweden)

    Ching-Ting Lin

    Full Text Available 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

  14. Dynamic fluctuations provide the basis of a conformational switch mechanism in apo cyclic AMP receptor protein.

    Directory of Open Access Journals (Sweden)

    Burcu Aykaç Fas

    Full Text Available Escherichia coli cyclic AMP Receptor Protein (CRP undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD simulations and Gaussian Network Model (GNM. The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

  15. Localized cyclic AMP-dependent protein kinase activity is required for myogenic cell fusion

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-01

    Multinucleated myotubes are formed by fusion of mononucleated myogenic progenitor cells (myoblasts) during terminal skeletal muscle differentiation. In addition, myoblasts fuse with myotubes, but terminally differentiated myotubes have not been shown to fuse with each other. We show here that an adenylate cyclase activator, forskolin, and other reagents that elevate intracellular cyclic AMP (cAMP) levels induced cell fusion between small bipolar myotubes in vitro. Then an extra-large myotube, designated a 'myosheet,' was produced by both primary and established mouse myogenic cells. Myotube-to-myotube fusion always occurred between the leading edge of lamellipodia at the polar end of one myotube and the lateral plasma membrane of the other. Forskolin enhanced the formation of lamellipodia where cAMP-dependent protein kinase (PKA) was accumulated. Blocking enzymatic activity or anchoring of PKA suppressed forskolin-enhanced lamellipodium formation and prevented fusion of multinucleated myotubes. Localized PKA activity was also required for fusion of mononucleated myoblasts. The present results suggest that localized PKA plays a pivotal role in the early steps of myogenic cell fusion, such as cell-to-cell contact/recognition through lamellipodium formation. Furthermore, the localized cAMP-PKA pathway might be involved in the specification of the fusion-competent areas of the plasma membrane in lamellipodia of myogenic cells

  16. Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP-cAMP Receptor Protein Signaling System.

    Directory of Open Access Journals (Sweden)

    M Shamim Hasan Zahid

    Full Text Available Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7th cholera pandemic. The expression of cholera toxin (CT and toxin coregulated pilus (TCP, the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP-cAMP receptor protein (CRP is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.

  17. Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System.

    Science.gov (United States)

    Zahid, M Shamim Hasan; Awasthi, Sharda Prasad; Asakura, Masahiro; Chatterjee, Shruti; Hinenoya, Atsushi; Faruque, Shah M; Yamasaki, Shinji

    2015-01-01

    Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.

  18. Cyclic GMP-AMP as an Endogenous Second Messenger in Innate Immune Signaling by Cytosolic DNA.

    Science.gov (United States)

    Kato, Kazuki; Omura, Hiroki; Ishitani, Ryuichiro; Nureki, Osamu

    2017-06-20

    The innate immune system functions as the first line of defense against invading bacteria and viruses. In this context, the cGAS/STING [cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase/STING] signaling axis perceives the nonself DNA associated with bacterial and viral infections, as well as the leakage of self DNA by cellular dysfunction and stresses, to elicit the host's immune responses. In this pathway, the noncanonical cyclic dinucleotide 2',3'-cyclic GMP-AMP (2',3'-cGAMP) functions as a second messenger for signal transduction: 2',3'-cGAMP is produced by the enzyme cGAS upon its recognition of double-stranded DNA, and then the 2',3'-cGAMP is recognized by the receptor STING to induce the phosphorylation of downstream factors, including TBK1 (TANK binding kinase 1) and IRF3 (interferon regulatory factor 3). Numerous crystal structures of the components of this cGAS/STING signaling axis have been reported and these clarify the structural basis for their signal transduction mechanisms. In this review, we summarize recent progress made in the structural dissection of this signaling pathway and indicate possible directions of forthcoming research.

  19. Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

    Science.gov (United States)

    Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

    2017-01-27

    The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. A Novel Indirect Sequence Readout Component in the E. coli Cyclic AMP Receptor Protein Operator

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter Eigil; Valentin-Hansen, Poul

    2014-01-01

    binding sites in the E. coli genome, but the exact role of the N6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N6 spacer region to demonstrate that CRP binding to the lacP1 site may be enhanced up to 14-fold......The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid...

  1. Mechanical control of cyclic AMP signalling and gene transcription through integrins

    Science.gov (United States)

    Meyer, C. J.; Alenghat, F. J.; Rim, P.; Fong, J. H.; Fabry, B.; Ingber, D. E.

    2000-01-01

    This study was carried out to discriminate between two alternative hypotheses as to how cells sense mechanical forces and transduce them into changes in gene transcription. Do cells sense mechanical signals through generalized membrane distortion or through specific transmembrane receptors, such as integrins? Here we show that mechanical stresses applied to the cell surface alter the cyclic AMP signalling cascade and downstream gene transcription by modulating local release of signals generated by activated integrin receptors in a G-protein-dependent manner, whereas distortion of integrins in the absence of receptor occupancy has no effect.

  2. Region-selective effects of long-term lithium and carbamazepine administration on cyclic AMP levels in rat brain

    International Nuclear Information System (INIS)

    Wiborg, Ove; Krueger, Tanja; Jakosen, Soeren N.

    1999-01-01

    The effect of lithium and carbamazepine in the treatment of bipolar affective disorder is well established. Althougt a number of biochemical effects have been found, the exact molecular mechanisms underlying their therapeutic actions have not been elucidated nor are the target regions in the brain identified. Taken into account the important role of the cyclic AMP second messenger system in the regulation of neuronal exitability and the indications of its involvement in the pathophysiology of bipolar affective disorder, we have focused on the drug effects on cyclic AMP levels. The objectives of this investigation were to measure the effects on basal cyclic AMP levels, and to locate target regions within the rat brain after long-term administration of lithium and carbamazepine. Drug treatments were carried out for a period of 28 days. After either drug treatment the cyclic AMP level was increased 3-4 times in frontal cortex but unchanged in hippocampus, hypothalamus, thalamus, amygdala and in cerebellum. In neostratum the cyclic AMP level was decreased to about 30% after treatment with lithium. We suggest the common region-selective effect, observed for both drugs in frontal cortex, to be essential for the therapeutic actions of lithium and carbamazepine. (au)

  3. Region-selective effects of long-term lithium and carbamazepine administration on cyclic AMP levels in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Wiborg, Ove; Krueger, Tanja; Jakosen, Soeren N. [Psychiatric Hospital, Dept. of Biological Psychiatry, Risskov (Denmark)

    1999-02-01

    The effect of lithium and carbamazepine in the treatment of bipolar affective disorder is well established. Althougt a number of biochemical effects have been found, the exact molecular mechanisms underlying their therapeutic actions have not been elucidated nor are the target regions in the brain identified. Taken into account the important role of the cyclic AMP second messenger system in the regulation of neuronal exitability and the indications of its involvement in the pathophysiology of bipolar affective disorder, we have focused on the drug effects on cyclic AMP levels. The objectives of this investigation were to measure the effects on basal cyclic AMP levels, and to locate target regions within the rat brain after long-term administration of lithium and carbamazepine. Drug treatments were carried out for a period of 28 days. After either drug treatment the cyclic AMP level was increased 3-4 times in frontal cortex but unchanged in hippocampus, hypothalamus, thalamus, amygdala and in cerebellum. In neostratum the cyclic AMP level was decreased to about 30% after treatment with lithium. We suggest the common region-selective effect, observed for both drugs in frontal cortex, to be essential for the therapeutic actions of lithium and carbamazepine. (au) 46 refs.

  4. A simple electrostatic switch important in the activation of type I protein kinase A by cyclic AMP.

    Science.gov (United States)

    Vigil, Dominico; Lin, Jung-Hsin; Sotriffer, Christoph A; Pennypacker, Juniper K; McCammon, J Andrew; Taylor, Susan S

    2006-01-01

    Cyclic AMP activates protein kinase A by binding to an inhibitory regulatory (R) subunit and releasing inhibition of the catalytic (C) subunit. Even though crystal structures of regulatory and catalytic subunits have been solved, the precise molecular mechanism by which cyclic AMP activates the kinase remains unknown. The dynamic properties of the cAMP binding domain in the absence of cAMP or C-subunit are also unknown. Here we report molecular-dynamics simulations and mutational studies of the RIalpha R-subunit that identify the C-helix as a highly dynamic switch which relays cAMP binding to the helical C-subunit binding regions. Furthermore, we identify an important salt bridge which links cAMP binding directly to the C-helix that is necessary for normal activation. Additional mutations show that a hydrophobic "hinge" region is not as critical for the cross-talk in PKA as it is in the homologous EPAC protein, illustrating how cAMP can control diverse functions using the evolutionarily conserved cAMP-binding domains.

  5. Salivary gland ultrastructure and cyclic AMP-dependent reactions in Spacelab 3 rats

    International Nuclear Information System (INIS)

    Mednieks, M.I.; Hand, A.R.

    1987-01-01

    Environmental stimuli influencing catecholamine levels induce changes in cyclic AMP-dependent reactions and cell morphology in the rat parotid. Responses of salivary glands to spaceflight were determined by measurement of cyclic AMP-mediated reactions in fresh-frozen salivary glands and by microscopic evaluation of ultrastructure in fixed parotid glands. Decreased cell-free protein phosphorylation, determined by autoradiography, occurred in parotid glands in three of five flight animals. Protein kinase activity ratios were decreased in the soluble and increased in the particulate fractions of Spacelab 3 (SL-3) rat sublingual glands, compared with ground controls. Biochemical analyses show that effects of space flight on salivary glands are similar to those induced experimentally by physiological manipulation or alteration of catecholamine levels. Morphological evaluation of three SL-3 rat parotid glands showed increased numbers of lysosomes, autophagic vacuoles containing degenerating secretory product, and accumulation of lipid droplets. Since these animals lost weight, consistent with disruption of food and water consumption, morphological changes may in part be due to decreased masticatory stimulation, as occurs with reduced food intake or a liquid diet. The observed changes may reflect physiological responses of the gastrointestinal and autonomic systems to effects of spaceflight

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

    Science.gov (United States)

    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

  7. The enzymatic preparation of [α-32P]nucleoside triphosphates, cyclic [32P]AMP, and cyclic [32P]GMP

    International Nuclear Information System (INIS)

    Walseth, T.F.; Johnson, R.A.

    1979-01-01

    A method has been developed for the enzymatic preparation of α- 32 P-labelled ribo- and deoxyribonucleoside triphosphates, cyclic [ 32 P]AMP, and cyclic [ 32 P]GMP of high specific radioactivity and in high yield from 32 Psub(i). The method also enables the preparation of [γ- 32 P]ATP, [γ- 32 P]GTP, [γ- 32 P]ITP, and [γ- 32 P]-dATP of very high specific activity and in high yield. (Auth.)

  8. Cyclic AMP-specific phosphodiesterase-4 as a target for the development of antidepressant drugs.

    Science.gov (United States)

    Zhang, Han-Ting

    2009-01-01

    Phosphodiesterase-4 (PDE4), one of eleven PDE enzyme families, specifically catalyzes hydrolysis of cyclic AMP (cAMP); it has four subtypes (PDE4A-D) with at least 25 splice variants. PDE4 plays a critical role in the control of intracellular cAMP concentrations. PDE4 inhibitors produce antidepressant actions in both animals and humans via enhancement of cAMP signaling in the brain. However, their clinical utility has been hampered by side effects, in particular nausea and emesis. While there is still a long way to go before PDE4 inhibitors with high therapeutic indices are available for treatment of depressive disorders, important advances have been made in the development of PDE4 inhibitors as antidepressants. First, limited, but significant studies point to PDE4D as the major PDE4 subtype responsible for antidepressant-like effects of PDE4 inhibitors, although the role of PDE4A cannot be excluded. Second, PDE4D may contribute to emesis, the major side effect of PDE4 inhibitors. For this reason, identification of roles of PDE4D splice variants in mediating antidepressant activity is particularly important. Recent studies using small interfering RNAs (siRNAs) have demonstrated the feasibility to identify cellular functions of individual PDE4 variants. Third, mixed inhibitors of PDE4 and PDE7 or PDE4 and serotonin reuptake have been developed and may be potential antidepressants with minimized side effects. Finally, relatively selective inhibitors of one or two PDE4 subtypes have been synthesized using structure- and scaffold-based design. This review also discusses the relationship between PDE4 and antidepressant activity based on structures, brain distributions, and pharmacological properties of PDE4 and its isoforms.

  9. Effects of exogenous cyclic AMP on growth characteristics and radiation response of Reuber H35 hepatoma cells

    International Nuclear Information System (INIS)

    van Rijn, J.; van Den Berg, J.; van Meeteren, A.; van Wijk, R.

    1983-01-01

    Reuber H35 rat hepatoma cells, clone KRC, were used to study the effect of cyclic AMP on radiation-induced cell death. Treatment of logarithmically growing cultures with 0.5 mM cAMP for 17 hr prior to irradiation resulted in a decreased cell survival. Similar results were obtained with cultures irradiated after treatment with Bt 2 cAMP. Treatment of H35 cells with cAMP or Bt 2 cAMP caused inhibition of their proliferation and resulted in an accumulation of cells in early S phase and depletion of G2-phase cells. In synchronized cultures cells were relatively radioresistant during their S phase. In addition to single-dose treatment with X rays, the effect of Bt 2 cAMP on radiation-induced cell death was studied during fractionated irradiation wtih 2.5 Gy per day. This fractionated irradiation resulted in a dose-reduction factor of 1.6 at the 10% survival level and a 10-fold decrease in the surviving cell population due to the cooperative effects of Bt 2 cAMP on growth rate and radiation survival. The effect of cAMP on radiation-induced mitotic delay was also studied. It appeared that where cAMP had on effect on the progression of G2 cells into mitosis, it prevented cells from recovery from the X-ray mitotic delay in G2

  10. Structural Basis for the Catalytic Mechanism of DncV, Bacterial Homolog of Cyclic GMP-AMP Synthase.

    Science.gov (United States)

    Kato, Kazuki; Ishii, Ryohei; Hirano, Seiichi; Ishitani, Ryuichiro; Nureki, Osamu

    2015-05-05

    Cyclic dinucleotides (CDNs) play key roles as second messengers and signaling molecules in bacteria and metazoans. The newly identified dinucleotide cyclase in Vibrio cholerae (DncV) produces three different CDNs containing two 3'-5' phosphodiester bonds, and its predominant product is cyclic GMP-AMP, whereas mammalian cyclic GMP-AMP synthase (cGAS) produces only cyclic GMP-AMP containing mixed 2'-5' phosphodiester bonds. We report the crystal structures of V. cholerae and Escherichia coli DncV in complex with various nucleotides in the pre-reaction states. The high-resolution structures revealed that DncV preferably recognizes ATP and GTP as acceptor and donor nucleotides, respectively, in the first nucleotidyl transfer reaction. Considering the recently reported intermediate structures, our pre-reaction state structures provide the precise mechanism of 3'-5' linked cyclic AMP-GMP production in bacteria. A comparison with cGAS in the pre-reaction states suggests that the orientation of the acceptor nucleotide primarily determines the distinct linkage specificities between DncV and cGAS. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Early effects of synthetic bovine parathyroid hormone and synthetic salmon calcitonin on urinary excretion of cyclic AMP, phosphate and calcium in man.

    Science.gov (United States)

    Caniggia, A; Gennari, C; Vattimo, A; Nardi, P; Nuti, R; Galli, M

    1976-04-20

    Bovine synthetic parathyroid hormone infused intravenously in man increased both the urinary excretion of cyclic AMP and the urinary excretion of phosphate whereas a Salmon synthetic calcitonin infusion increased the urinary excretion of phosphate without change in urinary excretion of cyclic AMP. These data are consistent with the hypothesis that different renal mechanisms are involved in the response to each hormone.

  12. An AP-2 element acts synergistically with the cyclic AMP- and Phorbol ester-inducible enhancer of the human proenkephalin gene

    Energy Technology Data Exchange (ETDEWEB)

    Hyman, S.E.; Comb, M.; Pearlberg, J.; Goodman, H.M.

    1989-01-01

    An enhancer with two DNA elements, one containing the sequence CGTCA, is required for cyclic AMP-and phorbol ester-inducible transcription of the human proenkephalin gene. The authors report that an AP-2 element located adjacent to the enhancer acts synergistically with it to confer maximal response to cyclic AMP and phorbol esters.

  13. Angiotensin II potentiates prostaglandin stimulation of cyclic AMP levels in intact bovine adrenal medulla cells but not adenylate cyclase in permeabilized cells.

    Science.gov (United States)

    Boarder, M R; Plevin, R; Marriott, D B

    1988-10-25

    The level of cyclic AMP in primary cultures of bovine adrenal medulla cells is elevated by prostaglandin E1. Angiotensin II is commonly reported to act on receptors linked to phosphoinositide metabolism or to inhibition of adenylate cyclase. We have investigated the effect of angiotensin II on prostaglandin E1-stimulated cyclic AMP levels in these primary cultures. Rather than reducing cyclic AMP levels, we have found that angiotensin II powerfully potentiates prostaglandin E1-stimulated cyclic AMP accumulation in intact cells, both in the presence and absence of phosphodiesterase inhibitors. The 50% maximal response was similar to that for stimulation of phosphoinositide breakdown by angiotensin II in these cultures. The potentiation of stimulated cyclic AMP levels was seen, although to a smaller maximum, with the protein kinase C (Ca2+/phospholipid-dependent enzyme) activating phorbol ester tetradecanoyl phorbolacetate and with the synthetic diacylglycerol 1-oleoyl-2-acetylglycerol; pretreatment (24 h) with active phorbol ester, which would be expected to diminish protein kinase C levels, attenuated the angiotensin II potentiation of cyclic AMP. Using digitonin-permeabilized cells we showed that adenylate cyclase activity was stimulated by prostaglandin E1 with the same dose-response relationship as was cyclic AMP accumulation in intact cells, but the permeabilized cells showed no response to angiotensin II. The results are discussed with respect to the hypothesis that the angiotensin II influence on cyclic AMP levels is mediated, in part, by diacylglycerol stimulation of protein kinase C.

  14. Theoretical Analysis of Allosteric and Operator Binding for Cyclic-AMP Receptor Protein Mutants

    Science.gov (United States)

    Einav, Tal; Duque, Julia; Phillips, Rob

    2018-02-01

    Allosteric transcription factors undergo binding events both at their inducer binding sites as well as at distinct DNA binding domains, and it is often difficult to disentangle the structural and functional consequences of these two classes of interactions. In this work, we compare the ability of two statistical mechanical models - the Monod-Wyman-Changeux (MWC) and the Koshland-N\\'emethy-Filmer (KNF) models of protein conformational change - to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. In light of these models, we examine data from a beautiful recent experiment that created a single-chain version of the CRP homodimer, thereby enabling each subunit to be mutated separately. Using this construct, six mutants were created using all possible combinations of the wild type subunit, a D53H mutant subunit, and an S62F mutant subunit. We demonstrate that both the MWC and KNF models can explain the behavior of all six mutants using a small, self-consistent set of parameters. In comparing the results, we find that the MWC model slightly outperforms the KNF model in the quality of its fits, but more importantly the parameters inferred by the MWC model are more in line with structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.

  15. Dibutyryl cyclic AMP induces differentiation of human neuroblastoma SH-SY5Y cells into a noradrenergic phenotype.

    Science.gov (United States)

    Kume, Toshiaki; Kawato, Yuka; Osakada, Fumitaka; Izumi, Yasuhiko; Katsuki, Hiroshi; Nakagawa, Takayuki; Kaneko, Shuji; Niidome, Tetsuhiro; Takada-Takatori, Yuki; Akaike, Akinori

    2008-10-10

    Dibutyryl cyclic AMP (dbcAMP) and retinoic acid (RA) have been demonstrated to be the inducers of morphological differentiation in SH-SY5Y cells, a human catecholaminergic neuroblastoma cell line. However, it remains unclear whether morphologically differentiated SH-SY5Y cells by these compounds acquire catecholaminergic properties. We focused on the alteration of tyrosine hydroxylase (TH) expression and intracellular content of noradrenaline (NA) as the indicators of functional differentiation. Three days treatment with dbcAMP (1mM) and RA (10microM) induced morphological changes and an increase of TH-positive cells using immunocytochemical analysis in SH-SY5Y cells. The percentage of TH-expressing cells in dbcAMP (1mM) treatment was larger than that in RA (10microM) treatment. In addition, dbcAMP increased intracellular NA content, whereas RA did not. The dbcAMP-induced increase in TH-expressing cells is partially inhibited by KT5720, a protein kinase A (PKA) inhibitor. We also investigated the effect of butyrate on SH-SY5Y cells, because dbcAMP is enzymatically degraded by intracellular esterase, thereby resulting in the formation of butyrate. Butyrate induced the increase of NA content at lower concentrations than dbcAMP, although the increase in TH-expressing cells by butyrate was smaller than that by dbcAMP. The dbcAMP (1mM)- and butyrate (0.3mM)-induced increase in NA content was completely suppressed by alpha-methyl-p-tyrosine (1mM), an inhibitor of TH. These results suggest that dbcAMP induces differentiation into the noradrenergic phenotype through both PKA activation and butyrate.

  16. Essential role for cyclic-AMP responsive element binding protein 1 (CREB) in the survival of acute lymphoblastic leukemia

    NARCIS (Netherlands)

    van der Sligte, Naomi E.; Kampen, Kim R.; ter Elst, Arja; Scherpen, Frank J. G.; Meeuwsen-de Boer, Tiny G. J.; Guryev, Victor; van Leeuwen, Frank N.; Kornblau, Steven M.; de Bont, Eveline S. J. M.

    2015-01-01

    Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric

  17. The effects of histamine and prostaglandin D2 on rat mast-cell cyclic AMP and mediator release

    International Nuclear Information System (INIS)

    Wescott, S.; Kaliner, M.

    1981-01-01

    The possibility that histamine may play a functional role in modulating mast-cell secretion, as has been suggested for basophil degranulation, has both physiologic and pharmacologic implications. Therefore the capacity of histamine to influence rat peritoneal mast-cell (RPMC) cyclic AMP levels and reversed anaphylatic degranulation as reflected in the release of 3H-serotonin (5-HT) was examined. To ascertain that RPMC were functionally responsive to exogenous hormonal stimulation, assessment of prostaglandin (PG) D2 effects on cyclic AMP and 5-HT release were determined in parallel. Although PGD2 (100 microM) increased cyclic AMP and inhibited 5-HT release in the presence of 50 microM aminophylline, histamine (up to 1000 microM) was ineffective was ineffective in both. However, 1000 microM histamine in the presence of 500 microM aminophylline was capable of transiently increasing RPMC cyclic AMP (for 15 to 30 sec) and under these conditions of suppressing 5-HT release. The receptor subtype involved in the suppressive actions of histamine appeared to be of the H-1 type as reflected in the capacity of specific H-1 agonists to reproduce the inhibition of 5-HT release, whereas neither H-2 agonists nor H-2 antagonists had any influence. Thus, under conditions in which phosphodiesterase enzymatic action is impaired, histamine in extremely high concentrations is able to modulate mast-cell secretion. However, it seems very unlikely that this action of histamine has any physiologic significance

  18. Cyclic GMP-AMP synthase is activated by double-stranded DNA-induced oligomerization.

    Science.gov (United States)

    Li, Xin; Shu, Chang; Yi, Guanghui; Chaton, Catherine T; Shelton, Catherine L; Diao, Jiasheng; Zuo, Xiaobing; Kao, C Cheng; Herr, Andrew B; Li, Pingwei

    2013-12-12

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor mediating innate antimicrobial immunity. It catalyzes the synthesis of a noncanonical cyclic dinucleotide, 2',5' cGAMP, that binds to STING and mediates the activation of TBK1 and IRF-3. Activated IRF-3 translocates to the nucleus and initiates the transcription of the IFN-β gene. The structure of mouse cGAS bound to an 18 bp dsDNA revealed that cGAS interacts with dsDNA through two binding sites, forming a 2:2 complex. Enzyme assays and IFN-β reporter assays of cGAS mutants demonstrated that interactions at both DNA binding sites are essential for cGAS activation. Mutagenesis and DNA binding studies showed that the two sites bind dsDNA cooperatively and that site B plays a critical role in DNA binding. The structure of mouse cGAS bound to dsDNA and 2',5' cGAMP provided insight into the catalytic mechanism of cGAS. These results demonstrated that cGAS is activated by dsDNA-induced oligomerization. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Cooperative DNA binding of heterologous proteins: Evidence for contact between the cyclic AMP receptor protein and RNA polymerase

    International Nuclear Information System (INIS)

    Ren, Y.L.; Garges, S.; Adhya, S.; Krakow, J.S.

    1988-01-01

    Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 can activate lacP + -directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection (footprinting) indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the [ 32 P]lacP + fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding

  20. Activation of PKA and Epac proteins by cyclic AMP depletes intracellular calcium stores and reduces calcium availability for vasoconstriction.

    Science.gov (United States)

    Cuíñas, Andrea; García-Morales, Verónica; Viña, Dolores; Gil-Longo, José; Campos-Toimil, Manuel

    2016-06-15

    We investigated the implication of PKA and Epac proteins in the endothelium-independent vasorelaxant effects of cyclic AMP (cAMP). Cytosolic Ca(2+) concentration ([Ca(2+)]c) was measured by fura-2 imaging in rat aortic smooth muscle cells (RASMC). Contraction-relaxation experiments were performed in rat aortic rings deprived of endothelium. In extracellular Ca(2+)-free solution, cAMP-elevating agents induced an increase in [Ca(2+)]c in RASMC that was reproduced by PKA and Epac activation and reduced after depletion of intracellular Ca(2+) reservoirs. Arginine-vasopressin (AVP)-evoked increase of [Ca(2+)]c and store-operated Ca(2+) entry (SOCE) were inhibited by cAMP-elevating agents, PKA or Epac activation in these cells. In aortic rings, the contractions induced by phenylephrine in absence of extracellular Ca(2+) were inhibited by cAMP-elevating agents, PKA or Epac activation. In these conditions, reintroduction of Ca(2+) induced a contraction that was inhibited by cAMP-elevating agents, an effect reduced by PKA inhibition and reproduced by PKA or Epac activators. Our results suggest that increased cAMP depletes intracellular, thapsigargin-sensitive Ca(2+) stores through activation of PKA and Epac in RASMC, thus reducing the amount of Ca(2+) released by IP3-generating agonists during the contraction of rat aorta. cAMP rise also inhibits the contraction induced by depletion of intracellular Ca(2+), an effect mediated by reduction of SOCE after PKA or Epac activation. Both effects participate in the cAMP-induced endothelium-independent vasorelaxation. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Expression of Cyclic GMP-AMP Synthase in Patients With Systemic Lupus Erythematosus.

    Science.gov (United States)

    An, Jie; Durcan, Laura; Karr, Reynold M; Briggs, Tracy A; Rice, Gillian I; Teal, Thomas H; Woodward, Joshua J; Elkon, Keith B

    2017-04-01

    Type I interferon (IFN) is implicated in the pathogenesis of systemic lupus erythematosus (SLE) and interferonopathies such as Aicardi-Goutières syndrome. A recently discovered DNA-activated type I IFN pathway, cyclic GMP-AMP synthase (cGAS), has been linked to Aicardi-Goutières syndrome and mouse models of lupus. The aim of this study was to determine whether the cGAS pathway contributes to type I IFN production in patients with SLE. SLE disease activity was measured by the Safety of Estrogens in Lupus Erythematosus National Assessment version of the Systemic Lupus Erythematosus Disease Activity Index. Expression of messenger RNA for cGAS and IFN-stimulated genes (ISGs) was determined by quantitative polymerase chain reaction analysis. Cyclic GMP-AMP (cGAMP) levels were examined by multiple reaction monitoring with ultra-performance liquid chromatography tandem mass spectrometry. Expression of cGAS in peripheral blood mononuclear cells (PBMCs) was significantly higher in SLE patients than in normal controls (n = 51 and n = 20 respectively; P < 0.01). There was a positive correlation between cGAS expression and the IFN score (P < 0.001). The expression of cGAS in PBMCs showed a dose response to type I IFN stimulation in vitro, consistent with it being an ISG. Targeted measurement of cGAMP by tandem mass spectrometry detected cGAMP in 15% of the SLE patients (7 of 48) but none of the normal (0 of 19) or rheumatoid arthritis (0 of 22) controls. Disease activity was higher in SLE patients with cGAMP versus those without cGAMP. Increased cGAS expression and cGAMP in a proportion of SLE patients indicates that the cGAS pathway should be considered as a contributor to type I IFN production. Whereas higher cGAS expression may be a consequence of exposure to type I IFN, detection of cGAMP in patients with increased disease activity indicates potential involvement of this pathway in disease expression. © 2016, American College of Rheumatology.

  2. Cyclic AMP-specific phosphodiesterase, PDE8A1, is activated by protein kinase A-mediated phosphorylation

    OpenAIRE

    Brown, Kim M.; Lee, Louisa C.Y; Findlay, Jane E.; Day, Jonathan P.; Baillie, George S.

    2012-01-01

    The cyclic AMP-specific phosphodiesterase PDE8 has been shown to play a pivotal role in important processes such as steroidogenesis, T cell adhesion, regulation of heart beat and chemotaxis. However, no information exists on how the activity of this enzyme is regulated. We show that under elevated cAMP conditions, PKA acts to phosphorylate PDE8A on serine 359 and this action serves to enhance the activity of the enzyme. This is the first indication that PDE8 activity can be modulated by a kin...

  3. Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment.

    Science.gov (United States)

    Girada, Shravan Babu; Kuna, Ramya S; Bele, Shilpak; Zhu, Zhimeng; Chakravarthi, N R; DiMarchi, Richard D; Mitra, Prasenjit

    2017-10-01

    Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein-protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  4. Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

    DEFF Research Database (Denmark)

    Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller

    2008-01-01

    AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho......-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of c......AMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response....

  5. Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses

    Directory of Open Access Journals (Sweden)

    Geneviève Pèépin

    2017-10-01

    Full Text Available Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1 by low-dose camptothecin (CPT can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40 large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development.

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

    Science.gov (United States)

    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.

  7. Maintenance of cyclic GMP-AMP homeostasis by ENPP1 is involved in pseudorabies virus infection.

    Science.gov (United States)

    Wang, Jiang; Lu, Shao-Fang; Wan, Bo; Ming, Sheng-Li; Li, Guo-Li; Su, Bing-Qian; Liu, Jiao-Yang; Wei, Yu-Shuang; Yang, Guo-Yu; Chu, Bei-Bei

    2018-03-01

    In a previous study, we demonstrated that porcine cyclic GMP-AMP (cGAMP) synthase (cGAS) catalyzes cGAMP production and is an important DNA sensor for the pseudorabies virus (PRV)-induced activation of interferon β (IFN-β). Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) has recently been identified as the hydrolase of cGAMP in rodents, but its role in porcine cells is not clear. Our recent study demonstrated that porcine ENPP1 is responsible for the homeostasis of cGAMP and is critical for PRV infection. Porcine ENPP1 mRNA is predominantly expressed in muscle. PRV infection was enhanced by ENPP1 overexpression and attenuated by silencing of ENPP1. During PRV infection, the activation of IFN-β and NF-κB was reduced in ENPP1 overexpressed cells and promoted in ENPP1 knockdown cells. Investigation of the molecular mechanisms of ENPP1 during PRV infection showed that ENPP1 hydrolyzed cGAMP in PRV-infected or cGAMP-transfected cells and inhibited IRF3 phosphorylation, reducing IFN-β secretion. These results, combined with those for porcine cGAS, demonstrate that ENPP1 acts coordinately with cGAS to maintain the reservoir of cGAMP and participates in PRV infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. The reduction of radiation-induced mitotic delay by caffeine: a test of the cyclic AMP hypothesis

    International Nuclear Information System (INIS)

    Oleinick, N.L.; Brewer, E.N.; Rustad, R.C.

    1978-01-01

    A study has been made of the reduction in γ-radiation-induced mitotic delay by caffeine in the naturally-synchronous plasmodial slime mould. Physarum polycephalum during late G 2 and early prophase, and the results compared with those obtained with other compounds of similar structure and/or physiological function. The reduction of radiation-induced mitotic delay was related to increasing concentrations of caffeine over at least two orders of magnitude. Pre-irradiation treatment with caffeine had no detectable effect. Caffeine had to be present for most, if not all, of the post-irradiation pre-mitotic period. Other chemicals which are reported to inhibit cyclic AMP phosphodiesterase either reduce or increase radiation-induced mitotic delay. The results therefore indicate that the reduction of mitotic delay by caffeine is not a result of altered cyclic AMP levels. (UK)

  9. Control of Saccharomyces cerevisiae catalase T gene (CTT1) expression by nutrient supply via the RAS-cyclic AMP pathway.

    Science.gov (United States)

    Bissinger, P H; Wieser, R; Hamilton, B; Ruis, H

    1989-03-01

    In Saccharomyces cerevisiae, lack of nutrients triggers a pleiotropic response characterized by accumulation of storage carbohydrates, early G1 arrest, and sporulation of a/alpha diploids. This response is thought to be mediated by RAS proteins, adenylate cyclase, and cyclic AMP (cAMP)-dependent protein kinases. This study shows that expression of the S. cerevisiae gene coding for a cytoplasmic catalase T (CTT1) is controlled by this pathway: it is regulated by the availability of nutrients. Lack of a nitrogen, sulfur, or phosphorus source causes a high-level expression of the gene. Studies with strains with mutations in the RAS-cAMP pathway and supplementation of a rca1 mutant with cAMP show that CTT1 expression is under negative control by a cAMP-dependent protein kinase and that nutrient control of CTT1 gene expression is mediated by this pathway. Strains containing a CTT1-Escherichia coli lacZ fusion gene have been used to isolate mutants with mutations in the pathway. Mutants characterized in this investigation fall into five complementation groups. Both cdc25 and ras2 alleles were identified among these mutants.

  10. Reversal of propranolol blockade of adrenergic receptors and related toxicity with drugs that increase cyclic AMP.

    Science.gov (United States)

    Whitehurst, V E; Vick, J A; Alleva, F R; Zhang, J; Joseph, X; Balazs, T

    1999-09-01

    An overdose of propranolol, a widely used nonselective beta-adrenergic receptor blocking agent, can result in hypotension and bradycardia leading to irreversible shock and death. In addition, the blockade of adrenergic receptors can lead to alterations in neurotransmitter receptors resulting in the interruption of the activity of other second messengers and the ultimate cellular responses. In the present experiment, three agents, aminophylline, amrinone, and forskolin were tested in an attempt to reverse the potential lethal effects of a propranolol overdose in dogs. Twenty-two anesthetized beagle dogs were given a 10-min infusion of propranolol at a dose of 1 mg/kg/min. Six of the dogs, treated only with intravenous saline, served as controls. Within 15-30 min all six control dogs exhibited profound hypotension and severe bradycardia that led to cardiogenic shock and death. Seven dogs were treated with intravenous aminophylline 20 mg/kg 5 min after the end of the propranolol infusion. Within 10-15 min heart rate and systemic arterial blood pressure returned to near control levels, and all seven dogs survived. Intravenous amrinone (2-3 mg/kg) given to five dogs, and forskolin (1-2 mg/kg) given to four dogs, also increased heart rate and systemic arterial blood pressure but the recovery of these parameters was appreciably slower than that seen with aminophylline. All of these animals also survived with no apparent adverse effects. Histopathologic evaluation of the hearts of the dogs treated with aminophylline showed less damage (vacuolization, inflammation, hemorrhage) than the hearts from animals given propranolol alone. Results of this study showed that these three drugs, all of which increase cyclic AMP, are capable of reversing the otherwise lethal effects of a propranolol overdose in dogs.

  11. Cyclic AMP Receptor Protein Acts as a Transcription Regulator in Response to Stresses in Deinococcus radiodurans.

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    Su Yang

    Full Text Available The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2 treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998, Lon proteases (dr0349 and dr1974, NADH-quinone oxidoreductase (dr1506, thiosulfate sulfurtransferase (dr2531, the DNA repair protein UvsE (dr1819, PprA (dra0346, and RecN (dr1447, are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways.

  12. Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses.

    Science.gov (United States)

    Pépin, Geneviève; Nejad, Charlotte; Ferrand, Jonathan; Thomas, Belinda J; Stunden, H James; Sanij, Elaine; Foo, Chwan-Hong; Stewart, Cameron R; Cain, Jason E; Bardin, Philip G; Williams, Bryan R G; Gantier, Michael P

    2017-10-03

    Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through cyclic GMP-AMP synthase (cGAS) detection of cytoplasmic DNA. This argues against CPT having only anti-inflammatory activity. Furthermore, expression of the simian virus 40 (SV40) large T antigen was paramount to the proinflammatory antiviral activity of CPT, as it potentiated cytoplasmic DNA leakage and subsequent cGAS recruitment in human and mouse cell lines. This work suggests that the capacity of Top1 inhibitors to blunt inflammatory responses can be counteracted by viral oncogenes and that this should be taken into account for their therapeutic development. IMPORTANCE Recent studies suggest that low-dose DNA-damaging compounds traditionally used in cancer therapy can have opposite effects on antiviral responses, either suppressing (with the example of CPT) or potentiating (with the example of doxorubicin) them. Our work demonstrates that the minor DNA damage promoted by low-dose CPT can also trigger strong antiviral responses, dependent on the presence of viral oncogenes. Taken together, these results call for caution in the therapeutic use of low-dose chemotherapy agents to modulate antiviral responses in humans. Copyright © 2017 Pépin et al.

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

    Science.gov (United States)

    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.

  14. Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia

    Energy Technology Data Exchange (ETDEWEB)

    Ohnishi, Masatoshi, E-mail: ohnishi@fupharm.fukuyama-u.ac.jp [Department of Pharmacotherapeutics, Graduate School of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Department of Pharmacotherapeutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Urasaki, Tomoka [Department of Pharmacotherapeutics, Graduate School of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Ochiai, Hiroyuki; Matsuoka, Kohei; Takeo, Shin; Harada, Tomoki; Ohsugi, Yoshihito [Department of Pharmacotherapeutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Inoue, Atsuko [Department of Pharmacotherapeutics, Graduate School of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan); Department of Pharmacotherapeutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292 (Japan)

    2015-11-13

    The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3′, 5’-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction. - Highlights: • Dibutyryl cAMP increased wnt4, but not wnt3a, 5a, 7a and 11, mRNA in mixed glia. • Wnt4 protein increased in astrocytes co-cultivated with microglia. • It took a long time to robustly increase wnt4 expression. • Rolipram

  15. Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia

    International Nuclear Information System (INIS)

    Ohnishi, Masatoshi; Urasaki, Tomoka; Ochiai, Hiroyuki; Matsuoka, Kohei; Takeo, Shin; Harada, Tomoki; Ohsugi, Yoshihito; Inoue, Atsuko

    2015-01-01

    The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3′, 5’-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), with dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction. - Highlights: • Dibutyryl cAMP increased wnt4, but not wnt3a, 5a, 7a and 11, mRNA in mixed glia. • Wnt4 protein increased in astrocytes co-cultivated with microglia. • It took a long time to robustly increase wnt4 expression. • Rolipram

  16. Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia

    Directory of Open Access Journals (Sweden)

    Kleberg Karen

    2012-06-01

    Full Text Available Abstract Background The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients with and without colorectal neoplasia. Methods Cyclic nucleotides were used as model substrates shared by some OATP- and ABC-transporters, which in part are responsible for clearance of metabolites and xenobiotics from the colonic epithelium. On colonic biopsies from patients with and without colorectal neoplasia, molecular transport was electrophysiologically registered in Ussing-chamber set-ups, mRNA level of selected transporters was quantified by rt-PCR, and subcellular location of transporters was determined by immunohistochemistry. Results Of four cyclic nucleotides, dibuturyl-cAMP induced the largest short circuit current in both patient groups. The induced short circuit current was significantly lower in neoplasia-patients (p = 0.024. The observed altered transport of dibuturyl-cAMP in neoplasia-patients could not be directly translated to an observed increased mRNA expression of OATP4A1 and OATP2B1 in neoplasia patients. All other examined transporters were expressed to similar extents in both patient groups. Conclusions OATP1C1, OATP4A1, OATP4C1 seem to be involved in the excretory system of human colon. ABCC4 is likely to be involved from an endoplasmic-Golgi complex and basolateral location in goblet cells. ABCC5 might be directly involved in the turnover of intracellular cAMP at the basolateral membrane of columnar epithelial cells, while OATP2B1 is indirectly related to the excretory system. Colorectal neoplasia is associated with lower transport or sensitivity to cyclic nucleotides and increased expression of OATP2B1 and OATP4A1 transporters, known to transport PGE2.

  17. Cyclic AMP signaling restricts activation and promotes maturation and antioxidant defenses in astrocytes.

    Science.gov (United States)

    Paco, Sonia; Hummel, Manuela; Plá, Virginia; Sumoy, Lauro; Aguado, Fernando

    2016-04-23

    cAMP signaling produces dramatic changes in astrocyte morphology and physiology. However, its involvement in phenotype acquisition and the transcriptionally mediated mechanisms of action are largely unknown. Here we analyzed the global transcriptome of cultured astroglial cells incubated with activators of cAMP pathways. A bulk of astroglial transcripts, 6221 annotated genes, were differentially regulated by cAMP signaling. cAMP analogs strongly upregulated genes involved in typical functions of mature astrocytes, such as homeostatic control, metabolic and structural support to neurons, antioxidant defense and communication, whereas they downregulated a considerable number of proliferating and immaturity-related transcripts. Moreover, numerous genes typically activated in reactive cells, such as scar components and immunological mediators, were repressed by cAMP. GSEA analysis contrasting gene expression profiles with transcriptome signatures of acutely isolated astrocytes and in situ evaluation of protein levels in these cells showed that cAMP signaling conferred mature and in vivo-like transcriptional features to cultured astrocytes. These results indicate that cAMP signaling is a key pathway promoting astrocyte maturation and restricting their developmental and activation features. Therefore, a positive modulation of cAMP signaling may promote the normal state of differentiated astrocytes and favor the protection and function of neuronal networks.

  18. Physiological and Molecular Effects of the Cyclic Nucleotides cAMP and cGMP on Arabidopsis thaliana

    KAUST Repository

    Herrera, Natalia M.

    2012-12-01

    The cyclic nucleotide monophosphates (CNs), cAMP and cGMP, are second messengers that participate in the regulation of development, metabolism and adaptive responses. In plants, CNs are associated with the control of pathogen responses, pollen tube orientation, abiotic stress response, membrane transport regulation, stomatal movement and light perception. In this study, we hypothesize that cAMP and cGMP promote changes in the transcription level of genes related to photosynthesis, high light and membrane transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time quantitative PCR was used to assess transcription levels of selected genes and infrared gas analyzers coupled to fluorescence sensors were used to measure the photosynthetic parameters. We present evidence that both cAMP and cGMP modulate foliar mRNA levels early after stimulation. The two CNs trigger different responses indicating that the signals have specificity. A comparison of proteomic and transcriptional changes suggest that both transcriptional and post-transcriptional mechanisms are modulated by CNs. cGMP up-regulates the mRNA levels of components of the photosynthesis and carbon metabolism. However, neither cAMP nor cGMP trigger differences in the rate of carbon assimilation, maximum efficiency of the photosystem II (PSII), or PSII operating efficiency. It was also demonstrated that CN regulate the expression of its own targets, the cyclic nucleotide gated channels - CNGC. Further studies are needed to identify the components of the signaling transduction pathway that mediate cellular changes and their respective regulatory and/or signaling roles.

  19. Cyclic AMP regulates the biosynthesis of cellobiohydrolase in Cellulomonas flavigena growing in sugar cane bagasse.

    Science.gov (United States)

    Herrera-Herrera, Jesús Antonio; Pérez-Avalos, Odilia; Salgado, Luis M; Ponce-Noyola, Teresa

    2009-10-01

    Cellulomonas flavigena produces a battery of cellulase components that act concertedly to degrade cellulose. The addition of cAMP to repressed C. flavigena cultures released catabolic repression, while addition of cAMP to induced C. flavigena cultures led to a cellobiohydrolase hyperproduction. Exogenous cAMP showed positive regulation on cellobiohydrolase production in C. flavigena grown on sugar cane bagasse. A C. flavigena cellobiohydrolase gene was cloned (named celA), which coded for a 71- kDa enzyme. Upstream, a repressor celR1, identified as a 38 kDa protein, was monitored by use of polyclonal antibodies.

  20. The DNA sensor, cyclic GMP-AMP synthase, is essential for induction of IFN-β during Chlamydia trachomatis infection.

    Science.gov (United States)

    Zhang, Yugen; Yeruva, Laxmi; Marinov, Anthony; Prantner, Daniel; Wyrick, Priscilla B; Lupashin, Vladimir; Nagarajan, Uma M

    2014-09-01

    IFN-β has been implicated as an effector of oviduct pathology resulting from genital chlamydial infection in the mouse model. In this study, we investigated the role of cytosolic DNA and engagement of DNA sensors in IFN-β expression during chlamydial infection. We determined that three-prime repair exonuclease-1, a host 3' to 5' exonuclease, reduced IFN-β expression significantly during chlamydial infection using small interfering RNA and gene knockout fibroblasts, implicating cytosolic DNA as a ligand for this response. The DNA sensor cyclic GMP-AMP synthase (cGAS) has been shown to bind cytosolic DNA to generate cyclic GMP-AMP, which binds to the signaling adaptor stimulator of IFN genes (STING) to induce IFN-β expression. We determined that cGAS is required for IFN-β expression during chlamydial infection in multiple cell types. Interestingly, although infected cells deficient for STING or cGAS alone failed to induce IFN-β, coculture of cells depleted for either STING or cGAS rescued IFN-β expression. These data demonstrate that cyclic GMP-AMP produced in infected cGAS(+)STING(-) cells can migrate into adjacent cells via gap junctions to function in trans in cGAS(-)STING(+) cells. Furthermore, we observed cGAS localized in punctate regions on the cytosolic side of the chlamydial inclusion membrane in association with STING, indicating that chlamydial DNA is most likely recognized outside the inclusion as infection progresses. These novel findings provide evidence that cGAS-mediated DNA sensing directs IFN-β expression during Chlamydia trachomatis infection and suggest that effectors from infected cells can directly upregulate IFN-β expression in adjacent uninfected cells during in vivo infection, contributing to pathogenesis. Copyright © 2014 by The American Association of Immunologists, Inc.

  1. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes

    OpenAIRE

    Wang, Yan; Su, Guo-Hua; Zhang, Fang; Chu, Jing-Xue; Wang, Yun-Shan

    2015-01-01

    Rheumatoid arthritis (RA) is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS) invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdow...

  2. Hepatitis C virus NS2 protein activates cellular cyclic AMP-dependent pathways

    International Nuclear Information System (INIS)

    Kim, Kyoung Mi; Kwon, Shi-Nae; Kang, Ju-Il; Lee, Song Hee; Jang, Sung Key; Ahn, Byung-Yoon; Kim, Yoon Ki

    2007-01-01

    Chronic infection of the hepatitis C virus (HCV) leads to liver cirrhosis and cancer. The mechanism leading to viral persistence and hepatocellular carcinoma, however, has not been fully understood. In this study, we show that the HCV infection activates cellular cAMP-dependent pathways. Expression of a luciferase reporter gene controlled by a basic promoter with the cAMP response element (CRE) was significantly elevated in human hepatoma Huh-7 cells infected with the HCV JFH1. Analysis with viral subgenomic replicons indicated that the HCV NS2 protein is responsible for the effect. Furthermore, the level of cellular transcripts whose stability is known to be regulated by cAMP was specifically reduced in cells harboring NS2-expressing replicons. These results allude to the HCV NS2 protein having a novel function of regulating cellular gene expression and proliferation through the cAMP-dependent pathway

  3. Cyclic-AMP mediated regulation of ABCB mRNA expression in mussel haemocytes.

    Directory of Open Access Journals (Sweden)

    Silvia Franzellitti

    Full Text Available BACKGROUND: The multixenobiotic resistance system (MXR allows aquatic organisms to cope with their habitat despite high pollution levels by over-expressing membrane and intracellular transporters, including the P-glycoprotein (Pgp. In mammals transcription of the ABCB1 gene encoding Pgp is under cAMP/PKA-mediated regulation; whether this is true in mollusks is not fully clarified. METHODOLOGY/PRINCIPAL FINDINGS: cAMP/PKA regulation and ABCB mRNA expression were assessed in haemocytes from Mediterranean mussels (Mytilus galloprovincialis exposed in vivo for 1 week to 0.3 ng/L fluoxetine (FX alone or in combination with 0.3 ng/L propranolol (PROP. FX significantly decreased cAMP levels and PKA activity, and induced ABCB mRNA down-regulation. FX effects were abolished in the presence of PROP. In vitro experiments using haemocytes treated with physiological agonists (noradrenaline and serotonin and pharmacological modulators (PROP, forskolin, dbcAMP, and H89 of the cAMP/PKA system were performed to obtain clear evidence about the involvement of the signaling pathway in the transcriptional regulation of ABCB. Serotonin (5-HT decreased cAMP levels, PKA activity and ABCB mRNA expression but increased the mRNA levels for a putative 5-HT1 receptor. Interestingly, 5-HT1 was also over-expressed after in vivo exposures to FX. 5-HT effects were counteracted by PROP. Forskolin and dbcAMP increased PKA activity as well as ABCB mRNA expression; the latter effect was abolished in the presence of the PKA inhibitor H89. CONCLUSIONS: This study provides the first direct evidence for the cAMP/PKA-mediated regulation of ABCB transcription in mussels.

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

    Science.gov (United States)

    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.

  5. Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.

    Science.gov (United States)

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L; Zillinger, Thomas; Serganov, Artem A; Liu, Yizhou; Jones, Roger A; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J

    2013-05-23

    Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2',5')pA and A(3',5')pG phosphodiester linkages, designated c[G(2',5')pA(3',5')p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2',5')pA(3',5')p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5'-pppG(2',5')pA prior to cyclization to c[G(2',5')pA(3',5')p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2',5')pA(3',5')p] as a founding member of a family of metazoan 2',5'-containing cyclic heterodinucleotide second messengers distinct from bacterial 3',5' cyclic dinucleotides. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Ecklonia cava Polyphenol Has a Protective Effect against Ethanol-Induced Liver Injury in a Cyclic AMP-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Haruka Yamashita

    2015-06-01

    Full Text Available 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.

  7. Effects of subacute and chronic lead treatment on glucose homestasis and renal cyclic AMP metabolism in rats

    Energy Technology Data Exchange (ETDEWEB)

    Stevenson, A; Merali, Z; Kacew, S; Singhal, R L

    1976-01-01

    The effects of chronic oral ingestion of lead in doses ranging from 20 to 80 ppM were compared with those seen after the subacute exposure of rats to a 10 mg/kg daily dose of the heavy metal for 7 days. Irrespective of the treatment regimen used, lead treatment significantly increased the activities of renal pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1,6-diphosphatase and glucose 6-phosphatase. The observed enhancement of kidney gluconeogenic enzymes in chronically treated animals was associated with a stimulation of the adenylate cyclase-cyclic AMP system, a rise in blood glucose and urea as well as a depression in hepatic glycogen and serum immunoreactive insulin (IRI) levels. In contrast, subacute exposure to lead failed to significantly alter cyclic AMP metabolism and the concentrations of liver glycogen, blood glucose, serum urea or IRI. Whereas the insulinogenic index (the ratio of serum IRI to blood glucose concentration) was markedly suppressed in chronically treated rats, this ratio remained within normal limits following subacute exposure to the heavy metal. However, a marked decrease in the insulinogenic index was observed in subacutely treated rats 15 min after the administration of a glucose load. The data provide evidence to show that increased glucose synthesis as well as suppressed pancreatic function may be responsible for lead-induced disturbances in glucose homeostasis.

  8. Regulation of the sodium/potassium/chloride cotransporter by calcium and cyclic AMP in cultured vascular smooth muscle cells

    International Nuclear Information System (INIS)

    Higgins, B.L.; Smith, L.; Smith, J.B.

    1987-01-01

    The activity of the Na/K/Cl cotransporter in smooth muscle cells cultured from rat aorta was assayed by measuring the initial rate of furosemide-inhibitable 86 Rb influx or efflux. Five uM furosemide or 0.2 uM bumetanide inhibited influx by 50%. Furosemide-inhibitable 86 Rb influx depended on the presence of all 3 ions in the external medium. The dependence on Na and K was hyperbolic with apparent Km values of 45 and 5 mM, respectively. The dependence on Cl was sigmoidal. Assuming a stoichiometry of 1:1:2 for Na:K:Cl, a Km for Cl of 60 mM was obtained from a Hofstee plot of the data. Rapidly growing cells had 3 fold higher cotransport activity than quiescent cells. Angiotensin II (ANG) stimulated furosemide-inhibitable 86 Rb efflux by 2 fold. An ANG receptor antagonist prevented ANG from increasing cotransport activity. Two calcium ionophores, A23187 and ionomycin, increased cotransport activity by 2 fold. Phorbol myristate acetate had no effect on cotransport activity. Isoproterenol, dibutyryl cyclic AMP, cholera toxin, or methylisobutylxanthine inhibited furosemide-sensitive 86 Rb influx by 35 to 50%. From these findings they conclude that increasing cytoplasmic free calcium stimulates cotransport activity, whereas increasing cellular cyclic AMP inhibits the cotransporter

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. An odor-specific threshold deficit implicates abnormal intracellular cyclic AMP signaling in schizophrenia.

    Science.gov (United States)

    Turetsky, Bruce I; Moberg, Paul J

    2009-02-01

    Although olfactory deficits are common in schizophrenia, their underlying pathophysiology remains unknown. Recent evidence has suggested that cAMP signaling may be disrupted in schizophrenia. Since cAMP mediates signal transduction in olfactory receptor neurons, this could contribute to the etiology of observed olfactory deficits. This study was designed to test this hypothesis by determining odor detection threshold sensitivities to two odorants that differ in their relative activations of this intracellular cAMP signaling cascade. Thirty schizophrenia patients, 25 healthy comparison subjects, and 19 unaffected first-degree relatives of schizophrenia patients were studied. Odor detection threshold sensitivities were measured for the two odorants citralva and lyral. Although both have fruity/floral scents, citralva strongly activates adenylyl cyclase to increase cAMP levels, while lyral is a very weak activator of adenylyl cyclase. There was a significant group-by-odor interaction. Both schizophrenia patients and unaffected first-degree relatives were impaired in their ability to detect lyral versus citralva. Comparison subjects were equally sensitive to both odorants. This selective deficit could not be explained by differences in age, sex, smoking, clinical symptom profile, or medication use. This study establishes the presence of an odor-specific hyposmia that may denote a disruption of cAMP-mediated signal transduction in schizophrenia. The presence of a parallel deficit in the patients' unaffected first-degree relatives suggests that this deficit is genetically mediated. Although additional physiological studies are needed to confirm the underlying mechanism, these results offer strong inferential support for the hypothesis that cAMP signaling is dysregulated in schizophrenia.

  11. Cyclic AMP is a key regulator of M1 to M2a phenotypic conversion of microglia in the presence of Th2 cytokines.

    Science.gov (United States)

    Ghosh, Mousumi; Xu, Yong; Pearse, Damien D

    2016-01-13

    Microglia and macrophages play a central role in neuroinflammation. Pro-inflammatory cytokines trigger their conversion to a classically activated (M1) phenotype, sustaining inflammation and producing a cytotoxic environment. Conversely, anti-inflammatory cytokines polarize the cells towards an alternatively activated (M2), tissue reparative phenotype. Elucidation of the signal transduction pathways involved in M1 to M2 phenotypic conversion may provide insight into how the innate immune response can be harnessed during distinct phases of disease or injury to mediate neuroprotection and neurorepair. Microglial cells (cell line and primary) were subjected to combined cyclic adenosine monophosphate (cyclic AMP) and IL-4, or either alone, in the presence of pro-inflammatory mediators, lipopolysaccharide (LPS), or tumor necrosis factor-α (TNF-α). Their effects on the expression of characteristic markers for M1 and M2 microglia were assessed. Similarly, the M1 and M2 phenotypes of microglia and macrophages within the lesion site were then evaluated following a contusive spinal cord injury (SCI) to the thoracic (T8) spinal cord of rats and mice when the agents were administered systemically. It was demonstrated that cyclic AMP functions synergistically with IL-4 to promote M1 to M2 conversion of microglia in culture. The combination of cyclic AMP and IL-4, but neither alone, induced an Arg-1(+)/iNOS(-)cell phenotype with concomitant expression of other M2-specific markers including TG2 and RELM-α. M2-converted microglia showed ameliorated production of pro-inflammatory cytokines (TNF-α and IP-10) and reactive oxygen species, with no alteration in phagocytic properties. M2a conversion required protein kinase A (PKA), but not the exchange protein directly activated by cyclic AMP (EPAC). Systemic delivery of cyclic AMP and IL-4 after experimental SCI also promoted a significant M1 to M2a phenotypic change in microglia and macrophage population dynamics in the lesion

  12. Comparison of phosphorylation of ribosomal proteins from HeLa and Krebs II ascites-tumour cells by cyclic AMP-dependent and cyclic GMP-dependent protein kinases

    DEFF Research Database (Denmark)

    Issinger, O G; Beier, H; Speichermann, N

    1980-01-01

    Phosphorylation of eukaryotic ribosomal proteins in vitro by essentially homogeneous preparations of cyclic AMP-dependent protein kinase catalytic subunit and cyclic GMP-dependent protein kinase was compared. Each protein kinase was added at a concentration of 30nM. Ribosomal proteins were...... by the cyclic AMP-dependent enzyme. Between 0.1 and 0.2 mol of phosphate was incorporated/mol of these phosphorylated proteins. With the exception of protein S7, the same proteins were also major substrates for the cyclic GMP-dependent protein kinase. Time courses of the phosphorylation of individual proteins...... from the small and large ribosomal subunits in the presence of either protein kinase suggested four types of phosphorylation reactions: (1) proteins S2, S10 and L5 were preferably phosphorylated by the cyclic GMP-dependent protein kinase; (2) proteins S3 and L6 were phosphorylated at very similar rates...

  13. Attempt to demonstrate an in vivo effect of mianserin hydrochloride on erythrocyte Na+-K+-ATPase activity and cyclic AMP concentration

    Science.gov (United States)

    Naylor, G. S.; Buckley, D. E.; Boardman, L. J.; Smith, A. H. W.; Moody, J. P.

    1978-01-01

    1 There is evidence that erythrocyte Na+-K+-ATPase activity and erythrocyte cyclic AMP change on recovery from a depressive illness. Mianserin is a recently introduced antidepressant but its mode of action is unknown. The present study was therefore designed to investigate in vivo the effect of mianserin on erythrocyte Na+-K+-ATPase and cyclic AMP. 2 Biochemical estimations were made on blood from depressed patients before beginning either mianserin or matched placebo treatment, after 1 week, and again after 2 weeks' treatment. 3 Neither the erythrocyte Na+-K+-ATPase, nor the erythrocyte cyclic AMP concentration, changed significantly in either the mianserin- or the placebo-treated group. 4 The study sheds no light on the possible mechanism of action of mianserin. PMID:203308

  14. Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: Mediation by cyclic AMP

    International Nuclear Information System (INIS)

    Egan, J.J.; Gronowicz, G.; Rodan, G.A.

    1991-01-01

    Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP

  15. Regulation of cyclic AMP metabolism by prostaglandins in rabbit cortical collecting tubule cells

    International Nuclear Information System (INIS)

    Sonnenburg, W.K.

    1987-01-01

    In the rabbit cortical collecting tubule (RCCT), prostaglandin E 1 (PGE 1 ) and prostaglandin E 2 (PGE 2 ) at 1 nM inhibit arginine-vasopressin (AVP)-induced water reabsorption, while 100 nM PGE 1 and PGE 2 alone stimulate water reabsorption. Reported here are studies designed to investigate the molecular basis for the biphasic physiological action of PGE 1 and PGE 2 in the collecting duct. In freshly isolated RCCT cells, PGE 1 , PGE 2 , and 16,16-dimethyl-PGE 2 (DM-PGE 2 ) stimulated cAMP synthesis at concentrations ranging from 0.1 to 10 M. Other prostaglandins including the synthetic PGE 2 analogue, sulprostone, failed to stimulate cAMP synthesis. Moreover, sulprostone did not antagonize PGE 2 -stimulated cAMP formation. In contrast, PGE 2 and sulprostone at concentrations ranging from 1 to 100 nM, inhibited AVP-induced cAMP accumulation in freshly isolated RCCT cells. PGE 2 , PGE 1 , DM-PGE 2 and sulprostone at 100 nM were equally effective in inhibiting AVP-induced cAMP formation. Moreover sulprostone inhibited AVP-stimulated adenylate cyclase activity. These results suggest that PGE derivatives mediate either inhibition or activation of adenylate cyclase by stimulating different PGE receptors. To further test this concept, PGE 2 binding to freshly isolated RCCT cell membranes was characterized. Two different classes of PGE 2 binding were detected. / 3 H/PGE 2 binding to the high affinity class of sites was increased by the GTP-analogue, GTP S, while pertussis toxin pretreatment blocked the stimulatory action. In contrast, / 3 H/ PGE 2 binding to the low affinity class of sites was decreased by GTP S; this inhibitory effect was not blocked by pertussis toxin pretreatment

  16. Export of cyclic AMP by avian red cells and inhibition by prostaglandin A1

    International Nuclear Information System (INIS)

    Heasley, L.E.

    1985-01-01

    The mechanism by which PGA 1 inhibits cAMP export by avian red cells was studied, to provide details on the molecular mechanism of a prostaglandin action and on the process of cAMP export itself. The interaction of PGA 1 with pigeon red cells is a multi-step process of uptake, metabolism and secretion. [ 3 H]PGA rapidly enters red cells and is promptly metabolized (V/sub max/ ≥ 1 nmol/min/10 7 cells) to a compound (5) that remains in the aqueous layer after ethyl acetate extraction. Chromatographic analyses, amino acid content and fast atom bombardment mass spectrometry reveal that the polar metabolite is conjugated with glutathione (PGA 1 -GSH) at C-11 via a thioether bond and is largely (80%) reduced to the C-9 hydroxyl derivative

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

    Science.gov (United States)

    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

  18. DNA-Mediated Cyclic GMP-AMP Synthase-Dependent and -Independent Regulation of Innate Immune Responses.

    Science.gov (United States)

    Motani, Kou; Ito, Shinji; Nagata, Shigekazu

    2015-05-15

    Cytoplasmic DNA activates cyclic GMP-AMP synthase (cGAS) to produce cyclic 2'-5'3'-5'GMP-AMP dinucleotide (2'5 'cGAMP). The binding of 2'5'cGAMP to an adaptor protein, stimulator of IFN genes (STING), activates a transcription factor, IFN regulatory factor 3, leading to the induction of IFN and chemokine gene expression. In this study, we found that the 2'5'cGAMP-dependent STING activation induced highly upregulated CXCL10 gene expression. Formation of a distinct STING dimer, which was detected by native PAGE, was induced by 2'5'cGAMP, but not 3'-5'3'-5'cGAMP. Analysis of DNase II(-/-) mice, which constitutively produce IFN-β and CXCL10, showed the accumulation of 2'5'cGAMP in their fetal livers and spleens, suggesting that the undigested DNA accumulating in DNase II(-/-) cells may have leaked from the lysosomes into the cytoplasm. The DNase II(-/-) mouse embryonic fibroblasts produced 2'5'cGAMP in a cGAS-dependent manner during apoptotic cell engulfment. However, cGAS deficiency did not impair the STING-dependent upregulation of CXCL10 in DNase II(-/-) mouse embryonic fibroblasts that was induced by apoptotic cell engulfment or DNA lipofection. These results suggest the involvement of a cGAS-independent additional DNA sensor(s) that induces the STING-dependent activation of innate immunity. Copyright © 2015 by The American Association of Immunologists, Inc.

  19. Enhanced phosphorylation of cyclic AMP response element binding protein in Brain of mice following repetitive hypoxic exposure

    International Nuclear Information System (INIS)

    Gao Yanan; Gao Ge; Long Caixia; Han Song; Zu Pengyu; Fang Li; Li Junfa

    2006-01-01

    Cerebral ischemic/hypoxic preconditioning (I/HPC) is a phenomenon of endogenous protection that renders Brain tolerant to sustained ischemia/hypoxia. This profound protection induced by I/HPC makes it an attractive target for developing potential clinical therapeutic approaches. However, the molecular mechanism of I/HPC is unclear. Cyclic AMP (cAMP) response element binding protein (CREB), a selective nuclear transcriptional factor, plays a key role in the neuronal functions. Phosphorylation of CREB on Ser-133 may facilitate its transcriptional activity in response to various stresses. In the current study, we observed the changes in CREB phosphorylation (Ser-133) and protein expression in Brain of auto-hypoxia-induced HPC mice by using Western blot analysis. We found that the levels of phosphorylated CREB (Ser-133), but not protein expression of CREB, increased significantly (p < 0.05) in the hippocampus and the frontal cortex of mice after repetitive hypoxic exposure (H2-H4, n = 6 for each group), when compared to that of the normoxic (H0, n = 6) or hypoxic exposure once group (H1, n = 6). In addition, a significant enhancement (p < 0.05) of CREB phosphorylation (Ser-133) could also be found in the nuclear extracts from the whole hippocampus of hypoxic preconditioned mice (H2-H4, n = 6 for each group). These results suggest that the phosphorylation of CREB might be involved in the development of cerebral hypoxic preconditioning

  20. Adrenomedullin stimulates cyclic AMP production in the airway epithelial cells of guinea-pigs and in the human epithelial cell line

    Directory of Open Access Journals (Sweden)

    Takashi Kawaguchi

    1999-01-01

    Full Text Available This study was designed to examine the effects of adrenomedullin (AM on airway epithelial cells. Primary cultures of guinea-pig tracheal epithelial cells and the human bronchiolar epithelial cell line NCI-H441 were used. Intracellular cyclic adenosine monophosphate (cAMP, cyclic guanosine monophosphate (cGMP, prostaglandin E2 (PGE2, and stable end-products of nitric oxide were assayed. Adrenomedullin (10−6 mol/L stimulated cAMP production in guinea-pig epithelial cells. Indomethacin (10−5 mol/L significantly decreased the basal level of intracellular cAMP in guinea-pig epithelial cells, but not in NCI-H441 cells. However, AM did not stimulate production of PGE2, a major product that can increase cAMP formation. In the case of NCI-H441 cells, AM (10−8 – 10−6 mol/L did not significantly affect intracellular cGMP levels or nitrite content in conditioned medium. Adrenomedullin and calcitonin gene-related peptide (CGRP each stimulated cAMP production in NCI-H441 cells, but AM-stimulated cAMP production was antagonized by the CGRP fragment CGRP8–37. These findings suggest that AM stimulates cAMP production and functionally competes with CGRP for binding sites in airway epithelial cells, at least in human epithelial cells, but that it does not stimulate the release of PGE2 and nitric oxide. Though cyclooxygenase products contribute to some extent to cAMP formation in guinea-pigs, AM independently stimulates intracellular cAMP formation in airway epithelial cells.

  1. Distribution and function of 3',5'-Cyclic-AMP phosphodiesterases in the human ovary

    DEFF Research Database (Denmark)

    Petersen, T S; Kristensen, S G; Jeppesen, J V

    2015-01-01

    The concentration of the important second messenger cAMP is regulated by phosphodiesterases (PDEs) and hence an attractive drug target. However, limited human data are available about the PDEs in the ovary. The aim of the present study was to describe and characterise the PDEs in the human ovary....... Results were obtained by analysis of mRNA microarray data from follicles and granulosa cells (GCs), combined RT-PCR and enzymatic activity analysis in GCs, immunohistochemical analysis of ovarian sections and by studying the effect of PDE inhibitors on progesterone production from cultured GCs. We found...

  2. Influence of sulphate on effects of ADP, 3′,5′-cyclic amp and citrate on human platelet phosphofructokinase activity

    NARCIS (Netherlands)

    Akkerman, Jan Willem N.; Gorter, G.; Sixma, J.J.; Staal, Gerard E.J.

    1974-01-01

    1. 1. Sulphate ions activate partially-purified human platelet phosphofructokinase. This is caused by suppression of the cooperativity of the enzyme with respect to fructose 6-phosphate. 2. 2. Sulphate therefore markedly affects the influences of allosteric modifiers such as ADP, 3′,5′-cyclic AMP

  3. Dual effects of muscarinic M2 acetylcholine receptors on the synthesis of cyclic AMP in CHO cells: dependence on time, receptor density and receptor agonists

    Czech Academy of Sciences Publication Activity Database

    Michal, Pavel; Lysíková, Michaela; Tuček, Stanislav

    2001-01-01

    Roč. 132, č. 6 (2001), s. 1217-1228 ISSN 0007-1188 R&D Projects: GA ČR GA309/99/0214; GA AV ČR IAA7011910 Institutional research plan: CEZ:AV0Z5011922 Keywords : cyclic AMP * muscarinic receptors * CHO cells Subject RIV: ED - Physiology Impact factor: 3.502, year: 2001

  4. Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

    International Nuclear Information System (INIS)

    Stein, J.C.

    1985-01-01

    Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several 37 P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the 32 P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex

  5. Effect of Increased Cyclic AMP Concentration on Muscle Protein Synthesis and Beta-Adrenergic Receptor Expression in Chicken Skeletal Muscle Cells in Culture

    Science.gov (United States)

    Young, R. B.; Vaughn, J. R.; Bridge, K. Y.; Smith, C. K.

    1998-01-01

    Analogies of epinephrine are known to cause hypertrophy of skeletal muscle when fed to animals. These compounds presumably exert their physiological action through interaction with the P-adrenergic receptor. Since the intracellular signal generated by the Beta-adrenergic receptor is cyclic AMP (cAMP), experiments were initiated in cell culture to determine if artificial elevation of cAMP by treatment with forskolin would alter muscle protein metabolism and P-adrenergic receptor expression. Chicken skeletal muscle cells after 7 days in culture were treated with 0.2-30 micrometers forskolin for a total of three days. At the end of the treatment period, both the concentration of cAMP and the quantity of myosin heavy chain (MHC) were measured. Concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. In contrast, the quantity of MHC was increased approximately 50% above control cells at 0.2 micrometers forskolin, but exhibited a gradual decline at higher levels of forskolin so that the quantity of MHC in cells treated with 30 micrometers forskolin was not significantly different from controls. Curiously, the intracellular concentration of cAMP which elicited the maximum increase in the quantity of MHC was only 40% higher than cAMP concentration in control cells.

  6. Reflections on: "A general role for adaptations in G-Proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function".

    Science.gov (United States)

    Nestler, Eric J

    2016-08-15

    In 1991 we demonstrated that chronic morphine exposure increased levels of adenylyl cyclase and protein kinase A (PKA) in several regions of the rat central nervous system as inferred from measures of enzyme activity in crude extracts (Terwilliger et al., 1991). These findings led us to hypothesize that a concerted upregulation of the cAMP pathway is a general mechanism of opiate tolerance and dependence. Moreover, in the same study we showed similar induction of adenylyl cyclase and PKA activity in nucleus accumbens (NAc) in response to chronic administration of cocaine, but not of several non-abused psychoactive drugs. Morphine and cocaine also induced equivalent changes in inhibitory G protein subunits in this brain region. We thus extended our hypothesis to suggest that, particularly within brain reward regions such as NAc, cAMP pathway upregulation represents a common mechanism of reward tolerance and dependence shared by several classes of drugs of abuse. Research since that time, by many laboratories, has provided substantial support for these hypotheses. Specifically, opiates in several CNS regions including NAc, and cocaine more selectively in NAc, induce expression of certain adenylyl cyclase isoforms and PKA subunits via the transcription factor, CREB, and these transcriptional adaptations serve a homeostatic function to oppose drug action. In certain brain regions, such as locus coeruleus, these adaptations mediate aspects of physical opiate dependence and withdrawal, whereas in NAc they mediate reward tolerance and dependence that drives increased drug self-administration. This work has had important implications for understanding the molecular basis of addiction. "A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function". Previous studies have shown that chronic morphine increases levels of the G-protein subunits Giα and Goα, adenylate cyclase, cyclic AMP

  7. Increase of Intracellular Cyclic AMP by PDE4 Inhibitors Affects HepG2 Cell Cycle Progression and Survival.

    Science.gov (United States)

    Massimi, Mara; Cardarelli, Silvia; Galli, Francesca; Giardi, Maria Federica; Ragusa, Federica; Panera, Nadia; Cinque, Benedetta; Cifone, Maria Grazia; Biagioni, Stefano; Giorgi, Mauro

    2017-06-01

    Type 4 cyclic nucleotide phosphodiesterases (PDE4) are major members of a superfamily of enzymes (PDE) involved in modulation of intracellular signaling mediated by cAMP. Broadly expressed in most human tissues and present in large amounts in the liver, PDEs have in the last decade been key therapeutic targets for several inflammatory diseases. Recently, a significant body of work has underscored their involvement in different kinds of cancer, but with no attention paid to liver cancer. The present study investigated the effects of two PDE4 inhibitors, rolipram and DC-TA-46, on the growth of human hepatoma HepG2 cells. Treatment with these inhibitors caused a marked increase of intracellular cAMP level and a dose- and time-dependent effect on cell growth. The concentrations of inhibitors that halved cell proliferation to about 50% were used for cell cycle experiments. Rolipram (10 μM) and DC-TA-46 (0.5 μM) produced a decrease of cyclin expression, in particular of cyclin A, as well as an increase in p21, p27 and p53, as evaluated by Western blot analysis. Changes in the intracellular localization of cyclin D1 were also observed after treatments. In addition, both inhibitors caused apoptosis, as demonstrated by an Annexin-V cytofluorimetric assay and analysis of caspase-3/7 activity. Results demonstrated that treatment with PDE4 inhibitors affected HepG2 cell cycle and survival, suggesting that they might be useful as potential adjuvant, chemotherapeutic or chemopreventive agents in hepatocellular carcinoma. J. Cell. Biochem. 118: 1401-1411, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Cyclic GMP-AMP synthase is an innate immune sensor of HIV and other retroviruses.

    Science.gov (United States)

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

    2013-08-23

    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 guanosine monophosphate-adenosine monophosphate (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, and simian immunodeficiency virus. These results indicate that cGAS is an innate immune sensor of HIV and other retroviruses.

  9. Acute morphine alters GABAergic transmission in the central amygdala during naloxone-precipitated morphine withdrawal: role of cyclic AMP

    Directory of Open Access Journals (Sweden)

    Michal eBajo

    2014-06-01

    Full Text Available The central amygdala (CeA plays an important role in opioid addiction. Therefore, we examined the effects of naloxone-precipitated morphine withdrawal (WD on GABAergic transmission in rat CeA neurons using whole-cell recordings with naloxone in the bath. The basal frequency of miniature inhibitory postsynaptic currents (mIPSCs increased in CeA neurons from WD compared to placebo rats. Acute morphine (10 M had mixed effects (> 20% change from baseline on mIPSCs in placebo and WD rats. In most CeA neurons (64% from placebo rats, morphine significantly decreased mIPSC frequency and amplitude. In 32% of placebo neurons, morphine significantly increased mIPSC amplitudes but had no effect on mIPSC frequency. In WD rats, acute morphine significantly increased mIPSC frequency but had no effect on mIPSC amplitude in 41% of CeA neurons. In 45% of cells, acute morphine significantly decreased mIPSC frequency and amplitude. Pre-treatment with the cyclic AMP inhibitor (R-adenosine, cyclic 3’,5’-(hydrogenphosphorothioate triethylammonium (RP, prevented acute morphine-induced potentiation of mIPSCs. Pre-treatment of slices with the Gi/o G-protein subunit inhibitor pertussis toxin (PTX did not prevent the acute morphine-induced enhancement or inhibition of mIPSCs. PTX and RP decreased basal mIPSC frequencies and amplitudes only in WD rats. The results suggest that inhibition of GABAergic transmission in the CeA by acute morphine is mediated by PTX-insensitive mechanisms, although PTX-sensitive mechanisms cannot be ruled out for non-morphine responsive cells; by contrast, potentiation of GABAergic transmission is mediated by activated cAMP signaling that also mediates the increased basal GABAergic transmission in WD rats. Our data indicate that during the acute phase of WD, the CeA opioid and GABAergic systems undergo neuroadaptative changes conditioned by a previous chronic morphine exposure and dependence.

  10. Embryonic cholesterol esterification is regulated by a cyclic AMP-dependent pathway in yolk sac membrane-derived endodermal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Siou-Huei Wang

    Full Text Available During avian embryonic development, endodermal epithelial cells (EECs absorb yolk through the yolk sac membrane. Sterol O-acyltransferase (SOAT is important for esterification and yolk lipid utilization during development. Because the major enzyme for yolk sac membrane cholesteryl ester synthesis is SOAT1, we cloned the avian SOAT1 promoter and elucidated the cellular functions of SOAT1. Treatments with either glucagon, isobutylmethylxanthine (IBMX, an adenylate cyclase activator (forskolin, a cAMP analog (dibutyryl-cAMP, or a low glucose concentration all increased SOAT1 mRNA accumulation in EECs from Japanese quail, suggesting that SOAT1 is regulated by nutrients and hormones through a cAMP-dependent pathway. Activity of protein kinase A (PKA was increased by IBMX, whereas co-treatment with the PKA inhibitor, H89 negated the increase in PKA activity. Cyclic AMP-induced EECs had greater cholesterol esterification than untreated EECs. By promoter deletion and point-mutation, the cAMP-response element (-349 to -341 bp was identified as critical in mediating transcription of SOAT1. In conclusion, expression of SOAT1 was regulated by a cAMP-dependent pathway and factors that increase PKA will increase SOAT1 to improve the utilization of lipids in the EECs and potentially modify embryonic growth.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. The small molecule triclabendazole decreases the intracellular level of cyclic AMP and increases resistance to stress in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yong Joo Lee

    Full Text Available The Ras-adenylyl cyclase-protein kinase A nutrient-sensing pathway controls metabolism, proliferation and resistance to stress in Saccharomyces cerevisiae. The genetic disruption of this pathway increases resistance to a variety of stresses. We show here that the pharmacological inhibition of this pathway by the drug triclabendazole increases resistance to oxidants, heat stress and extends the chronological life. Evidence is presented that triclabendazole decreases the intracellular level of cyclic AMP by inhibiting adenylyl cyclase and triggers the parallel rapid translocation of the stress-resistance transcription factor Msn2 from the cytosol into the nucleus, as deduced from experiments employing a strain in which MSN2 is replaced with MSN2-GFP (GFP, green fluorescent protein. Msn2 and Msn4 are responsible for activating the transcription of numerous genes that encode proteins that protect cells from stress. The results are consistent with triclabendazole either inhibiting the association of Ras with adenylyl cyclase or directly inhibiting adenylyl cyclase, which in turn triggers Msn2/4 to enter the nucleus and activate stress-responsible element gene expression.

  13. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes

    Directory of Open Access Journals (Sweden)

    Yan Wang

    2015-01-01

    Full Text Available Rheumatoid arthritis (RA is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdown of cGAS inhibited the proliferation of RA FLS. cGAS overexpression enhanced the production of proinflammatory cytokines and matrix metalloproteinases (MMPs as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. In contrast, cGAS silencing inhibited production of proinflammatory cytokines and matrix metalloproteinases (MMPs as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. These results suggest that cGAS activates the AKT and ERK pathways to promote the inflammatory response of RA FLS, and the development of strategies targeting cGAS may have therapeutic potential for human RA.

  14. The catalytic mechanism of cyclic GMP-AMP synthase (cGAS) and implications for innate immunity and inhibition.

    Science.gov (United States)

    Hall, Justin; Ralph, Erik C; Shanker, Suman; Wang, Hong; Byrnes, Laura J; Horst, Reto; Wong, Jimson; Brault, Amy; Dumlao, Darren; Smith, James F; Dakin, Leslie A; Schmitt, Daniel C; Trujillo, John; Vincent, Fabien; Griffor, Matt; Aulabaugh, Ann E

    2017-12-01

    Cyclic GMP-AMP synthase (cGAS) is activated by ds-DNA binding to produce the secondary messenger 2',3'-cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds-DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2',3'-cGAMP formation, and interestingly, describe a catalytic mechanism where 2',3'-cGAMP may be a minor product of cGAS compared with linear nucleotides. © 2017 The Authors Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.

  15. Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes.

    Science.gov (United States)

    Wang, Yan; Su, Guo-Hua; Zhang, Fang; Chu, Jing-Xue; Wang, Yun-Shan

    2015-01-01

    Rheumatoid arthritis (RA) is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS) invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdown of cGAS inhibited the proliferation of RA FLS. cGAS overexpression enhanced the production of proinflammatory cytokines and matrix metalloproteinases (MMPs) as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. In contrast, cGAS silencing inhibited production of proinflammatory cytokines and matrix metalloproteinases (MMPs) as well as AKT and ERK phosphorylation in TNFα-stimulated FLS. These results suggest that cGAS activates the AKT and ERK pathways to promote the inflammatory response of RA FLS, and the development of strategies targeting cGAS may have therapeutic potential for human RA.

  16. Cutting edge: Antimalarial drugs inhibit IFN-β production through blockade of cyclic GMP-AMP synthase-DNA interaction.

    Science.gov (United States)

    An, Jie; Woodward, Joshua J; Sasaki, Tomikazu; Minie, Mark; Elkon, Keith B

    2015-05-01

    Type I IFN is strongly implicated in the pathogenesis of systemic autoimmune diseases, such as lupus, and rare monogenic IFNopathies, including Aicardi-Goutières syndrome. Recently, a new DNA-activated pathway involving the enzyme cyclic GMP-AMP synthase (cGAS) was described and potentially linked to Aicardi-Goutières syndrome. To identify drugs that could potentially inhibit cGAS activity, we performed in silico screening of drug libraries. By computational analysis, we identified several antimalarial drugs (AMDs) that were predicted to interact with the cGAS/dsDNA complex. Our studies validated that several AMDs were effective inhibitors of IFN-β production and that they functioned by inhibiting dsDNA stimulation of cGAS. Because AMDs have been widely used in human diseases and have an excellent safety profile, our findings suggest new therapeutic strategies for the treatment of severe debilitating diseases associated with type I IFNs due to cGAS activation. Copyright © 2015 by The American Association of Immunologists, Inc.

  17. [Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells].

    Science.gov (United States)

    Xiaojun, Yang; Yongmei, Tan; Zhihui, Tian; Ting, Zhou; Wanghong, Zhao; Jin, Hou

    2017-04-01

    This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues. The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry. P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues. Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
.

  18. Regulation of Cancer Cell Responsiveness to Ionizing Radiation Treatment by Cyclic AMP Response Element Binding Nuclear Transcription Factor

    Directory of Open Access Journals (Sweden)

    Francesca D’Auria

    2017-05-01

    Full Text Available Cyclic AMP response element binding (CREB protein is a member of the CREB/activating transcription factor (ATF family of transcription factors that play an important role in the cell response to different environmental stimuli leading to proliferation, differentiation, apoptosis, and survival. A number of studies highlight the involvement of CREB in the resistance to ionizing radiation (IR therapy, demonstrating a relationship between IR-induced CREB family members’ activation and cell survival. Consistent with these observations, we have recently demonstrated that CREB and ATF-1 are expressed in leukemia cell lines and that low-dose radiation treatment can trigger CREB activation, leading to survival of erythro-leukemia cells (K562. On the other hand, a number of evidences highlight a proapoptotic role of CREB following IR treatment of cancer cells. Since the development of multiple mechanisms of resistance is one key problem of most malignancies, including those of hematological origin, it is highly desirable to identify biological markers of responsiveness/unresponsiveness useful to follow-up the individual response and to adjust anticancer treatments. Taking into account all these considerations, this mini-review will be focused on the involvement of CREB/ATF family members in response to IR therapy, to deepen our knowledge of this topic, and to pave the way to translation into a therapeutic context.

  19. Regulation of Cox-2 by Cyclic AMP Response Element Binding Protein in Prostate Cancer: Potential Role for Nexrutine

    Directory of Open Access Journals (Sweden)

    Rita Ghosh

    2007-11-01

    Full Text Available We recently showed that NexrutineR, a Phellodendron amurense bark extract, suppresses proliferation of prostate cancer cell lines and tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP model. Our data also indicate that the antiproliferative effects of NexrutineR are mediated in part by Akt and Cyclic AMP response element binding protein (CREB. Cyclooxygenase (Cox-2, a pro-inflammatory mediator, is a CREB target that induces prostaglandin E2 (PGE2 and suppresses apoptosis. Treatment of LNCaP cells with NexrutineR reduced tumor necrosis factor α-induced enzymatic as well as promoter activities of Cox-2. NexrutineR also reduced the expression and promoter activity of Cox-2 in PC-3 cells that express high constitutive levels of Cox-2. Deletion analysis coupled with mutational analysis of the Cox-2 promoter identified CRE as being sufficient for mediating NexrutineR response. Immunohistochemical analysis of human prostate tumors show increased expression of CREB and DNA binding activity in high-grade tumors (three-fold higher in human prostate tumors compared to normal prostate; P = .01. We have identified CREB-mediated activation of Cox-2 as a potential signaling pathway in prostate cancer which can be blocked with a nontoxic, cost-effective dietary supplement like NexrutineR, demonstrating a prospective for development of NexrutineR for prostate cancer management.

  20. Mechanisms of Altered Control of Proliferation by Cyclic Amp/Protein Kinase A During Mammary Tumor Progression

    National Research Council Canada - National Science Library

    Imagawa, Walter

    1999-01-01

    We hypothesize that alterations in the regulation of growth by growth factors and cAMP during mammary tumor progression are related to MAP kinase signaling pathways known to be affected by cAMP and pertussis toxin (PT...

  1. Synthesis of interleukin 6 (interferon-β2/B cell stimulatory factor 2) in human fibroblasts is triggered by an increase in intracellular cyclic AMP

    International Nuclear Information System (INIS)

    Zhange, Y.; Lin, J.X.; Vilcek, J.

    1988-01-01

    Interleukin 6 (IL-6; also referred to as interferon-β 2 , 26-kDa protein, and B cell stimulatory factor 2) is a cytokine whose actions include a stimulation of immunoglobulin synthesis, enhancement of B cell growth, and modulation of acute phase protein synthesis by hepatocytes. Synthesis of IL-6 is stimulated by interleukin 1 (IL-1), tumor necrosis factor (TNF), or platelet-derived growth factor. The authors examined the role of the cyclic AMP (cAMP)-dependent signal transduction pathway in IL-6 gene expression. Several activators of adenylate cyclase, including prostaglandin E1, forskolin, and cholera toxin, as well as the phosphodiesterase inhibitor isobutylmethylxanthine and the cAMP analog dibutyryl cAMP, shared the ability to cause a dramatic and sustained increase in IL-6 mRNA levels in human FS-4 fibroblasts. Actinomycin D treatment abolished this enhancement. Treatments that increased intracellular cAMP also stimulated the secretion of the IL-6 protein in a biologically active form. Increased intracellular cAMP appears to enhance IL-6 gene expression by a protein kinase C-independent mechanism because down-regulation of protein kinase C by a chronic exposure of cells to a high dose of 12-O-tetradecanoylphorbol 13-acetate did not abolish the enhancement of IL-6 expression by treatments that increase cAMP. IL-1 and TNF too increased IL-6 mRNA levels by a protein kinase C-independent mechanism. The results suggest a role for the cAMP-dependent pathway(s) in IL-6 gene activation by TNF and IL-1

  2. Cyclic AMP Regulates Bacterial Persistence through Repression of the Oxidative Stress Response and SOS-Dependent DNA Repair in Uropathogenic Escherichia coli.

    Science.gov (United States)

    Molina-Quiroz, Roberto C; Silva-Valenzuela, Cecilia; Brewster, Jennifer; Castro-Nallar, Eduardo; Levy, Stuart B; Camilli, Andrew

    2018-01-09

    Bacterial persistence is a transient, nonheritable physiological state that provides tolerance to bactericidal antibiotics. The stringent response, toxin-antitoxin modules, and stochastic processes, among other mechanisms, play roles in this phenomenon. How persistence is regulated is relatively ill defined. Here we show that cyclic AMP, a global regulator of carbon catabolism and other core processes, is a negative regulator of bacterial persistence in uropathogenic Escherichia coli , as measured by survival after exposure to a β-lactam antibiotic. This phenotype is regulated by a set of genes leading to an oxidative stress response and SOS-dependent DNA repair. Thus, persister cells tolerant to cell wall-acting antibiotics must cope with oxidative stress and DNA damage and these processes are regulated by cyclic AMP in uropathogenic E. coli IMPORTANCE Bacterial persister cells are important in relapsing infections in patients treated with antibiotics and also in the emergence of antibiotic resistance. Our results show that in uropathogenic E. coli , the second messenger cyclic AMP negatively regulates persister cell formation, since in its absence much more persister cells form that are tolerant to β-lactams antibiotics. We reveal the mechanism to be decreased levels of reactive oxygen species, specifically hydroxyl radicals, and SOS-dependent DNA repair. Our findings suggest that the oxidative stress response and DNA repair are relevant pathways to target in the design of persister-specific antibiotic compounds. Copyright © 2018 Molina-Quiroz et al.

  3. Kinetics of activation of the P4 promoter of pBR322 by the Escherichia coli cyclic AMP receptor protein.

    Science.gov (United States)

    Hoggett, J G; Brierley, I

    1992-11-01

    The activation of transcription initiation from the P4 promoter of pBR322 by the Escherichia coli cyclic AMP receptor protein (CRP) has been investigated using a fluorescence abortive initiation assay. The effect of the cyclic-AMP/CRP complex on the linear P4 promoter was to increase the initial binding (KB) of RNA polymerase to the promoter by about a factor of 10, but the rate of isomerization of closed to open complex (kf) was unaffected. One molecule of CRP per promoter was required for activation, and the concentration of cyclic AMP producing half-maximal stimulation was about 7-8 microM. Supercoiling caused a 2-3-fold increase in the rate of isomerization of the CRP-activated promoter, but weakened the initial binding of polymerase by about one order of magnitude. The unactivated supercoiled promoter was too weak to allow reliable assessment of kinetic parameters against the high background rate originating from the rest of the plasmid.

  4. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From

  5. Cyclic 3',5'-adenosine monophosphate (cAMP) signaling in the anterior pituitary gland in health and disease.

    Science.gov (United States)

    Hernández-Ramírez, Laura C; Trivellin, Giampaolo; Stratakis, Constantine A

    2018-03-05

    The cyclic 3',5'-adenosine monophosphate (cAMP) was the first among the so-called "second messengers" to be described. It is conserved in most organisms and functions as a signal transducer by mediating the intracellular effects of multiple hormones and neurotransmitters. In this review, we first delineate how different members of the cAMP pathway ensure its correct compartmentalization and activity, mediate the terminal intracellular effects, and allow the crosstalk with other signaling pathways. We then focus on the pituitary gland, where cAMP exerts a crucial function by controlling the responsiveness of the cells to hypothalamic hormones, neurotransmitters and peripheral factors. We discuss the most relevant physiological functions mediated by cAMP in the different pituitary cell types, and summarize the defects affecting this pathway that have been reported in the literature. We finally discuss how a deregulated cAMP pathway is involved in the pathogenesis of pituitary disorders and how it affects the response to therapy. Copyright © 2017. Published by Elsevier B.V.

  6. Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    McInnis, Brittney; Mitchell, Jessica; Marcus, Stevan

    2010-01-01

    Research highlights: → cAMP deficiency induces phosphorylation of PKA catalytic subunit (Pka1) in S. pombe. → Pka1 phosphorylation is further induced by physiological stresses. → Pka1 phosphorylation is not induced in cells lacking the PKA regulatory subunit. → Results suggest that cAMP-independent Pka1 phosphorylation is stimulatory in nature. -- Abstract: In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1 + or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.

  7. Induction of dopamine biosynthesis by l-DOPA in PC12 cells: implications of L-DOPA influx and cyclic AMP.

    Science.gov (United States)

    Jin, Chun Mei; Yang, Yoo Jung; Huang, Hai Shan; Lim, Sung Cil; Kai, Masaaki; Lee, Myung Koo

    2008-09-04

    The effects of 3,4-dihydroxyphenylalanine (l-DOPA) on dopamine biosynthesis and cytotoxicity were investigated in PC12 cells. l-DOPA treatment (20-200 microM) increased the levels of dopamine by 226%-504% after 3-6 h of treatment and enhanced the activities of tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC). l-DOPA (20-200 muM) treatment led to a 562%-937% increase in l-DOPA influx at 1 h, which inhibited the activity of TH, but not AADC, during the same period. The extracellular releases of dopamine were also increased by 231%-570% after treatment with 20 and 200 microM l-DOPA for 0.5-3 h. l-DOPA at a concentration of 100-200 microM, but not 20 microM, exerted apoptotic cytotoxicity towards PC12 cells for 24-48 h. l-DOPA (20-200 microM) increased the intracellular cyclic AMP levels by 318%-557% after 0.5-1 h in a concentration-dependent manner. However, the elevated cyclic AMP levels by l-DOPA could not protect against l-DOPA (100-200 microM)-induced cytotoxicity after 24-48 h. In addition, l-DOPA (20-200 microM)-induced increases in cyclic AMP and dopamine were significantly reduced by treatment with SCH23390 (dopamine D(1) receptor antagonist). The increased levels of dopamine by l-DOPA were also reduced by H89 (protein kinase A, PKA, inhibitor) and GF109203X (protein kinase C inhibitor); however, the reduction by GF109203X was not significant. l-DOPA at 20-200 microM stimulated the phosphorylation of PKA and cyclic AMP-response element binding protein and induced the biosynthesis of the TH protein. These results indicate that 20-200 microM l-DOPA induces dopamine biosynthesis by two pathways. One pathway involves l-DOPA directly entering the cells to convert dopamine through AADC activity (l-DOPA decarboxylation). The other pathway involves l-DOPA and/or released dopamine activating TH to enhance dopamine biosynthesis by the dopamine D(1) receptor-cyclic AMP-PKA signaling system (dopamine biosynthesis by TH).

  8. Inhibition of cyclic AMP response element-directed transcription by decoy oligonucleotides enhances tumor-specific radiosensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Park, Serk In, E-mail: serkin@korea.edu [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); The BK21 Plus Program for Biomedical Sciences, Korea University College of Medicine, Seoul (Korea, Republic of); Department of Medicine and Center for Bone Biology, Vanderbilt University School of Medicine, Nashville, TN (United States); Park, Sung-Jun [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); Laboratory of Obesity and Aging Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD (United States); Lee, Junghan; Kim, Hye Eun; Park, Su Jin; Sohn, Jeong-Won [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of); Park, Yun Gyu, E-mail: parkyg@korea.ac.kr [Department of Biochemistry and Molecular Biology, Korea University College of Medicine, Seoul (Korea, Republic of)

    2016-01-15

    The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while there was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach. - Highlights: • γ-Irradiation induced CREB phosphorylation and CRE-directed transcription in tumor. • γ-Irradiation-induced transcriptional activation of CREB was via p38 MAPK pathway. • CRE blockade increased radiosensitivity of tumor cells but not of normal cells. • CRE decoy oligonucleotides or p38 MAPK inhibitors can be used as radiosensitizers.

  9. Inhibition of cyclic AMP response element-directed transcription by decoy oligonucleotides enhances tumor-specific radiosensitivity

    International Nuclear Information System (INIS)

    Park, Serk In; Park, Sung-Jun; Lee, Junghan; Kim, Hye Eun; Park, Su Jin; Sohn, Jeong-Won; Park, Yun Gyu

    2016-01-01

    The radiation stress induces cytotoxic responses of cell death as well as cytoprotective responses of cell survival. Understanding exact cellular mechanism and signal transduction pathways is important in improving cancer radiotherapy. Increasing evidence suggests that cyclic AMP response element binding protein (CREB)/activating transcription factor (ATF) family proteins act as a survival factor and a signaling molecule in response to stress. We postulated that CREB inhibition via CRE decoy oligonucleotide increases tumor cell sensitization to γ-irradiation-induced cytotoxic stress. In the present study, we demonstrate that CREB phosphorylation and CREB DNA-protein complex formation increased in time- and radiation dose-dependent manners, while there was no significant change in total protein level of CREB. In addition, CREB was phosphorylated in response to γ-irradiation through p38 MAPK pathway. Further investigation revealed that CREB blockade by decoy oligonucleotides functionally inhibited transactivation of CREB, and significantly increased radiosensitivity of multiple human cancer cell lines including TP53- and/or RB-mutated cells with minimal effects on normal cells. We also demonstrate that tumor cells ectopically expressing dominant negative mutant CREB (KCREB) and the cells treated with p38 MAPK inhibitors were more sensitive to γ-irradiation than wild type parental cells or control-treated cells. Taken together, we conclude that CREB protects tumor cells from γ-irradiation, and combination of CREB inhibition plus ionizing radiation will be a promising radiotherapeutic approach. - Highlights: • γ-Irradiation induced CREB phosphorylation and CRE-directed transcription in tumor. • γ-Irradiation-induced transcriptional activation of CREB was via p38 MAPK pathway. • CRE blockade increased radiosensitivity of tumor cells but not of normal cells. • CRE decoy oligonucleotides or p38 MAPK inhibitors can be used as radiosensitizers.

  10. Presence of cyclic adenosine-3':5'-monophosphate (cAMP) in primary shoots of Zea mays L

    International Nuclear Information System (INIS)

    Edlich, W.; Graeser, H.

    1978-01-01

    The concentration of cAMP was determined in extracts of crude homogenates of maize seedlings, 3800 . g supernatants and isolated chloroplasts by the isotope dilution test. After extractpurification by precipitation with BaSO 4 and by chromatography on aluminium oxide column, a factor by means of which the binding of [8- 3 H]-cAMP with cAMP-dependent protein kinase was inhibited. The inhibitor was found inactive after treatment of the extracts with phosphodiesterase. In conclusion, this factor was identical with cAMP. It is suggested that cAMP-synthesis is localized in chloroplasts. Microbial contaminations which might disturb the detection of cAMP was excluded at least in the preparations of chloroplasts. (author)

  11. Cyclic di-adenosine monophosphate (c-di-AMP) is required for osmotic regulation in Staphylococcus aureus but dispensable for viability in anaerobic conditions.

    Science.gov (United States)

    Zeden, Merve S; Schuster, Christopher F; Bowman, Lisa; Zhong, Qiyun; Williams, Huw D; Gründling, Angelika

    2018-03-02

    Cyclic di-adenosine monophosphate (c-di-AMP) is a recently discovered signaling molecule important for the survival of Firmicutes, a large bacterial group that includes notable pathogens such as Staphylococcus aureus However, the exact role of this molecule has not been identified. dacA , the S. aureus gene encoding the diadenylate cyclase enzyme required for c-di-AMP production, cannot be deleted when bacterial cells are grown in rich medium, indicating that c-di-AMP is required for growth in this condition. Here, we report that an S. aureus dacA mutant can be generated in chemically defined medium. Consistent with previous findings, this mutant had a severe growth defect when cultured in rich medium. Using this growth defect in rich medium, we selected for suppressor strains with improved growth to identify c-di-AMP-requiring pathways. Mutations bypassing the essentiality of dacA were identified in alsT and opuD, encoding a predicted amino acid and osmolyte transporter, the latter of which we show here to be the main glycine betaine-uptake system in S. aureus. Inactivation of these transporters likely prevents the excessive osmolyte and amino acid accumulation in the cell, providing further evidence for a key role of c-di-AMP in osmotic regulation. Suppressor mutations were also obtained in hepS, hemB, ctaA, and qoxB, coding proteins required for respiration. Furthermore, we show that dacA is dispensable for growth in anaerobic conditions. Together, these findings reveal an essential role for the c-di-AMP signaling network in aerobic, but not anaerobic, respiration in S. aureus . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Lack of effect of the alpha2C-adrenoceptor Del322-325 polymorphism on inhibition of cyclic AMP production in HEK293 cells.

    Science.gov (United States)

    Montgomery, M D; Bylund, D B

    2010-02-01

    The alpha(2C)-adrenoceptor has multiple functions, including inhibiting release of noradrenaline from presynaptic nerve terminals. A human alpha(2C) polymorphism, Del322-325, a potential risk factor for heart failure, has been reported to exhibit reduced signalling in CHO cells. To further understand the role of the Del322-325 polymorphism on receptor signalling, we attempted to replicate and further study the reduced signalling in HEK293 cells. Human alpha(2C) wild-type (WT) and Del322-325 adrenoceptors were stably transfected into HEK293 cells. Radioligand binding was performed to determine affinities for both receptors. In intact cells, inhibition of forskolin-stimulated cyclic AMP production by WT and Del322-325 clones with a range of receptor densities (200-2320 fmol.mg(-1) protein) was measured following agonist treatment. Noradrenaline, brimonidine and clonidine exhibited similar binding affinities for WT and Del322-325. Brimonidine and clonidine also had similar efficacies and potencies for both receptors for the inhibition of cyclic AMP production at all receptor densities tested. A linear regression analysis comparing efficacy and potency with receptor expression levels showed no differences in slopes between WT and Del322-325. The alpha(2C) WT and Del322-325 adrenoceptors exhibited similar binding properties. Additionally, inhibition of cyclic AMP production by Del322-325 was similar to that of WT over a range of receptor densities. Therefore, in intact HEK293 cells, the alpha(2C)-Del322-325 polymorphism does not exhibit reduced signalling to adenylyl cyclase and may not represent a clinically important phenotype.

  13. Electrophysiological and biochemical studies of slow responses to serotonin and dopamine of snail identified neurons. Mediating role of the cyclic AMP

    International Nuclear Information System (INIS)

    Deterre, Philippe

    1983-01-01

    In this research thesis, the electrophysiological study of slow incoming currents induced in some identified neurons of the Helix aspersa snail by serotonin and dopamine shows that they are associated with a decrease of a potassium conductance involved in the modulation of the action potential duration. By means of enzymatic tests performed on a single cell, and of electrophysiological experiments, the author shows that the cyclic AMP is an intracellular mediator involved in the genesis of these slow responses. Moreover, the obtained results show that serotonin and dopamine act by binding to specific receptors, and that these receptors activate the adenylate-cyclase through a GTP binding protein [fr

  14. Type I interferon induction by Neisseria gonorrhoeae: Dual requirement of cyclic GMP-AMP synthase and Toll-like receptor 4

    OpenAIRE

    Andrade, Warrison A.; Agarwal, Sarika; Mo, Shunyan; Shaffer, Scott A.; Dillard, Joseph P.; Schmidt, Tobias; Hornung, Veit; Fitzgerald, Katherine A.; Kurt-Jones, Evelyn A.; Golenbock, Douglas T.

    2016-01-01

    The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC). Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN) production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS), which produces 2′3′-cGAMP and trigg...

  15. Regulation of rat hepatocyte function by P2Y receptors: focus on control of glycogen phosphorylase and cyclic AMP by 2-methylthioadenosine 5'-diphosphate.

    Science.gov (United States)

    Dixon, C Jane; Hall, John F; Webb, Tania E; Boarder, Michael R

    2004-10-01

    Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-diphosphate (2-MeSADP), an agonist at P2Y(1), P2Y(12), and P2Y(13) receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N(6)-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y(1) receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y(1) antagonists, adenosine-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](c)) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y(12) and P2Y(13) receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y(12) and P2Y(13) receptor antagonist, 2-propylthio-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y(1) receptors coupled to raised [Ca(2+)](c), and by inhibiting cyclic AMP levels by an unknown G(i)-coupled receptor subtype, distinct from P2Y(1), P2Y(12), or P2Y(13) receptors.

  16. Effect of glucagon on cyclic AMP, albumin metabolism and incorporation of 14C-leucine into proteins in isolated parenchymal rat liver cells

    DEFF Research Database (Denmark)

    Dich, J; Gluud, C N

    1976-01-01

    wet wt. This is about the rate found in the perfused liver, Glucagon (10(-8-10(-6) M) inhibited albumin secretion and the incorporation of 14C-leucine into albumin, into total proteins in the medium and into total proteins in the cell suspension. The effect of glucagon on albumin secretion...... is compatible with an effect on the rate of synthesis. A positive correlation existed between the maximal level of cyclic AMP after glucagon administration and the inhibition of both albumin secretion and the incorporation of 149leucine....

  17. Subcellular compartmentation, interdependency and dynamics of the cyclic AMP-dependent PKA subunits during pathogenic differentiation in rice blast.

    Science.gov (United States)

    Selvaraj, Poonguzhali; Tham, Hong Fai; Ramanujam, Ravikrishna; Naqvi, Naweed I

    2017-08-01

    The cAMP-dependent PKA signalling plays a central role in growth, asexual development and pathogenesis in fungal pathogens. Here, we functionally characterised RPKA, the regulatory subunit of cAMP/PKA and studied the dynamics and organisation of the PKA subunits in the rice blast pathogen Magnaporthe oryzae. The RPKA subunit was essential for proper vegetative growth, asexual sporulation and surface hydrophobicity in M. oryzae. A spontaneous suppressor mutation, SMR19, that restored growth and conidiation in the RPKA deletion mutant was isolated and characterised. SMR19 enhanced conidiation and appressorium formation but failed to suppress the pathogenesis defects in rpkAΔ. The PKA activity was undetectable in the mycelial extracts of SMR19, which showed a single mutation (val242leu) in the highly conserved active site of the catalytic subunit (CPKA) of cAMP/PKA. The two subunits of cAMP/PKA showed different subcellular localisation patterns with RpkA being predominantly nucleocytoplasmic in conidia, while CpkA was largely cytosolic and/or vesicular. The CpkA anchored RpkA in cytoplasmic vesicles, and localisation of PKA in the cytoplasm was governed by CpkA in a cAMP-dependant or independent manner. We show that there exists a tight regulation of PKA subunits at the level of transcription, and the cAMP signalling is differentially compartmentalised in a stage-specific manner in rice blast. © 2017 John Wiley & Sons Ltd.

  18. Biochemical studies on the DNA binding function of the cyclic-amp reactor protein of Escherichia coli

    International Nuclear Information System (INIS)

    Angulo, J.A.

    1986-01-01

    The cAMP receptor protein (CRP) is an allosteric protein in which binding of cAMP effects a conformational change with a consequent increased affinity for DNA. Binding of double-stranded deoxyribopolynucleotides and calf thymus DNA by cAMP-CRP confers protection against attack by trypsin, subtilisin, Staph. aureus V8 protease and clostripain. Of the single-stranded deoxy- and ribopolynucleotides tested, only r(I)/sub n/ and r(A)/sub n/ gave significant protection against attack by these proteases. In the absence of cAMP, CRP is resistant to proteolysis. Incubation of CRP-DNA with trypsin results in the accumulation of two novel fragments. CRP-DNA is partially sensitive to digestion by chymotrypsin but resistant to attack by subtilisin, the Staph. aureus V8 protease and clostripain. Cleavage of CRP-DNA to fragments is accompanied by the loss of 3 H-cAMP binding activity. Modification of the arginines with phenylglyoxal or butanedione results in loss of DNA binding activity. cAMP-CRP incorporates more 14 C-phenylglyoxal than unliganded CRP. Titration of the arginines with 14 C-phenylglyoxal to where over 90% of the DNA binding activity is lost results in incorporation of one mole of reagent per mole of subunit

  19. Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase

    OpenAIRE

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L.; Zillinger, Thomas; Serganov, Artem A.; Liu, Yizhou; Jones, Roger A.; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J.

    2013-01-01

    Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages, designated c[G(2′,5′) pA(3′,5′)p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, res...

  20. Purine 3':5'-cyclic nucleotides with the nucleobase in a syn orientation: cAMP, cGMP and cIMP.

    Science.gov (United States)

    Řlepokura, Katarzyna Anna

    2016-06-01

    Purine 3':5'-cyclic nucleotides are very well known for their role as the secondary messengers in hormone action and cellular signal transduction. Nonetheless, their solid-state conformational details still require investigation. Five crystals containing purine 3':5'-cyclic nucleotides have been obtained and structurally characterized, namely adenosine 3':5'-cyclic phosphate dihydrate, C10H12N5O6P·2H2O or cAMP·2H2O, (I), adenosine 3':5'-cyclic phosphate 0.3-hydrate, C10H12N5O6P·0.3H2O or cAMP·0.3H2O, (II), guanosine 3':5'-cyclic phosphate pentahydrate, C10H12N5O7P·5H2O or cGMP·5H2O, (III), sodium guanosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H11N5O7P(-)·4H2O or Na(cGMP)·4H2O, (IV), and sodium inosine 3':5'-cyclic phosphate tetrahydrate, Na(+)·C10H10N4O7P(-)·4H2O or Na(cIMP)·4H2O, (V). Most of the cyclic nucleotide zwitterions/anions [two from four cAMP present in total in (I) and (II), cGMP in (III), cGMP(-) in (IV) and cIMP(-) in (V)] are syn conformers about the N-glycosidic bond, and this nucleobase arrangement is accompanied by Crib-H...Npur hydrogen bonds (rib = ribose and pur = purine). The base orientation is tuned by the ribose pucker. An analysis of data obtained from the Cambridge Structural Database made in the context of syn-anti conformational preferences has revealed that among the syn conformers of various purine nucleotides, cyclic nucleotides and dinucleotides predominate significantly. The interactions stabilizing the syn conformation have been indicated. The inter-nucleotide contacts in (I)-(V) have been systematized in terms of the chemical groups involved. All five structures display three-dimensional hydrogen-bonded networks.

  1. Cyclic-AMP mediated drugs: differential or global reduction of eicosanoid synthesis in the isolated rat lung?

    Directory of Open Access Journals (Sweden)

    Mark J. Post

    1992-01-01

    Full Text Available In this study the question was addressed whether cAMP mediated drugs induce a differential reduction of branches of the arachidonic acid metabolism rather than a global reduction of eicosanoid synthesis. The isolated lungs of actively sensitized rats were employed to study prostaglandin and leukotriene release in the presence and absence of the cAMP mediated drugs theophylline, milrinone, sulmazole, isobutyl-methylxanthine and salbutamol. The release of eicosanoids as measured by RIA was predominantly basal and continuous, with a mild antigen induced stimulation only for TXB2 and the leukotrienes. All drugs reduced eicosanoid release globally. It is concluded that cAMP mediated drugs interfere with arachidonic acid metabolism at a site proximal to the branching into lipoxygenase and cyclo-oxygenase pathways.

  2. New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus.

    Science.gov (United States)

    Bowman, Lisa; Zeden, Merve S; Schuster, Christopher F; Kaever, Volkhard; Gründling, Angelika

    2016-12-30

    Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Cyclic AMP induces apoptosis in multiple myeloma cells and inhibits tumor development in a mouse myeloma model

    International Nuclear Information System (INIS)

    Follin-Arbelet, Virginie; Hofgaard, Peter O; Hauglin, Harald; Naderi, Soheil; Sundan, Anders; Blomhoff, Rune; Bogen, Bjarne; Blomhoff, Heidi K

    2011-01-01

    Multiple myeloma is an incurable disease requiring the development of effective therapies which can be used clinically. We have elucidated the potential for manipulating the cAMP signaling pathway as a target for inhibiting the growth of multiple myeloma cells. As a model system, we primarily used the murine multiple myeloma cell line MOPC315 which can be grown both in vivo and in vitro. Human multiple myeloma cell lines U266, INA-6 and the B-cell precursor acute lymphoblastic leukemia cell line Reh were used only for in vitro studies. Cell death was assessed by flow cytometry and western blot analysis after treatment with cAMP elevating agents (forskolin, prostaglandin E2 and rolipram) and cAMP analogs. We followed tumor growth in vivo after forskolin treatment by imaging DsRed-labelled MOPC315 cells transplanted subcutaneously in BALB/c nude mice. In contrast to the effect on Reh cells, 50 μM forskolin more than tripled the death of MOPC315 cells after 24 h in vitro. Forskolin induced cell death to a similar extent in the human myeloma cell lines U266 and INA-6. cAMP-mediated cell death had all the typical hallmarks of apoptosis, including changes in the mitochondrial membrane potential and cleavage of caspase 3, caspase 9 and PARP. Forskolin also inhibited the growth of multiple myeloma cells in a mouse model in vivo. Elevation of intracellular levels of cAMP kills multiple myeloma cells in vitro and inhibits development of multiple myeloma in vivo. This strongly suggests that compounds activating the cAMP signaling pathway may be useful in the field of multiple myeloma

  4. cAMP control of HCN2 channel Mg2+ block reveals loose coupling between the cyclic nucleotide-gating ring and the pore.

    Directory of Open Access Journals (Sweden)

    Alex K Lyashchenko

    Full Text Available Hyperpolarization-activated cyclic nucleotide-regulated HCN channels underlie the Na+-K+ permeable IH pacemaker current. As with other voltage-gated members of the 6-transmembrane KV channel superfamily, opening of HCN channels involves dilation of a helical bundle formed by the intracellular ends of S6 albeit this is promoted by inward, not outward, displacement of S4. Direct agonist binding to a ring of cyclic nucleotide-binding sites, one of which lies immediately distal to each S6 helix, imparts cAMP sensitivity to HCN channel opening. At depolarized potentials, HCN channels are further modulated by intracellular Mg2+ which blocks the open channel pore and blunts the inhibitory effect of outward K+ flux. Here, we show that cAMP binding to the gating ring enhances not only channel opening but also the kinetics of Mg2+ block. A combination of experimental and simulation studies demonstrates that agonist acceleration of block is mediated via acceleration of the blocking reaction itself rather than as a secondary consequence of the cAMP enhancement of channel opening. These results suggest that the activation status of the gating ring and the open state of the pore are not coupled in an obligate manner (as required by the often invoked Monod-Wyman-Changeux allosteric model but couple more loosely (as envisioned in a modular model of protein activation. Importantly, the emergence of second messenger sensitivity of open channel rectification suggests that loose coupling may have an unexpected consequence: it may endow these erstwhile "slow" channels with an ability to exert voltage and ligand-modulated control over cellular excitability on the fastest of physiologically relevant time scales.

  5. Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP signaling to inhibit steroidogenesis in human granulosa cells

    Directory of Open Access Journals (Sweden)

    HoYuen Basil

    2009-10-01

    Full Text Available Abstract Background Obesity has been linked to an increased risk of female infertility. Leptin, an adipocytokine which is elevated during obesity, may influence gonadal function through modulating steroidogenesis in granulosa cells. Methods The effect of leptin on progesterone production in simian virus 40 immortalized granulosa (SVOG cells was examined by Enzyme linked immunosorbent assay (ELISA. The effect of leptin on the expression of the steroidogenic enzymes (StAR, P450scc, 3betaHSD in SVOG cells was examined by real-time PCR and Western blotting. The mRNA expression of leptin receptor isoforms in SVOG cells were examined by using PCR. SVOG cells were co-treated with leptin and specific pharmacological inhibitors to identify the signaling pathways involved in leptin-reduced progesterone production. Silencing RNA against leptin receptor was used to determine that the inhibition of leptin on cAMP-induced steroidogenesis acts in a leptin receptor-dependent manner. Results and Conclusion In the present study, we investigated the cellular mechanisms underlying leptin-regulated steroidogenesis in human granulosa cells. We show that leptin inhibits 8-bromo cAMP-stimulated progesterone production in a concentration-dependent manner. Furthermore, we show that leptin inhibits expression of the cAMP-stimulated steroidogenic acute regulatory (StAR protein, the rate limiting de novo protein in progesterone synthesis. Leptin induces the activation of ERK1/2, p38 and JNK but only the ERK1/2 (PD98059 and p38 (SB203580 inhibitors attenuate the leptin-induced inhibition of cAMP-stimulated StAR protein expression and progesterone production. These data suggest that the leptin-induced MAPK signal transduction pathway interferes with cAMP/PKA-stimulated steroidogenesis in human granulosa cells. Moreover, siRNA mediated knock-down of the endogenous leptin receptor attenuates the effect of leptin on cAMP-induced StAR protein expression and progesterone

  6. Modulation of cyclic amp-dependent protein kinase isozyme expression associated with activation of a macrophage cell line

    International Nuclear Information System (INIS)

    Justement, L.B.; Aldrich, W.A.; Wenger, G.D.; O'Dorisio, M.S.; Zwilling, B.S.

    1986-01-01

    The RAW 264.7 macrophage (MO) cell line was used to study cAMPdPK isozymes during activation by lymphokine (LK) and lipopolysaccharide (LPS). Untreated cells were found to have two isozymes of cAMPdPK in their cytosol. PKI and PKII were differentiated based on the M/sub r/ of their regulatory subunits (RI, 45,500; and RII, 52,000, respectively) as determined by photoactivated incorporation of the cAMP analog 8-N 3 -[ 32 P]cAMP. Loss of the RI subunit of PKI occurred in association with activation of the cell line by suboptimal concentrations of LK and LPS. No modulation of the RII subunit of PKII was observed under these conditions. The addition of a suboptimal concentration of LPS after LK or a high dose of LPS alone was required for acquisition of cytolytic activity and loss of RI. The antitumor activity of the RAW 264.7 cell line was transiently expressed after activation. Cells no longer exhibited tumoricidal activity 48 hr after the removal of activating agents. It was observed that the loss of cytolytic function was accompanied by the reexpression of RI in the cytosol. This study provides evidence that modulation of cAMPdPK isozymes occurs during activation, suggesting a potential mechanism for controlling the effects of cAMP on the MO

  7. Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

    Science.gov (United States)

    Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

    1994-10-25

    DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.

  8. Inhibition of insulin release by cyproheptadine: Effects on 3',5'-cyclic-AMP-content and /sup 45/Ca-accumulation of incubated mouse islets

    Energy Technology Data Exchange (ETDEWEB)

    Joost, H G; Beckmann, J; Lenzen, S; Hasselblatt, A [Goettingen Univ. (F.R. Germany)

    1976-01-01

    Cyproheptadine (1, 10 and 100 ..mu..m) significantly reduced insulin release from isolated mouse islets in response to glucose. In contrast, 1 mM cyproheptadine induced a large release of insulin into the incubation medium probably due to islet cell damage, since the islets had lost a considerable amount of their protein content. 3',5'-cyclic-AMP-levels of the islets were not significantly affected by 10 ..mu..M cyproheptadine in the presence as well as in the absence of theophylline (10 mM). As the inhibitory effect of cyproheptadine on insulin release was correlated with reduced accumulation of calcium-45, the agent may inhibit insulin release by interfering with the calcium handling of the ..beta..-cell.

  9. Transporter function and cyclic AMP turnover in normal colonic mucosa from patients with and without colorectal neoplasia

    DEFF Research Database (Denmark)

    Kleberg, Karen; Jensen, Gerda Majgaard; Christensen, Dan Ploug

    2012-01-01

    The pathogenesis of colorectal neoplasia is still unresolved but has been associated with alterations in epithelial clearance of xenobiotics and metabolic waste products. The aim of this study was to functionally characterize the transport of cyclic nucleotides in colonic biopsies from patients...

  10. L-4F Inhibits Oxidized Low-density Lipoprotein-induced Inflammatory Adipokine Secretion via Cyclic AMP/Protein Kinase A-CCAAT/Enhancer Binding Protein β Signaling Pathway in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Xiang-Zhu Xie

    2016-01-01

    Conclusions: OxLDL induces C/EBPβ protein synthesis in a time-dependent manner and enhances MCP-1 secretion and expression in 3T3-L1 adipocytes. L-4F dose-dependently counterbalances the pro-inflammatory effect of oxLDL, and cyclic AMP/PKA-C/EBPβ signaling pathway may participate in it.

  11. A family of octopamine [corrected] receptors that specifically induce cyclic AMP production or Ca2+ release in Drosophila melanogaster.

    Science.gov (United States)

    Balfanz, Sabine; Strünker, Timo; Frings, Stephan; Baumann, Arnd

    2005-04-01

    In invertebrates, the biogenic-amine octopamine is an important physiological regulator. It controls and modulates neuronal development, circadian rhythm, locomotion, 'fight or flight' responses, as well as learning and memory. Octopamine mediates its effects by activation of different GTP-binding protein (G protein)-coupled receptor types, which induce either cAMP production or Ca(2+) release. Here we describe the functional characterization of two genes from Drosophila melanogaster that encode three octopamine receptors. The first gene (Dmoa1) codes for two polypeptides that are generated by alternative splicing. When heterologously expressed, both receptors cause oscillatory increases of the intracellular Ca(2+) concentration in response to applying nanomolar concentrations of octopamine. The second gene (Dmoa2) codes for a receptor that specifically activates adenylate cyclase and causes a rise of intracellular cAMP with an EC(50) of approximately 3 x 10(-8) m octopamine. Tyramine, the precursor of octopamine biosynthesis, activates all three receptors at > or = 100-fold higher concentrations, whereas dopamine and serotonin are non-effective. Developmental expression of Dmoa genes was assessed by RT-PCR. Overlapping but not identical expression patterns were observed for the individual transcripts. The genes characterized in this report encode unique receptors that display signature properties of native octopamine receptors.

  12. Interplay of the modified nucleotide phosphoadenosine 5'-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein.

    Science.gov (United States)

    Longo, Francesca; Motta, Sara; Mauri, Pierluigi; Landini, Paolo; Rossi, Elio

    2016-11-25

    In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. Secretion of [Met]enkephalyl-Arg6-Phe7-related peptides and catecholamines from bovine adrenal chromaffin cells: modification by changes in cyclic AMP and by treatment with reserpine.

    Science.gov (United States)

    Adams, M; Boarder, M R

    1987-07-01

    Investigations into the effects of culturing bovine adrenal chromaffin cells in the presence (72 h) of dibutyryl cyclic AMP, forskolin, and reserpine on the level and release of [Met]enkephalyl-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline are reported. The assay for [Met]enkephalyl-Arg6-Phe7 immunoreactivity recognises both peptide B, the 31-amino acid carboxy-terminal segment of proenkephalin, and its heptapeptide fragment, [Met]enkephalyl-Arg6-Phe7. Treatments that elevate cyclic AMP increase the amount of peptide immunoreactivity in these cells; this is predominantly peptide B-like immunoreactivity in both control cells and cyclic AMP-elevated cells. Treatment with reserpine gives no change in total immunoreactivity levels, but does not result in increased accumulation of the heptapeptide [Met]enkephalyl-Arg6-Phe7 at the expense of immunoreactivity that elutes with its immediate precursor, peptide B. Cyclic AMP treatment causes either no change or a decrease in levels of accumulated noradrenaline and adrenaline. However, the release of [Met]enkephalin-Arg6-Phe7 immunoreactivity, noradrenaline, and adrenaline is increased by 72-h pretreatment with forskolin or dibutyryl cyclic AMP, whether release is stimulated by nicotine or elevated potassium. In each case the molecular form of [Met]enkephalyl-Arg6-Phe7 immunoreactivity that is released approximately reflects the cell content. Pretreatment with reserpine has no effect on the total [Met]enkephalyl-Arg6-Phe7 immunoreactivity released, but does result in an increased release of the heptapeptide and a decrease in release of peptide B-like immunoreactivity. The studies suggest that the levels of [Met]enkephalyl-Arg6-Phe7 and peptide B available for release are controlled both at the level of proenkephalin synthesis and at the level of double-basic residue proteolysis.

  14. Evidence for a cyclic AMP-dependent pathway in angiotensin AT1-receptor activation of human omental arteries

    Directory of Open Access Journals (Sweden)

    Hoa Ytterberg

    2001-03-01

    Full Text Available Enhanced responses to vasoconstriction induced by neuropeptide Y and α2-adrenoceptor agonists have been seen following pharmacological activation of the adenylyl cyclase (AC system. Since preliminary studies revealed only minor responses to angiotensin II (Ang II in human omental arteries, we have investigated whether enhanced activity of AC may unravel further functional Ang II receptors. Human omental arteries were obtained in conjunction with elective gut surgery. After dissection of the vessel, the endothelium was removed by 10 sec of Triton X-100 treatment. Ring segments (1—2 mm long were mounted on a myograph and studied. Ang II produced small contractions, 27±5% relative to the response elicited by 60 mM K+. However, enhanced Ang II (105±10%, p<0.001 responses were seen during AC activation by forskolin (0.1—1 µM. This enhanced contractile response to Ang II was not inhibited by the angiotensin II type 2 (AT2-receptor antagonist PD 123319 (0.1 µM, but was blocked in an insurmountable way by the angiotensin II type 1 (AT1-receptor antagonist candesartan (1 nM and in a surmountable manner by losartan (0.1 µM and irbesartan (0.1 µM. Pertussis toxin (a Gi-protein blocker and the protein kinase C inhibitor, RO31—8220 (0.01, 0.1 and 1 µM, markedly reduced this response, while the protein kinase A inhibitor, H89 (1, 10 µM, had no effect. RT-PCR provided evidence for the presence of mRNA for both AT1- and AT2-receptors. The results suggest that both a cAMP-dependent and a cAMP-independent mechanism are involved in the contractile responses to Ang II in human omental arteries and that both responses are mediated via the AT1-receptor.

  15. Propofol reduced myocardial contraction of vertebrates partly by mediating the cyclic AMP-dependent protein kinase phosphorylation pathway

    International Nuclear Information System (INIS)

    Sun, Xiaotong; Zhang, Xinyu; Bo, Qiyu; Meng, Tao; Lei, Zhen; Li, Jingxin; Hou, Yonghao; Yu, Xiaoqian; Yu, Jingui

    2016-01-01

    Propofol inhibits myocardial contraction in a dose dependent manner. The present study is designed to examine the effect of propofol on PKA mediated myocardial contraction in the absence of adrenoreceptor agonist. The contraction of isolated rat heart was measured in the presence or absence of PKA inhibitor H89 or propofol, using a pressure transducer. The levels of cAMP and PKA kinase activity were detected by ELISA. The mRNA and total protein or phosphorylation level of PKA and downstream proteins were tested in the presence or absence of PKA inhibitor H89 or propofol, using RT-PCR, QPCR and western blotting. The phosphorylation level of PKA was examined thoroughly using immunofluorescence and PKA activity non-radioactive detection kit. Propofol induced a dose-dependent negative contractile response on the rat heart. The inhibitory effect of high concentration propofol (50 μM) with 45% decease of control could be partly reversed by the PKA inhibitor H89 (10 μM) and the depressant effect of propofol decreased from 45% to 10%. PKA kinase activity was inhibited by propofol in a dose-dependent manner. Propofol also induced a decrease in phosphorylation of PKA, which was also inhibited by H89, but did not alter the production of cAMP and the mRNA levels of PKA. The downstream proteins of PKA, PLN and RyR2 were phosphorylated to a lesser extent with propofol or H89 than control. These results demonstrated that propofol induced a negative myocardial contractile response partly by mediating the PKA phosphorylation pathway.

  16. Cyclic [G(2′,5′)pA(3′,5′)p] Is the Metazoan Second Messenger Produced by DNA-Activated Cyclic GMP-AMP Synthase

    Science.gov (United States)

    Gao, Pu; Ascano, Manuel; Wu, Yang; Barchet, Winfried; Gaffney, Barbara L.; Zillinger, Thomas; Serganov, Artem A.; Liu, Yizhou; Jones, Roger A.; Hartmann, Gunther; Tuschl, Thomas; Patel, Dinshaw J.

    2015-01-01

    SUMMARY Recent studies identified cyclic GMP-AMP (cGAMP) as a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAMP synthase (cGAS). We combined structural, chemical, biochemical, and cellular assays to demonstrate that this second messenger contains G(2′,5′)pA and A(3′,5′)pG phosphodiester linkages, designated c[G(2′,5′) pA(3′,5′)p]. We show that, upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide-binding pocket for generation of c[G(2′,5′)pA(3′,5′)p]. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5′-pppG(2′,5′)pA prior to cyclization to c[G(2′,5′)pA(3′,5′)p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity. Our studies have identified c[G(2′,5′)pA(3′,5′)p] as a founding member of a family of metazoan 2′,5′-containing cyclic heterodinucleotide second messengers distinct from bacterial 3′,5′ cyclic dinucleotides. PMID:23647843

  17. Cyclic AMP-dependent signaling system is a primary metabolic target for non-thermal effect of microwaves on heart muscle hydration.

    Science.gov (United States)

    Narinyan, Lilia; Ayrapetyan, Sinerik

    2017-01-01

    Previously, we have suggested that cell hydration is a universal and extra-sensitive sensor for the structural changes of cell aqua medium caused by the impact of weak chemical and physical factors. The aim of present work is to elucidate the nature of the metabolic messenger through which physiological solution (PS) treated by non-thermal (NT) microwaves (MW) could modulate heart muscle hydration of rats. For this purpose, the effects of NT MW-treated PS on heart muscle hydration, [ 3 H]-ouabain binding with cell membrane, 45 Ca 2+ uptake and intracellular cyclic nucleotides contents in vivo and in vitro experiments were studied. It is shown that intraperitoneal injections of both Sham-treated PS and NT MW-treated PS elevate heart muscle hydration. However, the effect of NT MW-treated PS on muscle hydration is more pronounced than the effect of Sham-treated PS. In vitro experiments NT MW-treated PS has dehydration effect on muscle, which is not changed by decreasing Na + gradients on membrane. Intraperitoneal injection of Sham- and NT MW-treated PS containing 45 Ca 2+ have similar dehydration effect on muscle, while NT MW-treated PS has activation effect on Na + /Ca 2+ exchange in reverse mode. The intraperitoneal injection of NT MW-treated PS depresses [ 3 H]-ouabain binding with its high-affinity membrane receptors, elevates intracellular cAMP and decreases cGMP contents. Based on the obtained data, it is suggested that cAMP-dependent signaling system serves as a primary metabolic target for NT MW effect on heart muscle hydration.

  18. Caffeine Modulates Vesicle Release and Recovery at Cerebellar Parallel Fibre Terminals, Independently of Calcium and Cyclic AMP Signalling

    Science.gov (United States)

    Dobson, Katharine L.; Jackson, Claire; Balakrishnan, Saju; Bellamy, Tomas C.

    2015-01-01

    Background Cerebellar parallel fibres release glutamate at both the synaptic active zone and at extrasynaptic sites—a process known as ectopic release. These sites exhibit different short-term and long-term plasticity, the basis of which is incompletely understood but depends on the efficiency of vesicle release and recycling. To investigate whether release of calcium from internal stores contributes to these differences in plasticity, we tested the effects of the ryanodine receptor agonist caffeine on both synaptic and ectopic transmission. Methods Whole cell patch clamp recordings from Purkinje neurons and Bergmann glia were carried out in transverse cerebellar slices from juvenile (P16-20) Wistar rats. Key Results Caffeine caused complex changes in transmission at both synaptic and ectopic sites. The amplitude of postsynaptic currents in Purkinje neurons and extrasynaptic currents in Bergmann glia were increased 2-fold and 4-fold respectively, but paired pulse ratio was substantially reduced, reversing the short-term facilitation observed under control conditions. Caffeine treatment also caused synaptic sites to depress during 1 Hz stimulation, consistent with inhibition of the usual mechanisms for replenishing vesicles at the active zone. Unexpectedly, pharmacological intervention at known targets for caffeine—intracellular calcium release, and cAMP signalling—had no impact on these effects. Conclusions We conclude that caffeine increases release probability and inhibits vesicle recovery at parallel fibre synapses, independently of known pharmacological targets. This complex effect would lead to potentiation of transmission at fibres firing at low frequencies, but depression of transmission at high frequency connections. PMID:25933382

  19. 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

    Directory of Open Access Journals (Sweden)

    Benjamin A. Diner

    2016-11-01

    Full Text Available 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.

  20. Protein kinases mediate increment of the phosphorylation of cyclic AMP -responsive element binding protein in spinal cord of rats following capsaicin injection

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    Li Junfa

    2005-09-01

    Full Text Available Abstract Background Strong noxious stimuli cause plastic changes in spinal nociceptive neurons. Intracellular signal transduction pathways from cellular membrane to nucleus, which may further regulate gene expression by critical transcription factors, convey peripheral stimulation. Cyclic AMP-responsive element binding protein (CREB is a well-characterized stimulus-induced transcription factor whose activation requires phosphorylation of the Serine-133 residue. Phospho-CREB can further induce gene transcription and strengthen synaptic transmission by the activation of the protein kinase cascades. However, little is known about the mechanisms by which CREB phosphorylation is regulated by protein kinases during nociception. This study was designed to use Western blot analysis to investigate the role of mitogen-activated protein (MAP/extracellular signal-regulated kinase (ERK kinase (MEK 1/2, PKA and PKC in regulating the phosphorylation of CREB in the spinal cord of rats following intraplantar capsaicin injection. Results We found that capsaicin injection significantly increased the phosphorylation level of CREB in the ipsilateral side of the spinal cord. Pharmacological manipulation of MEK 1/2, PKA and PKC with their inhibitors (U0126, H89 and NPC 15473, respectively significantly blocked this increment of CREB phosphorylation. However, the expression of CREB itself showed no change in any group. Conclusion These findings suggest that the activation of intracellular MAP kinase, PKA and PKC cascades may contribute to the regulation of phospho-CREB in central nociceptive neurons following peripheral painful stimuli.

  1. Type I Interferon Induction by Neisseria gonorrhoeae: Dual Requirement of Cyclic GMP-AMP Synthase and Toll-like Receptor 4.

    Science.gov (United States)

    Andrade, Warrison A; Agarwal, Sarika; Mo, Shunyan; Shaffer, Scott A; Dillard, Joseph P; Schmidt, Tobias; Hornung, Veit; Fitzgerald, Katherine A; Kurt-Jones, Evelyn A; Golenbock, Douglas T

    2016-06-14

    The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC). Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN) production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS), which produces 2'3'-cGAMP and triggers STING/TBK-1/IRF3 activation, resulting in type I IFN expression. Here, we reveal a cytosolic response to GC DNA that also contributes to type I IFN induction. We demonstrate that complete IFN-β induction by live GC depends on both cGAS and TLR4. Type I IFN is detrimental to the host, and dysregulation of iron homeostasis genes may explain lower bacteria survival in cGAS(-/-) and TLR4(-/-) cells. Collectively, these observations reveal cooperation between TLRs and cGAS in immunity to GC infection. Copyright © 2016. Published by Elsevier Inc.

  2. Cyclic GMP-AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria.

    Science.gov (United States)

    Gallego-Marin, Carolina; Schrum, Jacob E; Andrade, Warrison A; Shaffer, Scott A; Giraldo, Lina F; Lasso, Alvaro M; Kurt-Jones, Evelyn A; Fitzgerald, Katherine A; Golenbock, Douglas T

    2018-01-15

    Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-β induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites. Copyright © 2018 by The American Association of Immunologists, Inc.

  3. Type I Interferon Induction by Neisseria gonorrhoeae: Dual Requirement of Cyclic GMP-AMP Synthase and Toll-like Receptor 4

    Directory of Open Access Journals (Sweden)

    Warrison A. Andrade

    2016-06-01

    Full Text Available The innate immune system is the first line of defense against Neisseria gonorrhoeae (GC. Exposure of cells to GC lipooligosaccharides induces a strong immune response, leading to type I interferon (IFN production via TLR4/MD-2. In addition to living freely in the extracellular space, GC can invade the cytoplasm to evade detection and elimination. Double-stranded DNA introduced into the cytosol binds and activates the enzyme cyclic-GMP-AMP synthase (cGAS, which produces 2′3′-cGAMP and triggers STING/TBK-1/IRF3 activation, resulting in type I IFN expression. Here, we reveal a cytosolic response to GC DNA that also contributes to type I IFN induction. We demonstrate that complete IFN-β induction by live GC depends on both cGAS and TLR4. Type I IFN is detrimental to the host, and dysregulation of iron homeostasis genes may explain lower bacteria survival in cGAS−/− and TLR4−/− cells. Collectively, these observations reveal cooperation between TLRs and cGAS in immunity to GC infection.

  4. Binding of the cyclic AMP receptor protein of Escherichia coli and DNA bending at the P4 promoter of pBR322.

    Science.gov (United States)

    Brierley, I; Hoggett, J G

    1992-07-01

    The binding of the Escherichia coli cyclic AMP receptor protein (CRP) to its specific site on the P4 promoter of pBR322 has been studied by gel electrophoresis. Binding to the P4 site was about 40-50-fold weaker than to the principal CRP site on the lactose promoter at both low (0.01 M) and high (0.1 M) ionic strengths. CRP-induced bending at the P4 site was investigated from the mobilities of CRP bound to circularly permuted P4 fragments. The estimated bending angle, based on comparison with Zinkel & Crothers [(1990) Biopolymers 29, 29-38] A-tract bending standards, was found to be approximately 96 degrees, similar to that found for binding to the lac site. These observations suggest that there is not a simple relationship between strength of CRP binding and the extent of induced bending for different CRP sites. The apparent centre of bending in P4 is displaced about 6-8 bp away from the conserved TGTGA sequence and the P4 transcription start site.

  5. Cyclic AMP-dependent protein kinase interferes with GTP γS stimulated IP3 formation in differentiated HL-60 cell membranes

    International Nuclear Information System (INIS)

    Misaki, Naoyuki; Imaizumi, Taro; Watanabe, Yashuiro

    1989-01-01

    The effects of addition of activated cyclic AMP-dependent protein kinase (PKA) on the function of islet-activating protein (IAP)-sensitive GTP-binding (G) protein were studied in the plasma membranes of 3 H-inositol-labeled differentiated human leukemic (HL-60) cells. Pretreatment of the membranes with activated PKA in the presence of MgATP for 15 min. at 37 degree C decreased GTP γS-stimulated inositol trisphosphate (IP 3 ) formation by about 30%, but had no influence on Ca 2+ -stimulated IP 3 formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some 32 P incorporated bands, and among them one of the major bands showed the migration at 40 kDa supporting that the G protein coupling with PI response was phosphorylated by PKA. These results showed that pretreatment with activated PKA inhibited the mediating function of the G protein between the fMLP receptor and phospholipase C by its phosphorylation

  6. Met-enkephalyl-Arg6-Phe7 immunoreactivity in a human neuroblastoma cell line: effect of dibutyryl 3':5'-cyclic AMP and reserpine.

    Science.gov (United States)

    Boarder, M R; Marriott, D; Adams, M

    1986-12-30

    The carboxy terminal part of the proenkephalin A sequence is the 31 amino acid peptide B, which has as its final seven amino acids the sequence of the opioid peptide Met-enkephalyl-Arg6-Phe7. Using a radioimmunoassay which recognises both these peptides we have investigated the relative amounts of peptide B and Met-enkephalyl-Arg6-Phe7 in a human neuroblastoma cell line. We show that these cells contain peptide B-like immunoreactivity but not its heptapeptide fragment. This may be due to lack of proteolytic activity cleaving Met-enkephalyl-Arg6-Phe7 from its precursor, peptide B. On treatment with dibutyryl cyclic AMP the level of immunoreactivity approximately doubles, due to increased amounts of peptide B-like immunoreactivity. Treatment with reserpine, which increases conversion of peptide B to the heptapeptide in bovine chromaffin cells in culture does not stimulate the accumulation of Met-enkephalyl-Arg6-Phe7 in the human neuroblastoma cells. The results are discussed with respect to peptide processing.

  7. Regulation of the Vibrio vulnificus hupA gene by temperature alteration and cyclic AMP receptor protein and evaluation of its role in virulence.

    Science.gov (United States)

    Oh, Man Hwan; Lee, Sung Min; Lee, Dong Hwan; Choi, Sang Ho

    2009-03-01

    Availability of free iron is extremely limited in the mammalian host, and the acquisition of iron in the host is essential for successful infection by pathogenic bacteria. Expression of many genes involved in acquiring iron is regulated in response to the level of iron availability, and iron regulation is mediated by Fur. In this study, cellular levels of Vibrio vulnificus HupA, a heme receptor protein, and the hupA transcript were found to increase in cells grown at 40 degrees C compared to cells grown at 30 degrees C. The results suggested that change in growth temperature, in addition to iron availability, is an environmental cue controlling the expression of the hupA gene. The influence of global regulatory proteins on the expression of hupA was examined, and the cyclic AMP receptor protein (CRP) was found to activate the expression of hupA at the transcriptional level. CRP exerts its effects by directly binding to DNA upstream of the hupA promoter P(hupA), and a CRP binding site, centered at 174 bp upstream of the transcription start site, was identified by a DNase I protection assay. Finally, a hupA mutant showed reduced virulence in mice and in tissue cultures, in which growth of the hupA mutant was impaired, indicating that HupA of V. vulnificus is essential for survival and multiplication during infection.

  8. Regulation of the Vibrio vulnificus hupA Gene by Temperature Alteration and Cyclic AMP Receptor Protein and Evaluation of Its Role in Virulence▿

    Science.gov (United States)

    Oh, Man Hwan; Lee, Sung Min; Lee, Dong Hwan; Choi, Sang Ho

    2009-01-01

    Availability of free iron is extremely limited in the mammalian host, and the acquisition of iron in the host is essential for successful infection by pathogenic bacteria. Expression of many genes involved in acquiring iron is regulated in response to the level of iron availability, and iron regulation is mediated by Fur. In this study, cellular levels of Vibrio vulnificus HupA, a heme receptor protein, and the hupA transcript were found to increase in cells grown at 40°C compared to cells grown at 30°C. The results suggested that change in growth temperature, in addition to iron availability, is an environmental cue controlling the expression of the hupA gene. The influence of global regulatory proteins on the expression of hupA was examined, and the cyclic AMP receptor protein (CRP) was found to activate the expression of hupA at the transcriptional level. CRP exerts its effects by directly binding to DNA upstream of the hupA promoter PhupA, and a CRP binding site, centered at 174 bp upstream of the transcription start site, was identified by a DNase I protection assay. Finally, a hupA mutant showed reduced virulence in mice and in tissue cultures, in which growth of the hupA mutant was impaired, indicating that HupA of V. vulnificus is essential for survival and multiplication during infection. PMID:19139193

  9. 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.

    Science.gov (United States)

    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

  10. Bone Abnormalities in Mice with Protein Kinase A (PKA) Defects Reveal a Role of Cyclic AMP Signaling in Bone Stromal Cell-Dependent Tumor Development.

    Science.gov (United States)

    Liu, S; Shapiro, J M; Saloustros, E; Stratakis, C A

    2016-11-01

    Protein kinase A (PKA) is an important enzyme for all eukaryotic cells. PKA phosphorylates other proteins, thus, it is essential for the regulation of many diverse cellular functions, including cytoplasmic trafficking and signaling, organelle structure and mitochondrial oxidation, nuclear gene expression, the cell cycle, and cellular division. The PKA holoenzyme is composed of 2 regulatory and 2 catalytic subunits. Four regulatory (R1α, R1β, R2α, and R2β) and 4 catalytic subunits (Cα, Cβ, Cγ, and Prkx) have been identified, giving rise to mainly PKA-I (when the 2 regulatory subunits are either R1α or R1β), or PKA-II (when the 2 regulatory subunits are either R2α or R2β). Mutations in the PKA subunits can lead to altered total PKA activity or abnormal PKA-I to PKA-II ratio, leading to various abnormalities in both humans and mice. These effects can be tissue-specific. We studied the effect of PKA subunit defects on PKA activity and bone morphology of mice that were single or double heterozygous for null alleles of the various PKA subunit genes. Bone lesions including fibrous dysplasia, myxomas, osteo-sarcomas, -chondromas and -chondrosarcomas were found in these mice. Observational and molecular studies showed that these lesions were derived from bone stromal cells (BSCs). We conclude that haploinsufficiency for different PKA subunit genes affected bone lesion formation, new bone generation, organization, and mineralization in variable ways. This work identified a PKA subunit- and activity-dependent pathway of bone lesion formation from BSCs with important implications for understanding how cyclic AMP affects the skeleton and its tumorigenesis. © Georg Thieme Verlag KG Stuttgart · New York.

  11. The cyclic AMP receptor protein, CRP, is required for both virulence and expression of the minimal CRP regulon in Yersinia pestis biovar microtus.

    Science.gov (United States)

    Zhan, Lingjun; Han, Yanping; Yang, Lei; Geng, Jing; Li, Yingli; Gao, He; Guo, Zhaobiao; Fan, Wei; Li, Gang; Zhang, Lianfeng; Qin, Chuan; Zhou, Dongsheng; Yang, Ruifu

    2008-11-01

    The cyclic AMP receptor protein (CRP) is a bacterial regulator that controls more than 100 promoters, including those involved in catabolite repression. In the present study, a null deletion of the crp gene was constructed for Yersinia pestis bv. microtus strain 201. Microarray expression analysis disclosed that at least 6% of Y. pestis genes were affected by this mutation. Further reverse transcription-PCR and electrophoretic mobility shift assay analyses disclosed a set of 37 genes or putative operons to be the direct targets of CRP, and thus they constitute the minimal CRP regulon in Y. pestis. Subsequent primer extension and DNase I footprinting assays mapped transcriptional start sites, core promoter elements, and CRP binding sites within the DNA regions upstream of pla and pst, revealing positive and direct control of these two laterally acquired plasmid genes by CRP. The crp disruption affected both in vitro and in vivo growth of the mutant and led to a >15,000-fold loss of virulence after subcutaneous infection but a pestis and, particularly, is more important for infection by subcutaneous inoculation. It can further be concluded that the reduced in vivo growth phenotype of the crp mutant should contribute, at least partially, to its attenuation of virulence by both routes of infection. Consistent with a previous study of Y. pestis bv. medievalis, lacZ reporter fusion analysis indicated that the crp deletion resulted in the almost absolute loss of pla promoter activity. The plasminogen activator encoded by pla was previously shown to specifically promote Y. pestis dissemination from peripheral infection routes (subcutaneous infection [flea bite] or inhalation). The above evidence supports the notion that in addition to the reduced in vivo growth phenotype, the defect of pla expression in the crp mutant will greatly contribute to the huge loss of virulence of this mutant strain in subcutaneous infection.

  12. Presenilins Regulate Neurotrypsin Gene Expression and Neurotrypsin-dependent Agrin Cleavage via Cyclic AMP Response Element-binding Protein (CREB) Modulation*

    Science.gov (United States)

    Almenar-Queralt, Angels; Kim, Sonia N.; Benner, Christopher; Herrera, Cheryl M.; Kang, David E.; Garcia-Bassets, Ivan; Goldstein, Lawrence S. B.

    2013-01-01

    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment. PMID:24145027

  13. Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

    Science.gov (United States)

    Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

    2013-12-06

    Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

  14. Cyclic adenosine 3',5'-monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter.

    Science.gov (United States)

    Clem, Brian F; Hudson, Elizabeth A; Clark, Barbara J

    2005-03-01

    Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5'-TGACGTCA-3') within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.

  15. Cyclic AMP regulation of the human glycoprotein hormone α-subunit gene is mediated by an 18-base-pair element

    International Nuclear Information System (INIS)

    Silver, B.J.; Bokar, J.A.; Virgin, J.B.; Vallen, E.A.; Milsted, A.; Nilson, J.H.

    1987-01-01

    cAMP regulates transcription of the gene encoding the α-subunit of human chorionic gonadotropin (hCG) in the choriocarcinoma cells (BeWo). To define the sequences required for regulation by cAMP, the authors inserted fragments from the 5' flanking region of the α-subunit gene into a test vector containing the simian virus 40 early promoter (devoid of its enhancer) linked to the bacterial chloramphenicol acetyltransferase (CAT) gene. Results from transient expression assays in BeWo cells indicated that a 1500-base-pair (bp) fragment conferred cAMP responsiveness on the CAT gene regardless of position or orientation of the insert relative to the viral promoter. A subfragment extending from position -169 to position -100 had the same effect on cAMP-induced expression. Furthermore, the entire stimulatory effect could be achieved with an 18-bp synthetic oligodeoxynucleotide corresponding to a direct repeat between position -146 and -111. In the absence of cAMP, the α-subunit 5' flanking sequence also enhanced transcription from the simian virus 40 early promoter. They localized this enhancer activity to the same -169/-100 fragment containing the cAMP response element. The 18-bp element alone, however, had no effect on basal expression. Thus, this short DNA sequence serves as a cAMP response element and also functions independently of other promoter-regulatory elements located in the 5' flanking sequence of the α-subunit gene

  16. Insulin elevates leptin secretion and mRNA levels via cyclic AMP in 3T3-L1 adipocytes deprived of glucose

    Directory of Open Access Journals (Sweden)

    Tomomi Tsubai

    2016-11-01

    Conclusion: Insulin alone stimulates leptin secretion and elevates leptin mRNA levels via cAMP under the lack of glucose metabolism, while glucose is a significant and ambivalent effector on the insulin effects of leptin.

  17. Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model.

    Directory of Open Access Journals (Sweden)

    Sameer Udhane

    Full Text Available Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs. Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute versus long-term (chronic stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

  18. Increase in cyclic AMP concentration in a cerebral giant interneuron mimics part of a memory trace for conditioned taste aversion of the pond snail.

    Science.gov (United States)

    Otsuka, Emi; Matsunaga, Miho; Okada, Ryuichi; Yamagishi, Miki; Okuta, Akiko; Lukowiak, Ken; Ito, Etsuro

    2013-01-01

    Conditioned taste aversion (CTA) can be classically conditioned in the pond snail Lymnaea stagnalis and subsequently be consolidated into long-term memory (LTM). The neural trace that subserves CTA-LTM can be summarized as follows: A polysynaptic inhibitory postsynaptic potential recorded in the neuron 1 medial (N1M) cell in the conditioned snails as a result of activation of the cerebral giant cell (CGC) is larger and lasts longer than that in control snails. The N1M cell is ultimately activated by the CGC via the neuron 3 tonic (N3t) cell. That is, the inhibitory monosynaptic inputs from the N3t cell to the N1M cell are facilitated. The N1M and N3t cells are the members of feeding central pattern generator, whereas the CGC is a multimodal interneuron thought to play a key role in feeding behavior. Here we examined the involvement of a second messenger, cAMP, in the establishment of the memory trace. We injected cAMP into the CGC and monitored the potentials of the B3 motor neuron activated by the CGC. B3 activity is used as an index for the synaptic inputs from the N3t cell to the N1M cell. We found that the B3 potentials were transiently enlarged. Thus, when the cAMP concentration is increased in the CGC by taste aversion training, cAMP-induced changes may play a key role in the establishment of a memory trace in the N3t cell.

  19. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors

    International Nuclear Information System (INIS)

    Lund, Kaleb C.; Wallace, Kendall B.

    2008-01-01

    Nucleoside analog reverse transcriptase inhibitors (NRTIs) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study, we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3'-azido-3'-deoxythymidine (AZT; 10 and 50 μM), AZT monophosphate (150 μM), and 2',3'-dideoxycytidine (ddC; 1 μM) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2',3'-dideoxyinosine (ddI; 10 μM) and ddC (1 μM). In the presence of succinate + cAMP, AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-γ activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug

  20. Sweet taste receptor expressed in pancreatic beta-cells activates the calcium and cyclic AMP signaling systems and stimulates insulin secretion.

    Directory of Open Access Journals (Sweden)

    Yuko Nakagawa

    Full Text Available BACKGROUND: Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. METHODOLOGY/PRINCIPAL FINDINGS: The expression of the sweet taste receptor was determined by RT-PCR and immunohistochemistry. Changes in cytoplasmic Ca(2+ ([Ca(2+](c and cAMP ([cAMP](c were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca(2+](c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca(2+](c response. The effect of sucralose on [Ca(2+](c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a G(q inhibitor. Sucralose also induced sustained elevation of [cAMP](c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. CONCLUSIONS: Sweet taste receptor is expressed in beta-cells, and activation of this receptor induces insulin secretion by Ca(2+ and cAMP-dependent mechanisms.

  1. The freeze-thaw stress response of the yeast Saccharomyces cerevisiae is growth phase specific and is controlled by nutritional state via the RAS-cyclic AMP signal transduction pathway.

    Science.gov (United States)

    Park, J I; Grant, C M; Attfield, P V; Dawes, I W

    1997-10-01

    The ability of cells to survive freezing and thawing is expected to depend on the physiological conditions experienced prior to freezing. We examined factors affecting yeast cell survival during freeze-thaw stress, including those associated with growth phase, requirement for mitochondrial functions, and prior stress treatment(s), and the role played by relevant signal transduction pathways. The yeast Saccharomyces cerevisiae was frozen at -20 degrees C for 2 h (cooling rate, less than 4 degrees C min-1) and thawed on ice for 40 min. Supercooling occurred without reducing cell survival and was followed by freezing. Loss of viability was proportional to the freezing duration, indicating that freezing is the main determinant of freeze-thaw damage. Regardless of the carbon source used, the wild-type strain and an isogenic petite mutant ([rho 0]) showed the same pattern of freeze-thaw tolerance throughout growth, i.e., high resistance during lag phase and low resistance during log phase, indicating that the response to freeze-thaw stress is growth phase specific and not controlled by glucose repression. In addition, respiratory ability and functional mitochondria are necessary to confer full resistance to freeze-thaw stress. Both nitrogen and carbon source starvation led to freeze-thaw tolerance. The use of strains affected in the RAS-cyclic AMP (RAS-cAMP) pathway or supplementation of an rca1 mutant (defective in the cAMP phosphodiesterase gene) with cAMP showed that the freeze-thaw response of yeast is under the control of the RAS-cAMP pathway. Yeast did not adapt to freeze-thaw stress following repeated freeze-thaw treatment with or without a recovery period between freeze-thaw cycles, nor could it adapt following pretreatment by cold shock. However, freeze-thaw tolerance of yeast cells was induced during fermentative and respiratory growth by pretreatment with H2O2, cycloheximide, mild heat shock, or NaCl, indicating that cross protection between freeze-thaw stress

  2. FRET-based binding assay between a fluorescent cAMP analogue and a cyclic nucleotide-binding domain tagged with a CFP.

    Science.gov (United States)

    Romero, Francisco; Santana-Calvo, Carmen; Sánchez-Guevara, Yoloxochitl; Nishigaki, Takuya

    2017-09-01

    The cyclic nucleotide-binding domain (CNBD) functions as a regulatory domain of many proteins involved in cyclic nucleotide signalling. We developed a straightforward and reliable binding assay based on intermolecular fluorescence resonance energy transfer (FRET) between an adenosine-3', 5'-cyclic monophosphate analogue labelled with fluorescein and a recombinant CNBD of human EPAC1 tagged with a cyan fluorescence protein (CFP). The high FRET efficiency of this method (~ 80%) allowed us to perform several types of binding experiments with nanomolar range of sample using conventional equipment. In addition, the CFP tag on the CNBD enabled us to perform a specific binding experiment using an unpurified protein. Considering these advantages, this technique is useful to study poorly characterized CNBDs. © 2017 Federation of European Biochemical Societies.

  3. Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovine oocytes.

    Science.gov (United States)

    Bilodeau-Goeseels, Sylvie

    2011-01-01

    Meiotic maturation in mammalian oocytes is initiated during fetal development, and is then arrested at the dictyate stage - possibly for several years. Oocyte meiosis resumes in preovulatory follicles in response to the lutenizing hormone (LH) surge or spontaneously when competent oocytes are removed from follicles and cultured. The mechanisms involved in meiotic arrest and resumption in bovine oocytes are not fully understood, and several studies point to important differences between oocytes from rodent and livestock species. This paper reviews earlier and contemporary studies on the effects of cAMP-elevating agents and phosphodiesterase (PDE) enzyme inhibitors on the maintenance of meiotic arrest in bovine oocytes in vitro. Contrary to results obtained with mouse oocytes, bovine oocyte meiosis is inhibited by activators of the energy sensor adenosine monophosphate-activated protein kinase (AMPK, mammalian gene PRKA), which is activated by AMP, the degradation product of cAMP. It is not clear whether or not the effects were due to AMPK activation, and they may depend on culture conditions. Evidence suggests that other signaling pathways (for example, the cGMP/nitric oxide pathway) are involved in bovine oocyte meiotic arrest, but further studies are needed to understand the interactions between the signaling pathways that lead to maturation promoting factor (MPF) being inactive or active. An improved understanding of the mechanisms involved in the control of bovine oocyte meiosis will facilitate better control of the process in vitro, resulting in increased developmental competence and increased efficiency of in vitro embryo production procedures. Copyright © 2011 Wiley Periodicals, Inc.

  4. The μ opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway

    Directory of Open Access Journals (Sweden)

    Roberts-Thomson Sarah J

    2006-07-01

    Full Text Available Abstract Background The vanilloid receptor 1 (TRPV1 is critical in the development of inflammatory hyperalgesia. Several receptors including G-protein coupled prostaglandin receptors have been reported to functionally interact with the TRPV1 through a cAMP-dependent protein kinase A (PKA pathway to potentiate TRPV1-mediated capsaicin responses. Such regulation may have significance in inflammatory pain. However, few functional receptor interactions that inhibit PKA-mediated potentiation of TRPV1 responses have been described. Results In the present studies we investigated the hypothesis that the μ opioid receptor (MOP agonist morphine can modulate forskolin-potentiated capsaicin responses through a cAMP-dependent PKA pathway. HEK293 cells were stably transfected with TRPV1 and MOP, and calcium (Ca2+ responses to injection of the TRPV1 agonist capsaicin were monitored in Fluo-3-loaded cells. Pre-treatment with morphine did not inhibit unpotentiated capsaicin-induced Ca2+ responses but significantly altered capsaicin responses potentiated by forskolin. TRPV1-mediated Ca2+ responses potentiated by the direct PKA activator 8-Br-cAMP and the PKC activator Phorbol-12-myristate-13-acetatewere not modulated by morphine. Immunohistochemical studies confirmed that the TRPV1 and MOP are co-expressed on cultured Dorsal Root Ganglion neurones, pointing towards the existence of a functional relationship between the G-protein coupled MOP and nociceptive TRPV1. Conclusion The results presented here indicate that the opioid receptor agonist morphine acts via inhibition of adenylate cyclase to inhibit PKA-potentiated TRPV1 responses. Targeting of peripheral opioid receptors may therefore have therapeutic potential as an intervention to prevent potentiation of TRPV1 responses through the PKA pathway in inflammation.

  5. Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Škopová, Karolína; Tomalová, Barbora; Kanchev, Ivan; Rossmann, Pavel; Švédová, Martina; Adkins, Irena; Bíbová, Ilona; Tomala, Jakub; Mašín, Jiří; Guiso, N.; Osička, Radim; Sedláček, Radislav; Kovář, Marek; Šebo, Peter

    2017-01-01

    Roč. 85, č. 6 (2017), s. 1-22, č. článku e00937-16. ISSN 0019-9567 R&D Projects: GA MZd(CZ) NV16-28126A; GA ČR(CZ) GA13-14547S; GA ČR GA13-12885S; GA ČR GA15-09157S; GA ČR(CZ) GAP302/12/0460; GA MŠk(CZ) LM2015064; GA MŠk(CZ) LM2015040 Institutional support: RVO:61388971 ; RVO:68378050 Keywords : Bordetella pertussis * adenylate cyclase toxin-hemolysin * cAMP intoxication Subject RIV: EE - Microbiology, Virology; EE - Microbiology, Virology (UMG-J) OBOR OECD: Microbiology; Microbiology (UMG-J) Impact factor: 3.593, year: 2016

  6. Role of cyclic AMP sensor Epac1 in masseter muscle hypertrophy and myosin heavy chain transition induced by β2-adrenoceptor stimulation.

    Science.gov (United States)

    Ohnuki, Yoshiki; Umeki, Daisuke; Mototani, Yasumasa; Jin, Huiling; Cai, Wenqian; Shiozawa, Kouichi; Suita, Kenji; Saeki, Yasutake; Fujita, Takayuki; Ishikawa, Yoshihiro; Okumura, Satoshi

    2014-12-15

    The predominant isoform of β-adrenoceptor (β-AR) in skeletal muscle is β2-AR and that in the cardiac muscle is β1-AR. We have reported that Epac1 (exchange protein directly activated by cAMP 1), a new protein kinase A-independent cAMP sensor, does not affect cardiac hypertrophy in response to pressure overload or chronic isoproterenol (isoprenaline) infusion. However, the role of Epac1 in skeletal muscle hypertrophy remains poorly understood. We thus examined the effect of disruption of Epac1, the major Epac isoform in skeletal muscle, on masseter muscle hypertrophy induced by chronic β2-AR stimulation with clenbuterol (CB) in Epac1-null mice (Epac1KO). The masseter muscle weight/tibial length ratio was similar in wild-type (WT) and Epac1KO at baseline and was significantly increased in WT after CB infusion, but this increase was suppressed in Epac1KO. CB treatment significantly increased the proportion of myosin heavy chain (MHC) IIb at the expense of that of MHC IId/x in both WT and Epac1KO, indicating that Epac1 did not mediate the CB-induced MHC isoform transition towards the faster isoform. The mechanism of suppression of CB-mediated hypertrophy in Epac1KO is considered to involve decreased activation of Akt signalling. In addition, CB-induced histone deacetylase 4 (HDAC4) phosphorylation on serine 246 mediated by calmodulin kinase II (CaMKII), which plays a role in skeletal muscle hypertrophy, was suppressed in Epac1KO. Our findings suggest that Epac1 plays a role in β2-AR-mediated masseter muscle hypertrophy, probably through activation of both Akt signalling and CaMKII/HDAC4 signalling. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

  7. Cyclic-AMP metabolism in synaptic growth, strength and precision: Neural and behavioral phenotype-specific counterbalancing effects between dnc PDE and rut AC mutations

    Science.gov (United States)

    Ueda, Atsushi; Wu, Chun-Fang

    2012-01-01

    Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cAMP synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. We ask how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. Our study at larval neuromuscular junctions (NMJs) demonstrate that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29–30 °C) decreased synaptic transmission in rut, but did not alter dnc and WT. Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, we reviewed previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. We discuss factors that could contribute to the

  8. Inhibition of Cyclic Adenosine Monophosphate (cAMP-response Element-binding Protein (CREB-binding Protein (CBP/β-Catenin Reduces Liver Fibrosis in Mice

    Directory of Open Access Journals (Sweden)

    Yosuke Osawa

    2015-11-01

    Full Text Available Wnt/β-catenin is involved in every aspect of embryonic development and in the pathogenesis of many human diseases, and is also implicated in organ fibrosis. However, the role of β-catenin-mediated signaling on liver fibrosis remains unclear. To explore this issue, the effects of PRI-724, a selective inhibitor of the cAMP-response element-binding protein-binding protein (CBP/β-catenin interaction, on liver fibrosis were examined using carbon tetrachloride (CCl4- or bile duct ligation (BDL-induced mouse liver fibrosis models. Following repetitive CCl4 administrations, the nuclear translocation of β-catenin was observed only in the non-parenchymal cells in the liver. PRI-724 treatment reduced the fibrosis induced by CCl4 or BDL. C-82, an active form of PRI-724, inhibited the activation of isolated primary mouse quiescent hepatic stellate cells (HSCs and promoted cell death in culture-activated HSCs. During the fibrosis resolution period, an increase in F4/80+ CD11b+ and Ly6Clow CD11b+ macrophages was induced by CCl4 and was sustained for two weeks thereafter, even after having stopped CCl4 treatment. PRI-724 accelerated the resolution of CCl4-induced liver fibrosis, and this was accompanied by increased matrix metalloproteinase (MMP-9, MMP-2, and MMP-8 expression in intrahepatic leukocytes. In conclusion, targeting the CBP/β-catenin interaction may become a new therapeutic strategy in treating liver fibrosis.

  9. Regulation of Maltodextrin Phosphorylase Synthesis in Escherichia coli by Cyclic Adenosine 3′, 5′-Monophosphate and Glucose1

    Science.gov (United States)

    Chao, Julie; Weathersbee, Carolyn J.

    1974-01-01

    Cyclic adenosine 3′, 5′-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3′-AMP, 5′-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, α-methyl glucoside, 2-deoxyglucose, and pyridoxal 5′-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase. PMID:4358043

  10. Nitric oxide increases cyclic GMP levels, AMP-activated protein kinase (AMPK)alpha1-specific activity and glucose transport in human skeletal muscle

    DEFF Research Database (Denmark)

    Deshmukh, A S; Long, Y C; de Castro Barbosa, T

    2010-01-01

    -nitrosohydrazino)-1,2-ethylenediamine (spermine NONOate) would increase intracellular cyclic GMP (cGMP) levels and promote glucose transport. METHODS: Skeletal muscle strips were prepared from vastus lateralis muscle biopsies obtained from seven healthy men. Muscle strips were incubated in the absence or presence...... of 5 mmol/l spermine NONOate or 120 nmol/l insulin. The L6 muscle cells were treated with spermine NONOate (20 micromol/l) and incubated in the absence or presence of insulin (120 nmol/l). The direct effect of spermine NONOate and insulin on glucose transport, cGMP levels and signal transduction...... was determined. RESULTS: In human skeletal muscle, spermine NONOate increased glucose transport 2.4-fold (p GMP levels (80-fold, p

  11. CCAAT/enhancer-binding protein delta activates insulin-like growth factor-I gene transcription in osteoblasts. Identification of a novel cyclic AMP signaling pathway in bone

    Science.gov (United States)

    Umayahara, Y.; Ji, C.; Centrella, M.; Rotwein, P.; McCarthy, T. L.

    1997-01-01

    Insulin-like growth factor-I (IGF-I) plays a key role in skeletal growth by stimulating bone cell replication and differentiation. We previously showed that prostaglandin E2 (PGE2) and other cAMP-activating agents enhanced IGF-I gene transcription in cultured primary rat osteoblasts through promoter 1, the major IGF-I promoter, and identified a short segment of the promoter, termed HS3D, that was essential for hormonal regulation of IGF-I gene expression. We now demonstrate that CCAAT/enhancer-binding protein (C/EBP) delta is a major component of a PGE2-stimulated DNA-protein complex involving HS3D and find that C/EBPdelta transactivates IGF-I promoter 1 through this site. Competition gel shift studies first indicated that a core C/EBP half-site (GCAAT) was required for binding of a labeled HS3D oligomer to osteoblast nuclear proteins. Southwestern blotting and UV-cross-linking studies showed that the HS3D probe recognized a approximately 35-kDa nuclear protein, and antibody supershift assays indicated that C/EBPdelta comprised most of the PGE2-activated gel-shifted complex. C/EBPdelta was detected by Western immunoblotting in osteoblast nuclear extracts after treatment of cells with PGE2. An HS3D oligonucleotide competed effectively with a high affinity C/EBP site from the rat albumin gene for binding to osteoblast nuclear proteins. Co-transfection of osteoblast cell cultures with a C/EBPdelta expression plasmid enhanced basal and PGE2-activated IGF-I promoter 1-luciferase activity but did not stimulate a reporter gene lacking an HS3D site. By contrast, an expression plasmid for the related protein, C/EBPbeta, did not alter basal IGF-I gene activity but did increase the response to PGE2. In osteoblasts and in COS-7 cells, C/EBPdelta, but not C/EBPbeta, transactivated a reporter gene containing four tandem copies of HS3D fused to a minimal promoter; neither transcription factor stimulated a gene with four copies of an HS3D mutant that was unable to bind osteoblast

  12. Proteomic signatures implicate cAMP in light and temperature responses in Arabidopsis thaliana

    KAUST Repository

    Thomas, Ludivine; Marondedze, Claudius; Ederli, Luisa; Pasqualini, Stefania; Gehring, Christoph A

    2013-01-01

    The second messenger 3'-5'-cyclic adenosine monophosphate (cAMP) and adenylyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, are increasingly recognized as important signaling molecules in a number of physiological responses

  13. Effects of hypokinesia on cyclic nucleotides and hormonal regulation ...

    African Journals Online (AJOL)

    PTH), calcitonin (CT), cyclic nucleotides (cAMP, cGMP) and calcium in the blood of rats, while in urine - phosphate, calcium and cyclic nucleotides. Design: Laboratory based experiment. Setting: Laboratory in the Department of Biochemistry, ...

  14. Cyclic nucleotides and radioresistnace

    International Nuclear Information System (INIS)

    Kulinskij, V.I.; Mikheeva, G.A.; Zel'manovich, B.M.

    1982-01-01

    The addition of glucose to meat-peptone broth does not change the radiosensitizing effect (RSE) of cAMP at the logarithmic phase (LP) and the radioprotective effect (RPE) at the stationary phase (SP), but sensitization, characteristic of cGMP, disappears in SP and turns into RPE in LP. Introduction of glucose into the broth for 20 min eliminates all the effects of both cyclic nucleotides in the cya + strain while cya - mutant exhibits RSE. RSE of both cyclic nucleotides is only manifested on minimal media. These data brought confirmation of the dependence of the influence of cyclic media. These data brought confirmation of the dependence of the influence of cyclic nucleotides on radioresistance upon the metabolic status of the cell [ru

  15. Detection of Cyclic Dinucleotides by STING.

    Science.gov (United States)

    Du, Xiao-Xia; Su, Xiao-Dong

    2017-01-01

    STING (stimulator of interferon genes) is an essential signaling adaptor protein mediating cytosolic DNA-induced innate immunity for both microbial invasion and self-DNA leakage. STING is also a direct receptor for cytosolic cyclic dinucleotides (CDNs), including the microbial secondary messengers c-di-GMP (3',3'-cyclic di-GMP), 3',3'cGAMP (3',3'-cyclic GMP-AMP), and mammalian endogenous 2',3'cGAMP (2',3'-cyclic GMP-AMP) synthesized by cGAS (cyclic GMP-AMP synthase). Upon CDN binding, STING undergoes a conformational change to enable signal transduction by phosphorylation and finally to active IRF3 (Interferon regulatory factor 3) for type I interferon production. Here, we describe some experimental procedures such as Isothermal Titration Calorimetry and luciferase reporter assays to study the CDNs binding and activity by STING proteins.

  16. Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway.

    Science.gov (United States)

    Mottola, Giorgio; Chatterjee, Anuran; Wu, Bian; Chen, Mian; Conte, Michael S

    2017-01-01

    Resolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration. VSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity. Treatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor. Our results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

  17. Adenosine 3':5'-cyclic monophosphate in higher plants: Isolation and characterization of adenosine 3':5'-cyclic monophosphate from Kalanchoe and Agave.

    Science.gov (United States)

    Ashton, A R; Polya, G M

    1977-01-01

    1.3':5'-Cyclic AMP was extensively purified from Kalanchoe daigremontiana and Agave americana by neutral alumina and anion- and cation-exchange column chromatography. Inclusion of 3':5'-cyclic [8-3H]AMP from the point of tissue extraction permitted calculation of yields. The purification procedure removed contaminating material that was shown to interfere with the 3':5'-cyclic AMP estimation and characterization procedures. 2. The partially purified 3':5'-cyclic AMP was quantified by means of a radiochemical saturation assay using an ox heart 3':5'-cyclic AMP-binding protein and by an assay involving activation of a mammalian protein kinase. 3. The plant 3':5'-cyclic AMP co-migrated with 3':5'-cyclic [8-3H]AMP on cellulose chromatography, poly(ethyleneimine)-cellulose chromatography and silica-gel t.l.c. developed with several solvent systems. 4. The plant 3':5'-cyclic AMP was degraded by ox heart 3':5'-cyclic nucleotide phosphodiesterase at the same rates as authentic 3':5'-cyclic AMP. 1-Methyl-3-isobutylxanthine (1 mM), a specific inhibitor of the 3':5'-cyclic nucleotide phosphodieterase, completely inhibited such degradation. 5. The concentrations of 3':5'-cyclic AMP satisfying the above criteria in Kalanchoe and Agave were 2-6 and 1 pmol/g fresh wt. respectively. Possible bacterial contribution to these analyses was estimated to be less than 0.002pmol/g fresh wt. Evidence for the occurrence of 3':5'-cyclic AMP in plants is discussed. PMID:196595

  18. A multi-angular mass spectrometric view at cyclic nucleotide signaling proteins : Structure/function and protein interactions of cAMP- and cGMP-dependent protein kinase

    NARCIS (Netherlands)

    Scholten, A.

    2006-01-01

    The primary focus of this thesis is the two kinases PKA and PKG, cAMP and cGMP dependent protein kinase respectively. PKA and PKG are studied both at structure/function level as well as at the level of interaction with other proteins in tissue. Our primary methods are all based on mass spectrometry.

  19. Regulation of phospholipid synthesis in Mycobacterium smegmatis by cyclic adenosine monophosphate

    International Nuclear Information System (INIS)

    Sareen, Monica; Kaur, Harpinder; Khuller, G.K.

    1993-01-01

    Forskolin, an adenylate cyclase activator and a cyclic AMP analogue, dibutyryl cyclic AMP have been used to examine the relationship between intracellular levels of cyclic AMP and lipid synthesis in Mycobacterium smegmatis. Total phospholipid content was found to be increased in forskolin grown cells as a result of increased cyclic AMP levels caused by activation of adenylate cyclase. Increased phospholipid content was supported by increased [ 14 C]acetate incorporation as well as increased activity of glycerol-3-phosphate acyltransferase. Pretreatment of cells with dibutyryl cyclic AMP had similar effects on lipid synthesis. Taking all these observations together it is suggested that lipid synthesis is being controlled by cyclic AMP in mycobacteria. (author). 14 refs., 4 tabs

  20. Schwann Cells Metabolize Extracellular 2′,3′-cAMP to 2′-AMP

    Science.gov (United States)

    Verrier, Jonathan D.; Kochanek, Patrick M.

    2015-01-01

    The 3′,5′-cAMP–adenosine pathway (3′,5′-cAMP→5′-AMP→adenosine) and the 2′,3′-cAMP–adenosine pathway (2′,3′-cAMP→2′-AMP/3′-AMP→adenosine) are active in the brain. Oligodendrocytes participate in the brain 2′,3′-cAMP–adenosine pathway via their robust expression of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase; converts 2′,3′-cAMP to 2′-AMP). Because Schwann cells also express CNPase, it is conceivable that the 2′,3′-cAMP–adenosine pathway exists in the peripheral nervous system. To test this and to compare the 2′,3′-cAMP–adenosine pathway to the 3′,5′-cAMP–adenosine pathway in Schwann cells, we examined the metabolism of 2′,3′-cAMP, 2′-AMP, 3′-AMP, 3′,5′-cAMP, and 5′-AMP in primary rat Schwann cells in culture. Addition of 2′,3′-cAMP (3, 10, and 30 µM) to Schwann cells increased levels of 2′-AMP in the medium from 0.006 ± 0.002 to 21 ± 2, 70 ± 3, and 187 ± 10 nM/µg protein, respectively; in contrast, Schwann cells had little ability to convert 2′,3′-cAMP to 3′-AMP or 3′,5′-cAMP to either 3′-AMP or 5′-AMP. Although Schwann cells slightly converted 2′,3′-cAMP and 2′-AMP to adenosine, they did so at very modest rates (e.g., 5- and 3-fold, respectively, more slowly compared with our previously reported studies in oligodendrocytes). Using transected myelinated rat sciatic nerves in culture medium, we observed a time-related increase in endogenous intracellular 2′,3′-cAMP and extracellular 2′-AMP. These findings indicate that Schwann cells do not have a robust 3′,5′-cAMP–adenosine pathway but do have a 2′,3′-cAMP–adenosine pathway; however, because the pathway mostly involves 2′-AMP formation rather than 3′-AMP, and because the conversion of 2′-AMP to adenosine is slow, metabolism of 2′,3′-cAMP mostly results in the accumulation of 2′-AMP. Accumulation of 2′-AMP in peripheral nerves postinjury could have

  1. Direct Light-up of cAMP Derivatives in Living Cells by Click Reactions

    Directory of Open Access Journals (Sweden)

    Yan Xu

    2013-10-01

    Full Text Available 8-Azidoadenosine 3′,5′-cyclic monophosphate (8-azido cAMP was directly detected in living cells, by applying Cu-free azide-alkyne cycloaddition to probe cAMP derivatives by fluorescence light-up. Fluorescence emission was generated by two non-fluorescent molecules, 8-azido cAMP as a model target and difluorinated cyclooctyne (DIFO reagent as a probe. The azide-alkyne cycloaddition reaction between 8-azido cAMP and DIFO induces fluorescence in 8-azido cAMP. The fluorescence emission serves as a way to probe 8-azido cAMP in cells.

  2. Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element

    Science.gov (United States)

    McCarthy, T. L.; Thomas, M. J.; Centrella, M.; Rotwein, P.

    1995-01-01

    Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of

  3. Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

    Science.gov (United States)

    Katz, B M; Lundquist, L J; Walsh, D A; Glass, D B

    1989-06-01

    PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

  4. Comparison of thyroid stimulating activities measured by cyclic AMP production, those by radioiodine uptake in FRTL-5 cells and TSH-binding inhibitory activities in patients with hyperthyroid and euthyroid Graves' diseases

    International Nuclear Information System (INIS)

    Kasagi, Kanji; Hatabu, Hiroto; Tokuda, Yasutaka; Arai, Keisuke; Iida, Yasuhiro; Konishi, Junji

    1988-01-01

    By using an assay measuring cAMP production in FRTL-5 thyroid cells, thyroid stimulating antibodies (TSab) were detected in all of 15 patients with euthyroid Graves' disease (EG) and of 26 patients with hyperthyroid Graves' disease (HG). There was no signicant difference between TSab activities in Eg and in HG. In an effort to elucidate why EG patients remain euthyroid in spite of having TSab, we investigated the effect of the patient's crude immunoglobulin fractions 125 I uptake in FRTL-5 thyroid cells, one of the indices of stimulation subsequent to cAMP production. The 125 Iuptake stimulation (IUS) activity was positive in 46,7% (7/15) of EG patients and 88.5% (23/26) of HG patients, being significantly lower in the former than in the latter (P 99m Tc thyroid uptake (r = 0.401, P 99m Tc thyroid uptake in comparison to 19 HG patients with a similar range of IUS activities. There was a good correlation between thyroid weight and 99m Tc thyroid uptake (r = 8.827, P 99m Tc and presumably radioiodine in vivo, might be a factor responsible for keeping EG patients euthyroid despite the presence of TSab. (author)

  5. Cyclic Nucleotide Signalling in Kidney Fibrosis

    Directory of Open Access Journals (Sweden)

    Elisabeth Schinner

    2015-01-01

    Full Text Available Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure.

  6. Dopamine D1 receptors and phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein-32 in the medial preoptic area are involved in experience-induced enhancement of male sexual behavior in rats.

    Science.gov (United States)

    McHenry, Jenna A; Bell, Genevieve A; Parrish, Bradley P; Hull, Elaine M

    2012-08-01

    The medial preoptic area (MPOA) is an integral site for male sexual behavior. Dopamine is released in the MPOA before and during copulation and facilitates male rat sexual behavior. Repeated sexual experience and noncopulatory exposures to an estrous female facilitate subsequent copulation. However, the neurobiological mechanisms that mediate such enhancement remain unclear. Here, we examined the role of dopamine D₁ receptors in the MPOA in experience-induced enhancement of male sexual behavior in rats. In experiment 1, microinjections of the D₁ antagonist SCH-23390 into the MPOA before each of seven daily 30-min noncopulatory exposures to a receptive female impaired copulation on a drug-free test on Day 8, compared to vehicle-treated female-exposed animals. Copulatory performance in drug-treated animals was similar to that of vehicle-treated males that had not been preexposed to females. This effect was site specific. There were no group differences in locomotor activity in an open field on the copulation test day. In experiment 2, a separate cohort of animals was used to examine phosphorylation of dopamine- and cAMP-regulated phosphoprotein (DARPP-32) in the MPOA of animals with acute and/or chronic sexual experience. DARPP-32 is a downstream marker of D₁ receptor signaling and substrate of cAMP-dependent protein kinase (PKA). Western immunoblot analysis revealed that p-DARPP-32 expression was greatest in the MPOA of males that received both acute and chronic sexual experience, compared to all other mated conditions and naïve controls. These data suggest that D₁ receptors in the MPOA contribute to experience-induced enhancement of male sexual behavior, perhaps through a PKA regulated mechanism.

  7. MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

    DEFF Research Database (Denmark)

    Lundegaard, Pia R.; Anastasaki, Corina; Grant, Nicola J.

    2015-01-01

    Altered phosphodiesterase (PDE)-cyclic AMP (cAMP) activity is frequently associated with anxiety disorders, but current therapies act by reducing neuronal excitability rather than targeting PDE-cAMP-mediated signaling pathways. Here, we report the novel repositioning of anti-cancer MEK inhibitors...... as anxiolytics in a zebrafish model of anxiety-like behaviors. PDE inhibitors or activators of adenylate cyclase cause behaviors consistent with anxiety in larvae and adult zebrafish. Small-molecule screening identifies MEK inhibitors as potent suppressors of cAMP anxiety behaviors in both larvae and adult...... zebrafish, while causing no anxiolytic behavioral effects on their own. The mechanism underlying cAMP-induced anxiety is via crosstalk to activation of the RAS-MAPK signaling pathway. We propose that targeting crosstalk signaling pathways can be an effective strategy for mental health disorders, and advance...

  8. Imaging alterations of cardiomyocyte cAMP microdomains in disease

    Directory of Open Access Journals (Sweden)

    Alexander eFroese

    2015-08-01

    Full Text Available 3’,5’-cyclic adenosine monophosphate (cAMP is an important second messenger which regulates heart function by acting in distinct subcellular microdomains. Recent years have provided deeper mechanistic insights into compartmentalized cAMP signaling and its link to cardiac disease. In this mini review, we summarize newest developments in this field achieved by cutting-edge biochemical and biophysical techniques. We further compile the data from different studies into a bigger picture of so far uncovered alterations in cardiomyocyte cAMP microdomains which occur in compensated cardiac hypertrophy and chronic heart failure. Finally, future research directions and translational perspectives are briefly discussed.

  9. The Popeye Domain Containing Genes and cAMP Signaling

    Directory of Open Access Journals (Sweden)

    Thomas Brand

    2014-05-01

    Full Text Available 3'-5'-cyclic adenosine monophosphate (cAMP is a second messenger, which plays an important role in the heart. It is generated in response to activation of G-protein-coupled receptors (GPCRs. Initially, it was thought that protein kinase A (PKA exclusively mediates cAMP-induced cellular responses such as an increase in cardiac contractility, relaxation, and heart rate. With the identification of the exchange factor directly activated by cAMP (EPAC and hyperpolarizing cyclic nucleotide-gated (HCN channels as cAMP effector proteins it became clear that a protein network is involved in cAMP signaling. The Popeye domain containing (Popdc genes encode yet another family of cAMP-binding proteins, which are prominently expressed in the heart. Loss-of-function mutations in mice are associated with cardiac arrhythmia and impaired skeletal muscle regeneration. Interestingly, the cardiac phenotype, which is present in both, Popdc1 and Popdc2 null mutants, is characterized by a stress-induced sinus bradycardia, suggesting that Popdc proteins participate in cAMP signaling in the sinuatrial node. The identification of the two-pore channel TREK-1 and Caveolin 3 as Popdc-interacting proteins represents a first step into understanding the mechanisms of heart rate modulation triggered by Popdc proteins.

  10. Improvements in the automated radioimmunoassay for cAMP or cGMP

    International Nuclear Information System (INIS)

    Brooker, G.

    1988-01-01

    The work others in developing antibodies and the original radioimmunoassay for cyclic nucleotides provides the basis for these sensitive assays. The acetylation radioimmunoassay for cyclic nucleotides has enabled the measurement of cyclic AMP and cyclic GMP in very small biological samples. This is because accurate determinations can be made in samples containing less than 1 fmol of cyclic AMP or cyclic GMP. The Gamma-Flo automated radioimmunoassay system has been adapted to these assays such that cyclic nucleotides can be automatically measured at a rate of about 60 samples/hr. The Gamma-Flo instrument provides high-precision assays and eliminates human intervention in all steps of the radioimmunoassay. The automated assay has been in continuous operation in our laboratory over the last 10 years and this chapter summarizes the methodology and delineates improvements which have occurred over that time frame. Details for the preparation of the radioligands apply also to the manual acetylated radioimmunoassay for cyclic nucleotides

  11. cAMP signalling in the vasculature: the role of Epac (exchange protein directly activated by cAMP).

    Science.gov (United States)

    Roberts, Owain Llŷr; Dart, Caroline

    2014-02-01

    The second messenger cAMP plays a central role in mediating vascular smooth muscle relaxation in response to vasoactive transmitters and in strengthening endothelial cell-cell junctions that regulate the movement of solutes, cells and macromolecules between the blood and the surrounding tissue. The vasculature expresses three cAMP effector proteins: PKA (protein kinase A), CNG (cyclic-nucleotide-gated) ion channels, and the most recently discovered Epacs (exchange proteins directly activated by cAMP). Epacs are a family of GEFs (guanine-nucleotide-exchange factors) for the small Ras-related GTPases Rap1 and Rap2, and are being increasingly implicated as important mediators of cAMP signalling, both in their own right and in parallel with the prototypical cAMP target PKA. In the present paper, we review what is currently known about the role of Epac within blood vessels, particularly with regard to the regulation of vascular tone, endothelial barrier function and inflammation.

  12. Study on the change of cyclic nucleotide in mice with yang vacuity disease

    International Nuclear Information System (INIS)

    Zhu Xinhua; Shen Ling; Wang Shuguang

    2002-01-01

    To study the relation between Yang Vacuity disease happening, development and cyclic nucleotide response, and prove curative effects of some assisting Yang drug, the plasma cAMP, cGMP and cAMP/cGMP levels were detected by radioimmunoassay in the Yang Vacuity group and curing group. Results: showed: (1) Yang Vacuity group: the symptoms were clear, death rate was high, the plasma cAMP and cAMP/cGMP increased obviously, it suggests that cyclic nucleotide was imbalance. (2) Curing group: the symptoms of Yang Vacuity disease were improved obviously, death rate dropped, cAMP declined, cGMP increased, while cAMP/cGMP reached the normal level, it showed that cyclic nucleotide of the body had altered greatly. (3) It is a reference target for Yang Vacuity. (4) Assisting yang drug (Sini Decoction) had a close relation with correcting imbalance of cyclic nucleotide

  13. Cyclic adenosine 3:5-monophosphate binding proteins in Hartmannella culbertsoni

    International Nuclear Information System (INIS)

    Verma, A.K.; Krishna Murti, C.R.

    1976-01-01

    When 100, 000 g supernatant fractions of homogenates of Hartmannella culbertsoni were incubated with ('- 3 H)-cyclic adenosine 3 : 5 monophosphate and passed through a sephadex G-100 column, radioactivity appeared with protein fractions eluted after the void colume. About 75% radioactivity bound to these fractions was recovered as cyclic adenosine 3 : 5 monophosphate. Unlabelled cAMP diluted the amount of radioactivity bound. Adenosine, deoxyadenosine, 5-AMP, 3-AMP, ADP and ATP did not inhibit binding. (author)

  14. Conservation and divergence of the cyclic adenosine monophosphate-protein kinase A (cAMP–PKA) pathway in two plant-pathogenic fungi: Fusarium graminearum and F. verticillioides

    Science.gov (United States)

    The cyclic AMP (cAMP)-PKA pathway is a central signaling cascade that transmits extracellular stimuli and governs cell responses through the second messenger cAMP. The importance of cAMP signaling in fungal biology has been well documented. Two key conserved components, adenylate cyclase (AC) and ca...

  15. A Temporal-Specific and Transient cAMP Increase Characterizes Odorant Classical Conditioning

    Science.gov (United States)

    Cui, Wen; Smith, Andrew; Darby-King, Andrea; Harley, Carolyn W.; McLean, John H.

    2007-01-01

    Increases in cyclic adenosine monophosphate (cAMP) are proposed to initiate learning in a wide variety of species. Here, we measure changes in cAMP in the olfactory bulb prior to, during, and following a classically conditioned odor preference trial in rat pups. Measurements were taken up to the point of maximal CREB phosphorylation in olfactory…

  16. The cAMP Signaling and MAP Kinase Pathways in Plant Pathogenic Fungi

    NARCIS (Netherlands)

    Mehrabi, R.; Zhao, X.; Kim, Y.; Xu, J.R.

    2009-01-01

    The key components of the well conserved cyclic AMP signaling and MAP kinase pathways have been functionally characterized in the corn smut Ustilago maydis, rice blast fungus Magnaporthe grisea, and a few other fungal pathogens. In general, the cAMP signaling and the MAP kinase cascade homologous to

  17. Assisted Medical Procedures (AMP)

    Data.gov (United States)

    National Aeronautics and Space Administration — DOCUMENTATION, DEVELOPMENT, AND PROGRESS The AMP was initially being developed as part the Advanced Integrated Clinical System (AICS)-Guided Medical Procedure System...

  18. Antimicrobial Peptides (AMPs

    Directory of Open Access Journals (Sweden)

    Mehrzad Sadredinamin

    2016-04-01

    Full Text Available Antimicrobial peptides (AMPs are extensive group of molecules that produced by variety tissues of invertebrate, plants, and animal species which play an important role in their immunity response. AMPs have different classifications such as; biosynthetic machines, biological sources, biological functions, molecular properties, covalent bonding patterns, three dimensional structures, and molecular targets.These molecules have multidimensional properties including antimicrobial activity, antiviral activity, antifungal activity, anti-parasite activity, biofilm control, antitumor activity, mitogens activity and linking innate to adaptive immunity that making them promising agents for therapeutic drugs. In spite of this advantage of AMPs, their clinical developments have some limitation for commercial development. But some of AMPs are under clinical trials for the therapeutic purpose such as diabetic foot ulcers, different bacterial infections and tissue damage. In this review, we emphasized on the source, structure, multidimensional properties, limitation and therapeutic applications of various antimicrobial peptides.

  19. [Physiopathology of cAMP/PKA signaling in neurons].

    Science.gov (United States)

    Castro, Liliana; Yapo, Cedric; Vincent, Pierre

    2016-01-01

    Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, synaptic transmission, regulation of excitability or long term changes in the nucleus. Genetically-encoded optical biosensors for cAMP or PKA considerably improved our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progresses made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the subcellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus and axon. Combining this imaging approach with pharmacology or genetical models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly help understand the mechanism of action of current drugs as well as help in devising novel therapeutic strategies for neuropsychiatric diseases. © Société de Biologie, 2017.

  20. The Role of Cyclic Nucleotide Signaling Pathways in Cancer: Targets for Prevention and Treatment

    Energy Technology Data Exchange (ETDEWEB)

    Fajardo, Alexandra M.; Piazza, Gary A. [Drug Discovery Research Center, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Ave, Suite 3029, Mobile, AL 36604 (United States); Tinsley, Heather N., E-mail: htinsley@montevallo.edu [Department of Biology, Chemistry, and Mathematics, University of Montevallo, Station 6480, Montevallo, AL 35115 (United States)

    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.

  1. The solvent effect on the enthalpy of hydrolysis of cyclic adenosine 3',5'-monophosphate : a quantum chemical study

    NARCIS (Netherlands)

    Scheffers - Sap, Miek; Buck, H.M.

    1978-01-01

    The solvent effect on the enthalpy of hydrolysis has been studied by the Extended-Hückel method for the hydrolysis reactions of cyclic adenosine 3',5'-monophosphate (cyclic 3',5'-AMP) and related cyclic phosphate diesters. The results show that the difference in enthalpy of hydrolysis between cyclic

  2. Cardiac cAMP: production, hydrolysis, modulation and detection

    Directory of Open Access Journals (Sweden)

    Cédric eBOULARAN

    2015-10-01

    Full Text Available Cyclic adenosine 3’,5’-monophosphate (cAMP modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for β-adrenergic receptors’ signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefly discuss the complexity of cAMP synthesis and degradation in the cardiac context, describe the way to detect it and review the main pharmacological arsenal to modulate its availability.

  3. Critical Role of Nitric Oxide-cGMP Cascade in the Formation of cAMP-Dependent Long-Term Memory

    Science.gov (United States)

    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…

  4. EFFECT OF dbcAMP ON PROLIFERATION AND APOPTOSIS OF PORCINE GRANULOSA CELLS in vitro

    Directory of Open Access Journals (Sweden)

    Richard Alexa

    2013-02-01

    Full Text Available Cyclic nucleotide cAMP and its target protein kinase A (PKA dependent intracellular mechanisms can play an important role in regulation of ovarian cell function and in mediating gonadotropin action on these cells. The aim of the present study was to examine the effect of cAMP analogue, dibutyryl cyclic adenosine monophosphate (dbcAMP (0; 0.1; 1 and 10 µg/ml or FSH (0; 0,01; 1 IU/ml on proliferation and apoptosis of porcine granulosa cells in vitro. Indices of cell apoptosis (expression of apoptotic peptide bax and proliferation (expression of proliferation-associated peptide PCNA within ovarian granulosa cells were analysed by immunocytochemistry. It was observed that accumulation of PCNA was increased by dbcAMP and FSH at all doses added. The occurrence of bax was also stimulated by dbcAMP after exposition (at 0,1 and 1 µg/ml, but not at dose 10 µg/ml and by FSH (at all doses added. The stimulatory effect of both dbcAMP and FSH on both ovarian cell apoptosis and proliferation suggest, that these substances may promote ovarian follicular cell turnover. The similarity of dbcAMP and FSH effect may indicate that FSH can affect ovarian functions via cAMP-dependent intracellular mechanisms. The present data may provide new tools to regulate human and animal reproductive processes via cAMP-dependent mechanisms.

  5. AMP language reference manual

    International Nuclear Information System (INIS)

    Drouffe, J.M.

    1982-06-01

    The use of a program for symbolic calculations named AMP (Algebraic Manipulation Program) is described. Its main features are: high speed heart for usual algebraic calculations; conversational capability; derivation, substitutions, matricial calculus, expansions, non commutative algebrae...; possibility to define new symbols and associated rules; possibility to create and use external libraries; written for IBM/370 like computers

  6. The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

    Science.gov (United States)

    Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

    2010-01-01

    Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

  7. Further studies on the effect of adenosine cyclic monophosphate derivatives on cell proliferation in the jejunal crypts of rat.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1982-01-01

    1. Cell proliferation in the jejunal crypt epithelium of rat was measured using a stathmokinetic technique. 2. Sodium butyrate was found to promote jejunal crypt cell proliferation. 3. N6, O2'-Dibutyryl cyclic adenosine monophosphate (cAMP), N6-monobutyryl-cAMP and N6-monobutyryl-8-bromo-cAMP were found to inhibit cell proliferation when compared to sodium butyrate treated tissues. 4. 8-Chlorophenylthio-cAMP was found to inhibit cell division when compared to untreated animals. 5. O2'-Monobutyryl cAMP and 8-bromo-cAMP were not found to inhibit cell proliferation.

  8. Cyclic AMP response element binding protein and brain-derived ...

    Indian Academy of Sciences (India)

    Madhu

    learning tasks and memory (Bramham and Messaoudi 2005). Thus, BDNF is involved in structural remodeling, neuronal .... in hippocampal-dependent learning and memory (Tyler et al 2002; Mizuno and Giese 2005) may play a ..... neurons in the olfactory bulb; J. Neurosci. 25 10105–10118. Gould E and Tanapat P 1999 ...

  9. Non-Enzymatic Oligomerization of 3 ', 5 ' Cyclic AMP

    Czech Academy of Sciences Publication Activity Database

    Costanzo, G.; Pino, S.; Timperio, A.M.; Šponer, Judit E.; Sponer, Jiri; Nováková, Olga; Šedo, O.; Zdráhal, Z.; Di Mauro, E.

    2016-01-01

    Roč. 11, č. 11 (2016), č. článku e0165723. E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GA14-12010S; GA ČR GBP206/12/G151 Institutional support: RVO:68081707 Keywords : template-directed synthesis * nucleoside phosphorylation * montmorillonite catalysis * molecular-structure Subject RIV: BO - Biophysics Impact factor: 2.806, year: 2016

  10. Review Cyclic AMP response element binding protein and brain ...

    Indian Academy of Sciences (India)

    Madhu

    Several types of adult-onset stressors, including physical and psychosocial stressors ... stress-induced atrophy with antidepressant therapy in animal models (Watanabe et ..... (Strauss et al 2005) and geriatric depression (Hwang et al. 2005).

  11. Cyclic multiverses

    Science.gov (United States)

    Marosek, Konrad; Dąbrowski, Mariusz P.; Balcerzak, Adam

    2016-09-01

    Using the idea of regularization of singularities due to the variability of the fundamental constants in cosmology we study the cyclic universe models. We find two models of oscillating and non-singular mass density and pressure (`non-singular' bounce) regularized by varying gravitational constant G despite the scale factor evolution is oscillating and having sharp turning points (`singular' bounce). Both violating (big-bang) and non-violating (phantom) null energy condition models appear. Then, we extend this idea on to the multiverse containing cyclic individual universes with either growing or decreasing entropy though leaving the net entropy constant. In order to get an insight into the key idea, we consider the doubleverse with the same geometrical evolution of the two `parallel' universes with their physical evolution [physical coupling constants c(t) and G(t)] being different. An interesting point is that there is a possibility to exchange the universes at the point of maximum expansion - the fact which was already noticed in quantum cosmology. Similar scenario is also possible within the framework of Brans-Dicke theory where varying G(t) is replaced by the dynamical Brans-Dicke field φ(t) though these theories are slightly different.

  12. Role of AC-cAMP-PKA Cascade in Antidepressant Action of Electroacupuncture Treatment in Rats

    Directory of Open Access Journals (Sweden)

    Jian-hua Liu

    2012-01-01

    Full Text Available Adenylyl cyclase (AC-cyclic adenosine monophosphate (cAMP-cAMP-dependent protein kinase A (PKA cascade is considered to be associated with the pathogenesis and treatment of depression. The present study was conducted to explore the role of the cAMP cascade in antidepressant action of electroacupuncture (EA treatment for chronic mild stress (CMS-induced depression model rats. The results showed that EA improved significantly behavior symptoms in depression and dysfunction of AC-cAMP-PKA signal transduction pathway induced by CMS, which was as effective as fluoxetine. Moreover, the antidepressant effects of EA rather than Fluoxetine were completely abolished by H89, a specific PKA inhibitor. Consequently, EA has a significant antidepressant treatment in CMS-induced depression model rats, and AC-cAMP-PKA signal transduction pathway is crucial for it.

  13. The participation of elevated levels of cyclic GMP in the recovery from radiation-induced mitotic delay

    International Nuclear Information System (INIS)

    Daniel, J.W.; Oleinick, N.L.

    1984-01-01

    The levels of cyclic AMP and cyclic GMP have been measured in Physarum plasmodia before and after treatment with gamma-radiation, 2 mM caffeine, or combinations of the two agents compared to the length of the radiation-induced mitotic delay. Caffeine alone produces a rapid transient elevation of cyclic AMP and a slower delayed elevation of cyclic GMP. Irradiation elicits an immediate transient increase in cyclic AMP and a later cyclic GMP increase which accompanies or precedes the delayed mitosis. A composite pattern is produced by combinations of radiation and caffeine, a distinctive feature of which is an elevated level of cyclic GMP near the time of the radiation-delayed and caffeine-promoted mitosis. With pretreatment by caffeine, the least radiation-induced mitotic delay occurs when plasmodia are irradiated during the caffeine-elicited increase in cyclic GMP. The plasmodium becomes refractory to the reduction of mitotic delay by caffeine at approximately the time it becomes refractory to the further elevation of cyclic GMP by caffeine. The data support a role for cyclic AMP in the onset of and for cyclic GMP in the recovery from mitotic delay induced by ionizing radiation. (author)

  14. AMP (Activity Manipulation Program)

    International Nuclear Information System (INIS)

    Engle, W.W. Jr.

    1976-03-01

    AMP is a FORTRAN IV program written to handle energy-group structured activity factors such as sources, conversion factors, and response functions, as used by ANISN, DOT III, and other nuclear reactor and shielding codes. Activities may be retrieved from ANISN-type cross-section and activity sets found on cards and tapes, and from tabular-type sets on cards. They may be altered by change of group structure, multiplication by a constant, or multiplication by delta E (the group-energy interval), and then output to ANISN-type cards or tape and tabular-type cards. A full edit of input and output activities is always printed by group and activity number

  15. Involvement of cyclic nucleotides in locust flight muscle metabolism

    NARCIS (Netherlands)

    Worm, R.A.A.

    1980-01-01

    1. Flight had no significant effect on the levels of c-AMP of c-GMP in the flight muscles of Locusta migratoria. 2. Injections of 0.01 or 0.1 corpus cardiacum equivalents into the abdominal cavity did not elicit any effect on cyclic nucleotide levels either. 3. Injection of A23187 resulted in

  16. Targeting brain tumor cAMP: the case for sex-specific therapeutics

    Directory of Open Access Journals (Sweden)

    Nicole M Warrington

    2015-07-01

    Full Text Available A relationship between cyclic adenosine 3’, 5’-monophosphate (cAMP levels and brain tumor biology has been evident for nearly as long as cAMP and its synthetase, adenylate cyclase (ADCY have been known. The importance of the pathway in brain tumorigenesis has been demonstrated in vitro and in multiple animal models. Recently, we provided human validation for a cooperating oncogenic role for cAMP in brain tumorigenesis when we found that SNPs in ADCY8 were correlated with glioma (brain tumor risk in individuals with Neurofibromatosis type 1 (NF1. Together, these studies provide a strong rationale for targeting cAMP in brain tumor therapy. However, the cAMP pathway is well known to be sexually dimorphic, and SNPs in ADCY8 affected glioma risk in a sex-specific fashion, elevating the risk for females while protecting males. The cAMP pathway can be targeted at multiple levels in the regulation of its synthesis and degradation. Sex differences in response to drugs that target cAMP regulators indicate that successful targeting of the cAMP pathway for brain tumor patients is likely to require matching specific mechanisms of drug action with patient sex.

  17. Quantitative Proteomics Analysis of the cAMP/Protein Kinase A Signaling Pathway

    Science.gov (United States)

    2012-01-01

    To define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomics approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in the level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk with IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomics analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after incubation with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher level of basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress. PMID:23110364

  18. Cyclic adenosine monophosphate-dependent phosphorylation of mammalian mitochondrial proteins: enzyme and substrate characterization and functional role

    Czech Academy of Sciences Publication Activity Database

    Dobrová, Zuzana; Sardanelli, A. M.; Speranza, F.; Scacco, S.; Signorile, A.; Lorusso, V.; Papa, S.

    2001-01-01

    Roč. 40, - (2001), s. 13941-13947 ISSN 0006-2960 Institutional research plan: CEZ:AV0Z5020903 Keywords : cAMP * cyclic adenosine monophosphate Subject RIV: CE - Biochemistry Impact factor: 4.114, year: 2001

  19. Recent Advances in the Development of Antimicrobial Peptides (AMPs): Attempts for Sustainable Medicine?

    Science.gov (United States)

    Kokel, Anne; Torok, Marianna

    2018-01-17

    Since the first isolation of antimicrobial peptides (AMPs) they have attracted extensive interest in medicinal chemistry. However, only a few AMP-based drugs are currently available on the market. Despite their effectiveness, biodegradability, and versatile mode of action that is less likely to induce resistance compared to conventional antibiotics, AMPs suffer from major issues that need to be addressed to broaden their use. Notably, AMPs can lack selectivity leading to side effects and cytotoxicity, and also exhibit in vivo instability. Several strategies are being actively considered to overcome the limitations that restrain the success of AMPs. In the current work, recent strategies reported for improving AMPs in the context of drug design and delivery were surveyed, and also their possible impact on patients and the environment was assessed. As a major advantage AMPs possess an easily tunable skeleton offering opportunities to improve their properties. Strategic structural modifications and the beneficial properties of cyclic or branched AMPs in term of stability have been reported. The conjugation of AMPs with nanoparticles has also been explored to increase their in vivo stability. Other techniques such as the coupling of AMPs with specific antibodies aim to increase the selectivity of the potential drug towards the target. These strategies were evaluated for their effect on the environment highlighting green technologies. Although further research is needed taking into account both environmental and human health consequences of novel AMPs several of these compounds are promising drug candidates for use in sustainable medicine. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. Genetically-encoded tools for cAMP probing and modulation in living systems.

    Directory of Open Access Journals (Sweden)

    Valeriy M Paramonov

    2015-09-01

    Full Text Available Intracellular 3'-5'-cyclic adenosine monophosphate (cAMP is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming - all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells, underpin the ensuing limitations of the conventional cAMP assays: 1 genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; 2 inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control – something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs.

  1. Sleutelmag en amp

    Directory of Open Access Journals (Sweden)

    P. J. Rossouw

    1985-06-01

    Full Text Available As die Heidelbergse Kategismus (Sondag 31 handel oor die sleutels van die Koninkryk, dan word die betrokkenheid van die ampte slegs per implikasie veronderstel. Meer eksplisiet word die amp en sleutelmag in die Nederlandse Geloofsbelydenis gekoppel; “ons glo dat hierdie ware kerk ooreenkomstig die geestelike bestuurswyse wat ons Here ons in sy Woord geleer het, geregeer moet word... hulle (moet sorg dra dat die ware godsdiens onderhou (word, die ware leer orals versprei, die oortreders op geestelike wyse vermaan en in toom gehou (word... (N.G.B. art. 30 en: “Ons glo verder dat die regeerders van die kerk, al is dit nuttig en goed om onder mekaar ’n bepaalde orde tot instandhouding van die liggaam van die kerk in te stel en te handhaaf, tog noukeurig moet oppas om nie af te wyk van wat Christus, ons enigste Meester, vir ons ingestel het nie... Ons aanvaar derhalwe slegs wat kan dien om eendrag en eenheid te bewaar en te bevorder, en om alles in gehoorsaamheid van God te onderhou. Daarvoor is nodig die ban en alles wat daarmee saamhang, toegepas ooreenkomstig die Woord van God” (N.G.B. art. 32.

  2. Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling.

    Science.gov (United States)

    Inda, Carolina; Dos Santos Claro, Paula A; Bonfiglio, Juan J; Senin, Sergio A; Maccarrone, Giuseppina; Turck, Christoph W; Silberstein, Susana

    2016-07-18

    Corticotropin-releasing hormone receptor 1 (CRHR1) activates G protein-dependent and internalization-dependent signaling mechanisms. Here, we report that the cyclic AMP (cAMP) response of CRHR1 in physiologically relevant scenarios engages separate cAMP sources, involving the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). cAMP produced by tmACs and sAC is required for the acute phase of extracellular signal regulated kinase 1/2 activation triggered by CRH-stimulated CRHR1, but only sAC activity is essential for the sustained internalization-dependent phase. Thus, different cAMP sources are involved in different signaling mechanisms. Examination of the cAMP response revealed that CRH-activated CRHR1 generates cAMP after endocytosis. Characterizing CRHR1 signaling uncovered a specific link between CRH-activated CRHR1, sAC, and endosome-based signaling. We provide evidence of sAC being involved in an endocytosis-dependent cAMP response, strengthening the emerging model of GPCR signaling in which the cAMP response does not occur exclusively at the plasma membrane and introducing the notion of sAC as an alternative source of cAMP. © 2016 Inda et al.

  3. Decoding spatial and temporal features of neuronal cAMP/PKA signaling with FRET biosensors.

    Science.gov (United States)

    Castro, Liliana R V; Guiot, Elvire; Polito, Marina; Paupardin-Tritsch, Daniéle; Vincent, Pierre

    2014-02-01

    Cyclic adenosine monophosphate (cAMP) and the cyclic-AMP-dependent protein kinase (PKA) regulate a plethora of cellular functions in virtually all eukaryotic cells. In neurons, the cAMP/PKA signaling cascade controls a number of biological properties such as axonal growth, pathfinding, efficacy of synaptic transmission, regulation of excitability, or long term changes. Genetically encoded optical biosensors for cAMP or PKA are considerably improving our understanding of these processes by providing a real-time measurement in living neurons. In this review, we describe the recent progress made in the creation of biosensors for cAMP or PKA activity. These biosensors revealed profound differences in the amplitude of the cAMP signal evoked by neuromodulators between various neuronal preparations. These responses can be resolved at the level of individual neurons, also revealing differences related to the neuronal type. At the sub-cellular level, biosensors reported different signal dynamics in domains like dendrites, cell body, nucleus, and axon. Combining this imaging approach with pharmacology or genetic models points at phosphodiesterases and phosphatases as critical regulatory proteins. Biosensor imaging will certainly emerge as a forefront tool to decipher the subtle mechanics of intracellular signaling. This will certainly help us to understand the mechanism of action of current drugs and foster the development of novel molecules for neuropsychiatric diseases. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Protein kinase A and Epac activation by cAMP regulates the expression of glial fibrillary acidic protein in glial cells

    Directory of Open Access Journals (Sweden)

    Sugimoto Naotoshi

    2016-01-01

    Full Text Available Cyclic adenosine monophosphate (cAMP controls differentiation in several types of cells during brain development. However, the molecular mechanism of cAMP-controlled differentiation is not fully understood. We investigated the role of protein kinase A (PKA and exchange protein directly activated by cAMP (Epac on cAMP-induced glial fibrillary acidic protein (GFAP, an astrocyte marker, in cultured glial cells. B92 glial cells were treated with cAMP-elevating drugs, an activator of adenylate cyclase, phosphodiesterase inhibitor and a ß adrenal receptor agonist. These cAMP-elevating agents induced dramatic morphological changes and expression of GFAP. A cAMP analog, 8-Br-cAMP, which activates Epac as well as PKA, induced GFAP expression and morphological changes, while another cAMP analog, 8-CPT-cAMP, which activates Epac with greater efficacy when compared to PKA, induced GFAP expression but very weak morphological changes. Most importantly, the treatment with a PKA inhibitor partially reduced cAMP-induced GFAP expression. Taken together, these results indicate that cAMP-elevating drugs lead to the induction of GFAP via PKA and/or Epac activation in B92 glial cells.

  5. Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

    Science.gov (United States)

    Tuder, R M; Karasek, M A; Bensch, K G

    1990-02-01

    The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.

  6. cAMP and forskolin decrease γ-aminobutyric acid-gated chloride flux in rat brain synaptoneurosomes

    International Nuclear Information System (INIS)

    Heuschneider, G.; Schwartz, R.D.

    1989-01-01

    The effects of the cyclic nucleotide cAMP on γ-aminobutyric acid-gated chloride channel function were investigated. The membrane-permeant cAMP analog N 6 , O 2' -dibutyryladenosine 3',5'-cyclic monophosphate inhibited muscimol-induced 36 Cl - uptake into rat cerebral cortical synaptoneurosomes in a concentration-dependent manner. The inhibition was due to a decrease in the maximal effect of muscimol, with no change in potency. Similar effects were observed with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, 8-bromoadenosine 3',5'-cyclic monophosphate, and the phosphodiesterase inhibitor isobutylmethylxanthine. The effect of endogenous cAMP accumulation on the γ-aminobutyric acid-gated Cl - channel was studied with forskolin, an activator of adenylate cyclase. Under identical conditions, in the intact synaptoneurosomes, forskolin inhibited muscimol-induced 36 Cl - uptake and generated cAMP with similar potencies. Surprisingly, 1,9-dideoxyforskolin, which does not activate adenylate cyclase, also inhibited the muscimol response, suggesting that forskolin and its lipophilic derivatives may interact with the Cl - channel directly. The data suggest that γ-aminobutyric acid (GABA A ) receptor function in brain can be regulated by cAMP-dependent phosphorylation

  7. A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors

    DEFF Research Database (Denmark)

    Vedel, Line; Bräuner-Osborne, Hans; Mathiesen, Jesper Mosolff

    2015-01-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein-coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or ...... also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS....

  8. Effect of cholera toxin on cAMP levels and Na+ influx in isolated intestinal epithelial cells

    International Nuclear Information System (INIS)

    Hyun, C.S.; Kimmich, G.A.

    1982-01-01

    Freshly isolated chicken intestinal cells contain approximately 20 pmol adenosine 3',5'-cyclic monophosphate (cAMP)/mg cellular protein. Incubation with 3 μg/ml cholera toxin (CT) at 37 0 C induces an elevation of cellular cAMP beginning 10-15 min after initial exposure. The response is linear with time for 40-50 min and causes a six- to eightfold increase over control levels at steady state. Dibutyryl cAMP and agents that increase cAMP production inhibit Na + influx into the isolated enterocytes. Chlorpromazine completely abolishes the toxin-induced elevation of cAMP in the isolated cells and also reverses the effect on Na + entry. The data provide evidence for a cAMP-mediated control of intestinal cell Na + uptake, which may represent the mechanistic basis for the antiabsorptive effect of CT on Na + during induction of intestinal secretory activity. Studies on the time-dependent effects of chlorpromazine on both intracellular cAMP concentration and Na + influx suggest that the reactivation of the Na + transport system after cAMP-induced inhibition is slow relative to the disappearance of cAMP

  9. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    International Nuclear Information System (INIS)

    Bhattacharjee, Rajesh; Xiang, Wenpei; Wang, Yinna; Zhang, Xiaoying; Billiar, Timothy R.

    2012-01-01

    Highlights: ► cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. ► cAMP blocks NF-κB activation induced by TNF and actinomycin D. ► cAMP blocks DISC formation following TNF and actinomycin D exposure. ► cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor α (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found that cAMP exerts its affect at the proximal level of TNF signaling by inhibiting the formation of the DISC

  10. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Rajesh [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Xiang, Wenpei [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People' s Republic of China (China); Wang, Yinna [Vascular Medicine Institute, University of Pittsburgh School of Medicine, 10051-5A BST 3, 3501 Fifth Avenue, Pittsburgh, PA 15261 (United States); Zhang, Xiaoying [Department of Medicine/Endocrinology Division, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213 (United States); Billiar, Timothy R., E-mail: billiartr@upmc.edu [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  11. Studies on c-AMP contents in sea urchin eggs fertilized with normal and x-irradiated sperm

    International Nuclear Information System (INIS)

    Kimura, Hiroshi

    1975-01-01

    Intracellular levels of cyclic 3', 5'-adenosine monophosphate (c-AMP) seemed to remain constant through the first cleavage cycle of sea urchin eggs. X-irradiation to the sperm, which induced the first cleavage delay, did not change this level. Although it was shown in the previous paper that X-ray-induced cleavage delay was reduced by caffeine but not by aminophyline, both caffeine and aminophyline caused an increase in c-AMP levels. These results indicated the possibility that c-AMP does not mediate this caffeine effect on cleavage delay. (auth.)

  12. Inhibition of cAMP-Activated Intestinal Chloride Secretion by Diclofenac: Cellular Mechanism and Potential Application in Cholera

    OpenAIRE

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-01-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell...

  13. A conjugate of decyltriphenylphosphonium with plastoquinone can carry cyclic adenosine monophosphate, but not cyclic guanosine monophosphate, across artificial and natural membranes.

    Science.gov (United States)

    Firsov, Alexander M; Rybalkina, Irina G; Kotova, Elena A; Rokitskaya, Tatyana I; Tashlitsky, Vadim N; Korshunova, Galina A; Rybalkin, Sergei D; Antonenko, Yuri N

    2018-02-01

    The present study demonstrated for the first time the interaction between adenosine 3',5'-cyclic monophosphate (cAMP), one of the most important signaling compounds in living organisms, and the mitochondria-targeted antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1). The data obtained on model liquid membranes and human platelets revealed the ability of SkQ1 to selectively transport cAMP, but not guanosine 3',5'-cyclic monophosphate (cGMP), across both artificial and natural membranes. In particular, SkQ1 elicited translocation of cAMP from the source to the receiving phase of a Pressman-type cell, while showing low activity with cGMP. Importantly, only conjugate with plastoquinone, but not dodecyl-triphenylphosphonium, was effective in carrying cAMP. In human platelets, SkQ1 also appeared to serve as a carrier of cAMP, but not cGMP, from outside to inside the cell, as measured by phosphorylation of the vasodilator stimulated phosphoprotein. The SkQ1-induced transfer of cAMP across the plasma membrane found here can be tentatively suggested to interfere with cAMP signaling pathways in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Ru (amp)(bipy)Cl

    Indian Academy of Sciences (India)

    Administrator

    [RuV(amp)(bipy)O]+ intermediate complex which leads to the high affinity for hydrogen atom/hydride abstraction. Acknowledgement. We gratefully acknowledge the financial support from the Department of Science &. Technology, Government of India. We are thankful to Shri Hardyal Singh for his encouragement. Reference.

  15. Atrazine acts as an endocrine disrupter by inhibiting cAMP-specific phosphodiesterase-4

    International Nuclear Information System (INIS)

    Kucka, Marek; Pogrmic-Majkic, Kristina; Fa, Svetlana; Stojilkovic, Stanko S.; Kovacevic, Radmila

    2012-01-01

    Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In this study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells in a concentration-dependent manner, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin (an activator of adenylyl cyclase)- and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate prolactin release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in pituitary and Leydig cells together with mRNA for PDE8A. In contrast, immortalized lacto-somatotrophs showed low expression of these mRNA transcripts and several fold higher cAMP levels compared to normal pituitary cells, and atrazine was unable to further increase cAMP levels. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s. -- Highlights: ► Atrazine stimulates cAMP accumulation in pituitary and Leydig cells. ► Atrazine also stimulates PRL and androgens secretion. ► Stimulatory effects of atrazine were abolished in cells with IBMX-inhibited PDEs. ► Atrazine specificity toward cAMP

  16. Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

    Directory of Open Access Journals (Sweden)

    Viacheslav O. Nikolaev

    2013-04-01

    Full Text Available 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.

  17. Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium.

    Science.gov (United States)

    Schlageter, N; Janis, R A; Gualtieri, R T; Hechter, O

    1980-03-01

    The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.

  18. The effect of ultraviolet light on the cyclic nucleotide system of human fibroblasts

    International Nuclear Information System (INIS)

    Fertel, R.H.; Tejwani, G.A.; Albrightson, C.R.; Hart, R.W.

    1981-01-01

    The concentrations of cyclic AMP and cyclic GMP in in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m 2 , and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, the results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may effect the extranuclear functions of irradiated cells. (author)

  19. Polarized Th1 and Th2 cells are less responsive to negative feedback by receptors coupled to the AC/cAMP system compared to freshly isolated T cells

    NARCIS (Netherlands)

    Heijink, Irene H; Vellenga, Edo; Borger, Peter; Postma, Dirkje S; Monchy, Jan G R de; Kauffman, Henk F

    1 The adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP) system is known to negatively regulate transcriptional activity of T cells, thereby possibly modulating T-cell-mediated responses at the sites of inflammation. Effects of cAMP have been widely studied in freshly isolated T cells and

  20. Characterization of a crp* mutant of the E. coli cAMP receptor protein

    International Nuclear Information System (INIS)

    Ren, Y.L.; Garges, S.; Adhya, S.; Krakow, J.S.

    1987-01-01

    One of the crp* mutants previously isolated to activate lac promoter in vivo has been characterized with regard to its biochemical properties. CRP*592 shows a more open conformation than CRP as indicated by its sensitivity to proteolytic attack. Dithionitrobenzoic acid mediated intersubunit crosslinking of CRP requires cAMP; this reaction occurs with unliganded CRP*592. Binding of CRP to its site on the lac promoter and activation of abortive initiation is effected by cAMP but not by cGMP. CRP*592 can activate abortive initiation in the presence of cAMP or cGMP and also at a high CRP*592 concentration in the absence of cyclic nucleotide. DNase I footprinting shows that cAMP-CRP* binds to its site on lac P + while unliganded CRP* and cGMP-CRP* form a stable complex with the [ 32 P]lac P + only in the presence of RNA polymerase. While cGMP binds to CRP it cannot replace cAMP in effecting the conformation necessary for site specific promoter binding; the weakly active unliganded CRP*592 can be shifted to a functional conformation by cAMP, cGMP and RNA polymerase

  1. Understanding cAMP-dependent allostery by NMR spectroscopy: comparative analysis of the EPAC1 cAMP-binding domain in its apo and cAMP-bound states.

    Science.gov (United States)

    Mazhab-Jafari, Mohammad T; Das, Rahul; Fotheringham, Steven A; SilDas, Soumita; Chowdhury, Somenath; Melacini, Giuseppe

    2007-11-21

    cAMP (adenosine 3',5'-cyclic monophosphate) is a ubiquitous second messenger that activates a multitude of essential cellular responses. Two key receptors for cAMP in eukaryotes are protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC), which is a recently discovered guanine nucleotide exchange factor (GEF) for the small GTPases Rap1 and Rap2. Previous attempts to investigate the mechanism of allosteric activation of eukaryotic cAMP-binding domains (CBDs) at atomic or residue resolution have been hampered by the instability of the apo form, which requires the use of mixed apo/holo systems, that have provided only a partial picture of the CBD apo state and of the allosteric networks controlled by cAMP. Here, we show that, unlike other eukaryotic CBDs, both apo and cAMP-bound states of the EPAC1 CBD are stable under our experimental conditions, providing a unique opportunity to define at an unprecedented level of detail the allosteric interactions linking two critical functional sites of this CBD. These are the phosphate binding cassette (PBC), where cAMP binds, and the N-terminal helical bundle (NTHB), which is the site of the inhibitory interactions between the regulatory and catalytic regions of EPAC. Specifically, the combined analysis of the cAMP-dependent changes in chemical shifts, 2 degrees structure probabilities, hydrogen/hydrogen exchange (H/H) and hydrogen/deuterium exchange (H/D) protection factors reveals that the long-range communication between the PBC and the NTHB is implemented by two distinct intramolecular cAMP-signaling pathways, respectively, mediated by the beta2-beta3 loop and the alpha6 helix. Docking of cAMP into the PBC perturbs the NTHB inner core packing and the helical probabilities of selected NTHB residues. The proposed model is consistent with the allosteric role previously hypothesized for L273 and F300 based on site-directed mutagenesis; however, our data show that such a contact is part of a

  2. Synthesis and degradation of cyclic nucleotides in brain after a high dose of ionizing radiation

    International Nuclear Information System (INIS)

    Hunt, W.A.; Dalton, T.K.

    1981-01-01

    Previous data from our laboratory have indicated that a high dose of ionizing radiation can deplete the cyclic nucleotides guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) on several areas of the rat brain. cGMP is more sensitive to radiation than cAMP and does not recover for at least 24 h after irradiation. The response of cAMP is transient and recovery occurs within 4 h. The purpose of the present paper is to determine whether alternations in the activity of the synthetic and degradative enzymes that regulate cyclic nucleotide levels could account for the observed effects. Guanylate and adenylate cyclase and cGMP and cAMP phosphodiesterase activities were determined 10 min after irradiation with 10,000 rad of high-energy electrons. No alteration was detected under these experimental conditions. The data suggest that the reduction in cyclic nucleotides is not a direct effect on their metabolic enzymes and is probably secondary to some as yet-undefined action of radiation on the brain

  3. Effect of cAMP on short-circuit current in isolated human ciliary body.

    Science.gov (United States)

    Wu, Ren-yi; Ma, Ning; Hu, Qian-qian

    2013-07-01

    Cyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current, Isc) across the ciliary processes in pigs. The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change. In an Ussing-type chamber system, the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed. The involvement of Cl(-) component in the bath solution was investigated. The effect of Cl(-) channel (10 µmol/L niflumic acid and 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)), K(+) channel (10 mmol/L tetraethylammonium chloride (TEA)), or Na(+) channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied. Dose-dependently, 8-bromo-cAMP (10 nmol/L-30 µmol/L) or forskolin (10 nmol/L-3 µmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue. Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side. When the tissue was bathed in low Cl(-) solutions, the Isc increase was significantly inhibited. Finally, niflumic acid and DIDS, but not TEA or amiloride, significantly prevented the Isc increase induced by 8-bromo-cAMP. cAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes. Chloride is likely to be among the ions, the transportation of which across the tissue is triggered by cAMP, suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.

  4. Immunohistochemical distribution of cAMP- and cGMP-phosphodiesterase (PDE) isoenzymes in the human prostate

    NARCIS (Netherlands)

    Uckert, Stefan; Oelke, Matthias; Stief, Christian G.; Andersson, K.-E.; Jonas, Udo; Hedlund, Petter

    2006-01-01

    With the introduction of sildenafil citrate (Viagra), the concept of phosphodiesterase (PDE) inhibition has gained tremendous interest in the field of urology. Cyclic nucleotide second messengers cGMP and cAMP have been assumed to be involved in the control of the normal function of the prostate.

  5. Changes in calmodulin concentration and cyclic 3',5'-nucleotide phosphodiesterase activity in skeletal muscle of hyper- and hypothyroid rats.

    Science.gov (United States)

    Mano, T; Iwase, K; Yoshimochi, I; Sawai, Y; Oda, N; Nishida, Y; Mokuno, T; Kotake, M; Nakai, A; Hayakawa, N

    1995-08-01

    Hyper- and hypothyroid states occasionally induce skeletal muscle dysfunction i.e. periodic paralysis and thyroid myopathy. The etiology of these diseases remains unclear, but several findings suggest that the catecholamine-beta-receptor-cAMP system or other messenger systems are disturbed in these diseases. In this context, we evaluated changes in the cyclic 3',5'-nucleotide metabolic enzyme, cyclic 3',5'-nucleotide phosphodiesterase (PDE) and calmodulin concentrations in skeletal muscles of hyper- and hypothyroid rats. Activities of cyclic AMP-PDE were low in skeletal muscle both from hyper- and hypothyroid rats, and calmodulin concentration was high in hyperthyroid and low in hypothyroid rats, as compared with normal rats. DE-52 column chromatographic analysis showed that the cGMP hydrolytic activity in peak I and the cAMP hydrolytic activity in peak II were decreased in hypothyroid rats, whereas cAMP hydrolytic activity in peak III was unchanged. The cAMP hydrolytic activity in peak III was decreased in hyperthyroid rats, but the activities in peaks I and II were unchanged. These findings indicate that cAMP and calmodulin may have some role in skeletal muscle function in the hyperthyroid state, and that cAMP and calmodulin-dependent metabolism may be suppressed in the hypothyroid state.

  6. Plasma levels of cAMP, cGMP and CGRP in sildenafil-induced headache

    DEFF Research Database (Denmark)

    Kruuse, Christina Rostrup; Frandsen, E; Schifter, S

    2004-01-01

    Sildenafil, a selective inhibitor of the cyclic guanosine monophosphate (cGMP) degrading phosphodiestrase 5 (PDE5), induced migraine without aura in 10 of 12 migraine patients and in healthy subjects it induced significantly more headache than placebo. The aim of the present study was to determine...... whether the pain-inducing effects of sildenafil would be reflected in plasma levels of important signalling molecules in migraine: cGMP, cyclic adenosine monophosphate (cAMP) and calcitonin gene-related peptide (CGRP). Ten healthy subjects (four women, six men) and 12 patients (12 women) suffering from...... migraine without aura were included in two separate double-blind, placebo-controlled, cross-over studies in which placebo or sildenafil 100 mg was administered orally. Plasma levels of CGRP, cAMP and cGMP were determined in blood from the antecubital vein. Despite the ability of sildenafil to induce...

  7. Optimization of cAMP fluorescence dataset from ACTOne cannabinoid receptor 1 cell line

    Directory of Open Access Journals (Sweden)

    Chaela S. Presley

    2016-06-01

    Full Text Available The ACTOne cannabinoid receptor 1 functional system is comprised of transfected HEK cells with the parental cyclic nucleotide gated channel (CNG co-transfected with cannabinoid receptor 1 (CB1. The ACTOne CB1 cell line was evaluated for cAMP driven fluorescence by optimizing experimental conditions for sensitivity to forskolin and CP 55,940, reading time point, reliability of cell passage number, and pertussis inactivation of Gi/o.

  8. Mycobacterium tuberculosis cAMP Receptor Protein (Rv3676) Differs from the Escherichia coli Paradigm in Its cAMP Binding and DNA Binding Properties and Transcription Activation Properties*

    Science.gov (United States)

    Stapleton, Melanie; Haq, Ihtshamul; Hunt, Debbie M.; Arnvig, Kristine B.; Artymiuk, Peter J.; Buxton, Roger S.; Green, Jeffrey

    2010-01-01

    The pathogen Mycobacterium tuberculosis produces a burst of cAMP upon infection of macrophages. Bacterial cyclic AMP receptor proteins (CRP) are transcription factors that respond to cAMP by binding at target promoters when cAMP concentrations increase. Rv3676 (CRPMt) is a CRP family protein that regulates expression of genes (rpfA and whiB1) that are potentially involved in M. tuberculosis persistence and/or emergence from the dormant state. Here, the CRPMt homodimer is shown to bind two molecules of cAMP (one per protomer) at noninteracting sites. Furthermore, cAMP binding by CRPMt was relatively weak, entropy driven, and resulted in a relatively small enhancement in DNA binding. Tandem CRPMt-binding sites (CRP1 at −58.5 and CRP2 at −37.5) were identified at the whiB1 promoter (PwhiB1). In vitro transcription reactions showed that CRP1 is an activating site and that CRP2, which was only occupied in the presence of cAMP or at high CRPMt concentrations in the absence of cAMP, is a repressing site. Binding of CRPMt to CRP1 was not essential for open complex formation but was required for transcription activation. Thus, these data suggest that binding of CRPMt to the PwhiB1 CRP1 site activates transcription at a step after open complex formation. In contrast, high cAMP concentrations allowed occupation of both CRP1 and CRP2 sites, resulting in inhibition of open complex formation. Thus, M. tuberculosis CRP has evolved several distinct characteristics, compared with the Escherichia coli CRP paradigm, to allow it to regulate gene expression against a background of high concentrations of cAMP. PMID:20028978

  9. In Vivo Cardiovascular Pharmacology of 2′,3′-cAMP, 2′-AMP, and 3′-AMP in the Rat

    Science.gov (United States)

    Mi, Zaichuan

    2013-01-01

    The naturally occurring purine 2′,3′-cAMP is metabolized in vitro to 2′-AMP and 3′-AMP, which are subsequently metabolized to adenosine. Whether in vivo 2′,3′-cAMP, 2′-AMP, or 3′-AMP are rapidly converted to adenosine and exert rapid effects via adenosine receptors is unknown. To address this question, we compared the cardiovascular and renal effects of 2′,3′-cAMP, 2′-AMP, 3′-AMP, 3′,5′-cAMP, 5′-AMP, and adenosine in vivo in the rat. Purines were infused intravenously while monitoring mean arterial blood pressure (MABP), heart rate (HR), cardiac output, and renal and mesenteric blood flows. Total peripheral (TPR), renal vascular (RVR), and mesenteric vascular (MVR) resistances were calculated. Urine was collected for determination of urine excretion rate [urine volume (UV)]. When sufficient urine was available, the sodium excretion rate (Na+ER) and glomerular filtration rate (GFR) were determined. 2′,3′-cAMP, 2′-AMP, and 3′-AMP dose-dependently and profoundly reduced MABP, HR, TPR, and MVR with efficacy and potency similar to adenosine and 5′-AMP. These effects of 2′,3′-cAMP, 2′-AMP, and 3′-AMP were attenuated by blockade of adenosine receptors with 1,3-dipropyl-8-(p-sulfophenyl)xanthine. 2′,3′-cAMP, 2′-AMP, 3′-AMP, adenosine, and 5′-AMP variably affected RVR, but profoundly (nearly 100%) decreased UV at higher doses. GFR and Na+ER could be measured at the lower doses and were suppressed by 2′,3′-cAMP, 2′-AMP, and 3′-AMP, but not by adenosine or 5′-AMP. 2′,3′-cAMP increased urinary excretion rates of 2′-AMP, 3′-AMP, and adenosine. 3′,5′-cAMP exerted no adverse hemodynamic effects yet increased urinary adenosine as efficiently as 2′,3′-cAMP. Conclusions: In vivo 2′,3′-cAMP is rapidly converted to adenosine. Because both cAMPs increase adenosine in the urinary compartment, these agents may provide unique therapeutic opportunities. PMID:23759508

  10. Effects of fenspiride on human bronchial cyclic nucleotide phosphodiesterase isoenzymes: functional and biochemical study.

    Science.gov (United States)

    Cortijo, J; Naline, E; Ortiz, J L; Berto, L; Girard, V; Malbezin, M; Advenier, C; Morcillo, E J

    1998-01-02

    We have investigated the role of human bronchial cyclic nucleotide phosphodiesterases in the effects of fenspiride, a drug endowed with bronchodilator and anti-inflammatory properties. Functional studies on human isolated bronchi showed that fenspiride (10(-6)-3 x 10(-3) M, 30 min) induced a shift to the left of the concentration-response curves for isoprenaline and sodium nitroprusside with -logEC50 values of 4.1+/-0.1 (n = 7) and 3.5+/-0.2 (n = 8), respectively. Biochemical studies were carried out on three human bronchi in which separation of cyclic nucleotide phosphodiesterase isoenzymes was performed by ion exchange chromatography followed by determination of phosphodiesterase activity with a radioisotopic method. Phosphodiesterase 4 (cyclic AMP-specific) and phosphodiesterase 5 (cyclic GMP-specific) were the major phosphodiesterase isoforms present in the human bronchial tissue. The presence of phosphodiesterase 1 (Ca2+/calmodulin-stimulated), phosphodiesterase 2 (cyclic GMP-stimulated) and, in two cases, phosphodiesterase 3 (cyclic GMP-inhibited) was also identified. Fenspiride inhibited phosphodiesterase 4 and phosphodiesterase 3 activities with -logIC50 values of 4.16+/-0.09 and 3.44+/-0.12, respectively. Phosphodiesterase 5 activity was also inhibited with a -logIC50 value of approximately 3.8. Fenspiride (fenspiride is an effective inhibitor of both cyclic AMP and cyclic GMP hydrolytic activity in human bronchial tissues and this action may contribute to its airway effects.

  11. Effect of cAMP derivates on assembly and maintenance of tight junctions in human umbilical vein endothelial cells

    Directory of Open Access Journals (Sweden)

    Beese Michaela

    2010-09-01

    Full Text Available Abstract Background Endothelial tight and adherens junctions control a variety of physiological processes like adhesion, paracellular transport of solutes or trafficking of activated leukocytes. Formation and maintenance of endothelial junctions largely depend on the microenvironment of the specific vascular bed and on interactions of the endothelium with adjacent cell types. Consequently, primary cultures of endothelial cells often lose their specific junctional pattern and fail to establish tight monolayer in vitro. This is also true for endothelial cells isolated from the vein of human umbilical cords (HUVEC which are widely used as model for endothelial cell-related studies. Results We here compared the effect of cyclic 3'-5'-adenosine monophosphate (cAMP and its derivates on formation and stabilization of tight junctions and on alterations in paracellular permeability in HUVEC. We demonstrated by light and confocal laser microscopy that for shorter time periods the sodium salt of 8-bromoadenosine-cAMP (8-Br-cAMP/Na and for longer incubation periods 8-(4-chlorophenylthio-cAMP (pCPT-cAMP exerted the greatest effects of all compounds tested here on formation of continuous tight junction strands in HUVEC. We further demonstrated that although all compounds induced protein kinase A-dependent expression of the tight junction proteins claudin-5 and occludin only pCPT-cAMP slightly enhanced paracellular barrier functions. Moreover, we showed that pCPT-cAMP and 8-Br-cAMP/Na induced expression and membrane translocation of tricellulin. Conclusions pCPT-cAMP and, to a lesser extend, 8-Br-cAMP/Na improved formation of continuous tight junction strands and decreased paracellular permeability in primary HUVEC. We concluded that under these conditions HUVEC represent a feasible in vitro model to study formation and disassembly of endothelial tight junctions and to characterize tight junction-associated proteins

  12. A bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis.

    Science.gov (United States)

    Dey, Bappaditya; Dey, Ruchi Jain; Cheung, Laurene S; Pokkali, Supriya; Guo, Haidan; Lee, Jong-Hee; Bishai, William R

    2015-04-01

    Detection of cyclic-di-adenosine monophosphate (c-di-AMP), a bacterial second messenger, by the host cytoplasmic surveillance pathway (CSP) is known to elicit type I interferon (IFN) responses, which are crucial to antimicrobial defense. However, the mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis virulence remain unclear. Here we show that resistance to tuberculosis requires CSP-mediated detection of c-di-AMP produced by M. tuberculosis and that levels of c-di-AMP modulate the fate of infection. We found that a di-adenylate cyclase (disA or dacA)-overexpressing M. tuberculosis strain that secretes excess c-di-AMP activates the interferon regulatory factor (IRF) pathway with enhanced levels of IFN-β, elicits increased macrophage autophagy, and exhibits substantial virulence attenuation in mice. We show that c-di-AMP-mediated IFN-β induction during M. tuberculosis infection requires stimulator of interferon genes (STING)-signaling. We observed that c-di-AMP induction of IFN-β is independent of the cytosolic nucleic acid receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), but cGAS nevertheless contributes substantially to the overall IFN-β response to M. tuberculosis infection. In sum, our results reveal c-di-AMP to be a key mycobacterial pathogen-associated molecular pattern (PAMP) driving host type I IFN responses and autophagy. These findings suggest that modulating the levels of this small molecule may lead to novel immunotherapeutic strategies against tuberculosis.

  13. Modulatory effects of cAMP and PKC activation on gap junctional intercellular communication among thymic epithelial cells

    Directory of Open Access Journals (Sweden)

    Neves-dos-Santos Sandra

    2010-01-01

    Full Text Available Abstract Background We investigated the effects of the signaling molecules, cyclic AMP (cAMP and protein-kinase C (PKC, on gap junctional intercellular communication (GJIC between thymic epithelial cells (TEC. Results Treatment with 8-Br-cAMP, a cAMP analog; or forskolin, which stimulates cAMP production, resulted in an increase in dye transfer between adjacent TEC, inducing a three-fold enhancement in the mean fluorescence of coupled cells, ascertained by flow cytometry after calcein transfer. These treatments also increased Cx43 mRNA expression, and stimulated Cx43 protein accumulation in regions of intercellular contacts. VIP, adenosine, and epinephrine which may also signal through cyclic nucleotides were tested. The first two molecules did not mimic the effects of 8-Br-cAMP, however epinephrine was able to increase GJIC suggesting that this molecule functions as an endogenous inter-TEC GJIC modulators. Stimulation of PKC by phorbol-myristate-acetate inhibited inter-TEC GJIC. Importantly, both the enhancing and the decreasing effects, respectively induced by cAMP and PKC, were observed in both mouse and human TEC preparations. Lastly, experiments using mouse thymocyte/TEC heterocellular co-cultures suggested that the presence of thymocytes does not affect the degree of inter-TEC GJIC. Conclusions Overall, our data indicate that cAMP and PKC intracellular pathways are involved in the homeostatic control of the gap junction-mediated communication in the thymic epithelium, exerting respectively a positive and negative role upon cell coupling. This control is phylogenetically conserved in the thymus, since it was seen in both mouse and human TEC preparations. Lastly, our work provides new clues for a better understanding of how the thymic epithelial network can work as a physiological syncytium.

  14. Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603.

    Science.gov (United States)

    Goulding, Joelle; May, Lauren T; Hill, Stephen J

    2018-01-01

    Endogenous adenosine A 2B receptors (A 2B AR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A 2B AR antagonist PSB 603 in HEK 293 cells. The A 2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A 2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A 2A AR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA E MAX with no significant effect on EC 50 values. Kinetics analysis of the effect of PSB 603 on the A 2B AR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A 2B AR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Cyclic mononucleotides modulate potassium and calcium flux responses to H2O2 in Arabidopsis roots

    KAUST Repository

    Ordoñ ez, Natalia Maria; Marondedze, Claudius; Thomas, Ludivine; Pasqualini, Stefania; Shabala, Lana; Shabala, Sergey; Gehring, Christoph A

    2014-01-01

    Cyclic mononucleotides are messengers in plant stress responses. Here we show that hydrogen peroxide (H2O2) induces rapid net K+-efflux and Ca2+-influx in Arabidopsis roots. Pre-treatment with either 10 μM cAMP or cGMP for 1 or 24 h does

  16. Rac1 controls Schwann cell myelination through cAMP and NF2/merlin

    Science.gov (United States)

    Guo, Li; Moon, Chandra; Niehaus, Karen; Zheng, Yi; Ratner, Nancy

    2013-01-01

    During peripheral nervous system development, Schwann cells (SCs) surrounding single large axons differentiate into myelinating SCs. Previous studies implicate RhoGTPases in SC myelination, but the mechanisms involved in RhoGTPase regulation of SC myelination are unknown. Here, we show that SC myelination is arrested in Rac1 conditional knockout (Rac1-CKO) mice. Rac1 knockout abrogated phosphorylation of the effector p21-activated kinase (PAK) and decreased NF2/merlin phosphorylation. Mutation of NF2/merlin rescued the myelin deficit in Rac1-CKO mice in vivo, and the shortened processes in cultured Rac1-CKO SCs in vitro. Mechanistically, cyclic adenosine monophosphate (cAMP) levels and E-cadherin expression were decreased in the absence of Rac1, and both were restored by mutation of NF2/merlin. Reduced cAMP is a cause of the myelin deficiency in Rac1-CKO mice, as elevation of cAMP by rolipram in Rac1-CKO mice in vivo allowed myelin formation. Thus NF2/merlin and cAMP function downstream of Rac1 signaling in SC myelination, and cAMP levels control Rac1-regulated SC myelination. PMID:23197717

  17. Regulation of cAMP on the first mitotic cell cycle of mouse embryos.

    Science.gov (United States)

    Yu, Aiming; Zhang, Zhe; Bi, Qiang; Sun, Bingqi; Su, Wenhui; Guan, Yifu; Mu, Runqing; Miao, Changsheng; Zhang, Jie; Yu, Bingzhi

    2008-03-01

    Mitosis promoting factor (MPF) plays a central role during the first mitosis of mouse embryo. We demonstrated that MPF activity increased when one-cell stage mouse embryo initiated G2/M transition following the decrease of cyclic adenosine 3', 5'-monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) activity. When cAMP and PKA activity increases again, MPF activity decreases and mouse embryo starts metaphase-anaphase transition. In the downstream of cAMP/PKA, there are some effectors such as polo-like kinase 1 (Plk1), Cdc25, Mos (mitogen-activated protein kinase kinase kinase), MEK (mitogen-activated protein kinase kinase), mitogen-activated protein kinase (MAPK), Wee1, anaphase-promoting complex (APC), and phosphoprotein phosphatase that are involved in the regulation of MPF activity. Here, we demonstrated that following activation of MPF, MAPK activity was steady, whereas Plk1 activity fluctuated during the first cell cycle. Plk1 activity was the highest at metaphase and decreased at metaphase-anaphase transition. Further, we established a mathematical model using Gepasi algorithm and the simulation was in agreement with the experimental data. Above all the evidences, we suggested that cAMP and PKA might be the upstream factors which were included in the regulation of the first cell cycle development of mouse embryo. Copyright 2007 Wiley-Liss, Inc.

  18. Proteomic signatures implicate cAMP in light and temperature responses in Arabidopsis thaliana

    KAUST Repository

    Thomas, Ludivine

    2013-05-01

    The second messenger 3\\'-5\\'-cyclic adenosine monophosphate (cAMP) and adenylyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, are increasingly recognized as important signaling molecules in a number of physiological responses in higher plants. Here we used proteomics to identify cAMP-dependent protein signatures in Arabidopsis thaliana and identify a number of differentially expressed proteins with a role in light- and temperature-dependent responses, notably photosystem II subunit P-1, plasma membrane associated cation-binding protein and chaperonin 60 β. Based on these proteomics results we conclude that, much like in cyanobacteria, algae and fungi, cAMP may have a role in light signaling and the regulation of photosynthesis as well as responses to temperature and we speculate that ACs could act as light and/or temperature sensors in higher plants. Biological significance: This current study is significant since it presents the first proteomic response to cAMP, a novel and key second messenger in plants. It will be relevant to researchers in plant physiology and in particular those with an interest in second messengers and their role in biotic and abiotic stress responses. © 2013 Elsevier B.V.

  19. cAMP level modulates scleral collagen remodeling, a critical step in the development of myopia.

    Directory of Open Access Journals (Sweden)

    Yijin Tao

    Full Text Available The development of myopia is associated with decreased ocular scleral collagen synthesis in humans and animal models. Collagen synthesis is, in part, under the influence of cyclic adenosine monophosphate (cAMP. We investigated the associations between cAMP, myopia development in guinea pigs, and collagen synthesis by human scleral fibroblasts (HSFs. Form-deprived myopia (FDM was induced by unilateral masking of guinea pig eyes. Scleral cAMP levels increased selectively in the FDM eyes and returned to normal levels after unmasking and recovery. Unilateral subconjunctival treatment with the adenylyl cyclase (AC activator forskolin resulted in a myopic shift accompanied by reduced collagen mRNA levels, but it did not affect retinal electroretinograms. The AC inhibitor SQ22536 attenuated the progression of FDM. Moreover, forskolin inhibited collagen mRNA levels and collagen secretion by HSFs. The inhibition was reversed by SQ22536. These results demonstrate a critical role of cAMP in control of myopia development. Selective regulation of cAMP to control scleral collagen synthesis may be a novel therapeutic strategy for preventing and treating myopia.

  20. Release from Xenopus oocyte prophase I meiotic arrest is independent of a decrease in cAMP levels or PKA activity.

    Science.gov (United States)

    Nader, Nancy; Courjaret, Raphael; Dib, Maya; Kulkarni, Rashmi P; Machaca, Khaled

    2016-06-01

    Vertebrate oocytes arrest at prophase of meiosis I as a result of high levels of cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) activity. In Xenopus, progesterone is believed to release meiotic arrest by inhibiting adenylate cyclase, lowering cAMP levels and repressing PKA. However, the exact timing and extent of the cAMP decrease is unclear, with conflicting reports in the literature. Using various in vivo reporters for cAMP and PKA at the single-cell level in real time, we fail to detect any significant changes in cAMP or PKA in response to progesterone. More interestingly, there was no correlation between the levels of PKA inhibition and the release of meiotic arrest. Furthermore, we devised conditions whereby meiotic arrest could be released in the presence of sustained high levels of cAMP. Consistently, lowering endogenous cAMP levels by >65% for prolonged time periods failed to induce spontaneous maturation. These results argue that the release of oocyte meiotic arrest in Xenopus is independent of a reduction in either cAMP levels or PKA activity, but rather proceeds through a parallel cAMP/PKA-independent pathway. © 2016. Published by The Company of Biologists Ltd.

  1. Competitive cAMP Antagonists for cAMP-Receptor Proteins

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Driel, Roel van; Jastorff, Bernd; Baraniak, Janina; Stec, Wojciech J.; Wit, René J.W. de

    1984-01-01

    The two exocyclic oxygen atoms at phosphorus of cAMP have been replaced by a sulfur atom or by a dimethylamino group. These substitutions introduce chirality at the phosphorus atom; therefore, two diastereoisomers are known for each derivative: (SP)-cAMPS, (RP)-cAMPS, (SP)-cAMPN(CH3)2, and

  2. Effect of ionizing radiation on calcium and cyclic nucleotides metabolism in rats of different age

    International Nuclear Information System (INIS)

    Efimova, N.I.; Libenson, S.V.

    1982-01-01

    Some features of mechanism of calcium homeostasis and cyclic nucleotide exchange breakage in case of acute radiation injury of rats of various age were studied. It is established that calcium level in blood in nonpuberal animals, calcium and cAMP excretion with urine are minimal and reach maximum at puberal age. cGMP excretion with urine and concentrational levels of cAMP and cGMP in blood do not change with age. It is shown that calcium excretion with urine decreases adaptively in conditions of acute radiation injury in rats of all age groups. Maximal shifts in cAMP/cGMP ratio were noted in nonpuberal animals, whereas maximal adaptive-compensatory abilities in the regulation system of calcium homeostasis and cyclic nucleotides are typical to adolescent puberal animals

  3. Nuclease-resistant c-di-AMP derivatives that differentially recognize RNA and protein receptors

    Science.gov (United States)

    Meehan, Robert E.; Torgerson, Chad D.; Gaffney, Barbara L.; Jones, Roger A.; Strobel, Scott A.

    2016-01-01

    The ability of bacteria to sense environmental cues and adapt is essential for their survival. The use of second-messenger signaling molecules to translate these cues into a physiological response is a common mechanism employed by bacteria. The second messenger 3’-5’-cyclic diadenosine monophosphate (c-di-AMP) has been linked to a diverse set of biological processes involved in maintaining cell viability and homeostasis, as well as pathogenicity. A complex network of both protein and RNA receptors inside the cell activate specific pathways and mediate phenotypic outputs in response to c-di-AMP. Structural analysis of these RNA and protein receptors has revealed the different recognition elements employed by these effectors to bind the same small molecule. Herein, using a series of c-di-AMP analogs, we probed the interactions made with a riboswitch and a phosphodiesterase protein to identify the features important for c-di-AMP binding and recognition. We found that the ydaO riboswitch binds c-di-AMP in two discrete sites with near identical affinity and a Hill coefficient of 1.6. The ydaO riboswitch distinguishes between c-di-AMP and structurally related second messengers by discriminating against an amine at the C2 position, more than a carbonyl at the C6 position. We also identified phosphate-modified analogs that bind both the ydaO RNA and GdpP protein with high affinity, while symmetrically-modified ribose analogs exhibited a substantial decrease in ydaO affinity, but retained high affinity for GdpP. These ligand modifications resulted in increased resistance to enzyme-catalyzed hydrolysis by the GdpP enzyme. Together, these data suggest that these c-di-AMP analogs could be useful as chemical tools to specifically target subsections of the second-messenger signaling pathways. PMID:26789423

  4. The Anti-Warburg Effect Elicited by the cAMP-PGC1α Pathway Drives Differentiation of Glioblastoma Cells into Astrocytes

    Directory of Open Access Journals (Sweden)

    Fan Xing

    2017-01-01

    Full Text Available Summary: Glioblastoma multiforme (GBM is among the most aggressive of human cancers. Although differentiation therapy has been proposed as a potential approach to treat GBM, the mechanisms of induced differentiation remain poorly defined. Here, we established an induced differentiation model of GBM using cAMP activators that specifically directed GBM differentiation into astroglia. Transcriptomic and proteomic analyses revealed that oxidative phosphorylation and mitochondrial biogenesis are involved in induced differentiation of GBM. Dibutyryl cyclic AMP (dbcAMP reverses the Warburg effect, as evidenced by increased oxygen consumption and reduced lactate production. Mitochondrial biogenesis induced by activation of the CREB-PGC1α pathway triggers metabolic shift and differentiation. Blocking mitochondrial biogenesis using mdivi1 or by silencing PGC1α abrogates differentiation; conversely, overexpression of PGC1α elicits differentiation. In GBM xenograft models and patient-derived GBM samples, cAMP activators also induce tumor growth inhibition and differentiation. Our data show that mitochondrial biogenesis and metabolic switch to oxidative phosphorylation drive the differentiation of tumor cells. : Xing et al. show that the metabolic shift from glycolysis to oxidative phosphorylation drives differentiation of GBM cells into astrocytes by cAMP activation. Mechanistically, the cAMP-CREB-PGC1α signal mediates mitochondrial biogenesis, which leads to metabolic reprogramming, induced differentiation, and tumor growth inhibition. Keywords: glioblastoma, induced differentiation, Warburg effect, metabolic reprogramming, oxidative phosphorylation, glycolysis, mitochondrial biogenesis, cyclic adenosine monophosphate, cAMP, PPARγ coactivator-1α, PGC1α

  5. Cyclic nucleotide specific phosphodiesterases of Leishmania major

    Directory of Open Access Journals (Sweden)

    Linder Markus

    2006-03-01

    Full Text Available Abstract Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range

  6. The Arabidopsis thaliana Cyclic-Nucleotide-Dependent Response – a Quantitative Proteomic and Phosphoproteomic Analysis

    KAUST Repository

    Alqurashi, May M.

    2013-11-01

    Protein phosphorylation governs many regulatory pathways and an increasing number of kinases, proteins that transfer phosphate groups, are in turn activated by cyclic nucleotides. One of the cyclic nucleotides, cyclic adenosine monophosphate (cAMP), has been shown to be a second messenger in abiotic and biotic stress responses. However, little is known about the precise role of cAMP in plants and in the down-stream activation of kinases, and hence cAMP-dependent phosphorylation. To increase our understanding of the role of cAMP, proteomic and phosphoproteomic profiles of Arabidopsis thaliana suspension culture cells were analyzed before and after treatment of cells with two different concentrations of 8-Bromo-cAMP (1 µM and 100 nM) and over a time-course of one hour. A comparative quantitative analysis was undertaken using two- dimensional gel electrophoresis and the Delta 2D software (DECODON) followed by protein spot identification by tandem mass spectrometry combined with Mascot and Scaffold. Differentially expressed proteins and regulated phosphoproteins were categorized according to their biological function using bioinformatics tools. The results revealed that the treatment with 1 µM and 100 nM 8-Bromo-cAMP was sufficient to induce specific concentration- and time-dependent changes at the proteome and phosphoproteome levels. In particular, different phosphorylation patterns were observed overtime preferentially affecting proteins in a number of functional categories, notably phosphatases, proteins that remove phosphate groups. This suggests that cAMP both transiently activates and deactivates proteins through specific phosphorylation events and provides new insight into biological mechanisms and functions at the systems level.

  7. High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein β-galactosidase expression.

    Science.gov (United States)

    Lin, Hsiao-Hsien; Lee, Tsung-Yih; Liu, Ting-Wei; Tseng, Ching-Ping

    2017-07-01

    Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (∼300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27 kip by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PKA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERK phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the abovementioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce β-galactosidase (β-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the β-Gal production (from 2.8- to 4.5-fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. Copyright © 2017. Published by Elsevier B.V.

  8. Novel adenosine 3',5'-cyclic monophosphate dependent protein kinases in a marine diatom

    International Nuclear Information System (INIS)

    Lin, P.P.C.; Volcani, B.E.

    1989-01-01

    Two novel adenosine 3',5'-cyclic monophosphate (cAMP) dependent protein kinases have been isolated from the diatom Cylindrotheca fusiformis. The kinases, designated I and II, are eluted from DEAE-Sephacel at 0.10 and 0.15 M NaCl. They have a high affinity for cAMP and are activated by micromolar cAMP. They exhibit maximal activity at 5 mM Mg 2+ and pH 8 with the preferred phosphate donor ATP and phosphate acceptor histone H1. They phosphorylate sea urchin sperm histone H1 on a single serine site in the sequence Arg-Lys-Gly-Ser( 32 P)-Ser-Asn-Ala-Arg and have an apparent M r of 75,000 as determined by gel filtration and sucrose density sedimentation. In the kinase I preparation a single protein band with an apparent M r of about 78,000 is photolabeled with 8-azido[ 32 P]cAMP and is also phosphorylated with [γ- 32 P]ATP in a cAMP-dependent manner, after autoradiography following sodium dodecyl sulfate gel electrophoresis. The rate of phosphorylation of the 78,000-dalton band is independent of the enzyme concentration. The results indicate that (i) these diatom cAMP-dependent protein kinases are monomeric proteins, possessing both the cAMP-binding regulatory and catalytic domains on the same polypeptide chain, (ii) the enzymes do not dissociate into smaller species upon activation by binding cAMP, and (iii) self-phosphorylation of the enzymes by an intrapeptide reaction is cAMP dependent. The two diatom cAMP kinases are refractory to the heat-stable protein kinase modulator from rabbit muscle, but they respond differently to proteolytic degradation and to inhibition by arachidonic acid and several microbial alkaloids

  9. The camp analogue, dbcAMP can stimulate rabbit reproductive functions: I. Effect on ovarian folliculogenesis, ovulation and embryo production

    Directory of Open Access Journals (Sweden)

    Chrenek P.

    2012-01-01

    Full Text Available The aim of our study was to examine the influence of administration of N6,2’-dibutyryladenosine 3’5’-cyclic monophosphate (dbcAMP, a cAMP agonist, on ovarian folliculogenesis and atresia, as well as on reproductive efficiency in rabbits, whose ovarian cycle and ovulation was induced by gonadotropins. Ovarian cycle and ovulation of control rabbits were induced by 20 IU/kg PMSG followed by 35 IU/kg hCG administration. Experimental animals received PMSG and hCG together with dbcAMP (at 5, 25 or 50 μg/animal. After ovulation and insemination, the animals were sacrificed. Ovaries were weighted, histological sections of ovaries were prepared, and the presence of ovulated and not ovulated follicles and different stages of atresia was evaluated by light microscopy. The eggs were flushed from the oviducts after insemination and cultured up to blastocyst cell stage. Numbers of ovarian Corpora lutea, ovulated oocytes and oocyte-derived zygotes and embryos reaching hatched blastocyst stage were determined. Administration of dbcAMP (at doses 25 or 50 μg/animal, but not at 5 μg/animal was able to increase the proportion of follicles with cystic and luteinization-related atresia. Furthermore, dbcAMP (50 μg/animal, but not lower doses increased the ovarian mass, number of Corpora lutea, number of harvested oocytes, zygotes and embryos at blastocyst stage derived from these zygotes after culture. These data demonstrate that dbcAMP can stimulate rabbit ovarian follicle atresia, ovulation, oocyte, zygote and embryo yield and development. Furthermore, they confirm in the involvement of cyclic nucleotide-dependent intracellular mechanisms in the control of rabbit reproductive functions and potential practical usefulness of dbcAMP in improving animal reproduction and fertility.

  10. CSF concentrations of cAMP and cGMP are lower in patients with Creutzfeldt-Jakob disease but not Parkinson's disease and amyotrophic lateral sclerosis.

    Directory of Open Access Journals (Sweden)

    Patrick Oeckl

    Full Text Available 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.

  11. Generalized Wideband Cyclic MUSIC

    Directory of Open Access Journals (Sweden)

    Zhang-Meng Liu

    2009-01-01

    Full Text Available The method of Spectral Correlation-Signal Subspace Fitting (SC-SSF fails to separate wideband cyclostationary signals with coherent second-order cyclic statistics (SOCS. Averaged Cyclic MUSIC (ACM method made up for the drawback to some degree via temporally averaging the cyclic cross-correlation of the array output. This paper interprets ACM from another perspective and proposes a new DOA estimation method by generalizing ACM for wideband cyclostationary signals. The proposed method successfully makes up for the aforementioned drawback of SC-SSF and obtains a more satisfying performance than ACM. It is also demonstrated that ACM is a simplified form of the proposed method when only a single spectral frequency is exploited, and the integration of the frequencies within the signal bandwidth helps the new method to outperform ACM.

  12. Timing is critical for effective glucocorticoid receptor mediated repression of the cAMP-induced CRH gene.

    Directory of Open Access Journals (Sweden)

    Siem van der Laan

    Full Text Available Glucocorticoid negative feedback of the hypothalamus-pituitary-adrenal axis is mediated in part by direct repression of gene transcription in glucocorticoid receptor (GR expressing cells. We have investigated the cross talk between the two main signaling pathways involved in activation and repression of corticotrophin releasing hormone (CRH mRNA expression: cyclic AMP (cAMP and GR. We report that in the At-T20 cell-line the glucocorticoid-mediated repression of the cAMP-induced human CRH proximal promoter activity depends on the relative timing of activation of both signaling pathways. Activation of the GR prior to or in conjunction with cAMP signaling results in an effective repression of the cAMP-induced transcription of the CRH gene. In contrast, activation of the GR 10 minutes after onset of cAMP treatment, results in a significant loss of GR-mediated repression. In addition, translocation of ligand-activated GR to the nucleus was found as early as 10 minutes after glucocorticoid treatment. Interestingly, while both signaling cascades counteract each other on the CRH proximal promoter, they synergize on a synthetic promoter containing 'positive' response elements. Since the order of activation of both signaling pathways may vary considerably in vivo, we conclude that a critical time-window exists for effective repression of the CRH gene by glucocorticoids.

  13. Intrusion Detection amp Prevention Systems - Sourcefire Snort

    Directory of Open Access Journals (Sweden)

    Rajesh Vuppala

    2015-08-01

    Full Text Available Information security is a challenging issue for all business organizations today amidst increasing cyber threats. While there are many alternative intrusion detection amp prevention systems available to choose from selecting the best solution to implement to detect amp prevent cyber-attacks is a difficult task. The best solution is of the one that gets the best reviews and suits the organizations needs amp budget. In this review paper we summarize various classes of intrusion detection and prevention systems compare features of alternative solutions and make recommendation for implementation of one as the best solution for business organization in Fiji.

  14. The cAMP effectors PKA and Epac activate endothelial NO synthase through PI3K/Akt pathway in human endothelial cells.

    Science.gov (United States)

    García-Morales, Verónica; Luaces-Regueira, María; Campos-Toimil, Manuel

    2017-12-01

    3',5'-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2'-O-Me-cAMP) increased cytoplasmic Ca 2+ concentration ([Ca 2+ ] c ) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca 2+ ] c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca 2+ , was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca 2+ ] c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Warmer amps for the LHC

    CERN Multimedia

    Anaïs Schaeffer

    2012-01-01

    CERN is working together with an Italian company to develop superconducting cables that can function at temperatures of up to 25 K (-248°C). This will make it possible to move LHC magnet power supplies out of the tunnel, protecting them from exposure to the showers of very high-energy particles produced by the accelerator.   Figure 1: devices of this type, which measure approximately 10 metres in length, are inserted between the accelerating magnets at different points along the LHC. When it comes to consuming electricity, the magnets that steer particles through large accelerators can be characterised with just one word: greedy. For the LHC, the total current can reach 1.5 million amps. At the present time, this current is brought in via copper cables of up to 10 cm in diameter. In the tunnel, these cables connect the current leads - which provide the transition between the ambient-temperature cables and the magnets in their bath of superfluid helium - to the power supply. In the a...

  16. Bordetella pertussis Adenylate Cyclase Toxin Blocks Induction of Bactericidal Nitric Oxide in Macrophages through cAMP-Dependent Activation of the SHP-1 Phosphatase

    Czech Academy of Sciences Publication Activity Database

    Černý, Ondřej; Kamanová, Jana; Mašín, Jiří; Bíbová, Ilona; Škopová, Karolína; Šebo, Peter

    2015-01-01

    Roč. 194, č. 10 (2015), s. 4901-4913 ISSN 0022-1767 R&D Projects: GA ČR GAP302/12/0460; GA ČR GA13-14547S Institutional support: RVO:61388971 Keywords : CYCLIC-AMP * MURINE MACROPHAGE S * IFN-GAMMA Subject RIV: EE - Microbiology, Virology Impact factor: 4.985, year: 2015

  17. Hepatic overexpression of cAMP-responsive element modulator α induces a regulatory T-cell response in a murine model of chronic liver disease

    NARCIS (Netherlands)

    Kuttkat, Nadine; Mohs, Antje; Ohl, Kim; Hooiveld, Guido; Longerich, Thomas; Tenbrock, Klaus; Cubero, Francisco Javier; Trautwein, Christian

    2016-01-01


    Objective Th17 cells are a subset of CD4+ T-helper cells characterised by interleukin 17 (IL-17) production, a cytokine that plays a crucial role in inflammation-associated diseases. The cyclic AMP-responsive element modulator-α (CREMα) is a central mediator of T-cell pathogenesis, which

  18. cAMP Signaling of Adenylate Cyclase Toxin Blocks the Oxidative Burst of Neutrophils through Epac-Mediated Inhibition of Phospholipase C Activity

    Czech Academy of Sciences Publication Activity Database

    Černý, Ondřej; Anderson, K.E.; Stephens, L.R.; Hawkins, P.T.; Šebo, Peter

    2017-01-01

    Roč. 198, č. 3 (2017), s. 1285-1296 ISSN 0022-1767 R&D Projects: GA ČR(CZ) GA13-14547S; GA MŠk(CZ) LM2015064 Institutional support: RVO:61388971 Keywords : BORDETELLA-PERTUSSIS * NADPH OXIDASE * CYCLIC-AMP Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.856, year: 2016

  19. New kids on the block: The Popeye domain containing (POPDC) protein family acting as a novel class of cAMP effector proteins in striated muscle.

    Science.gov (United States)

    Brand, Thomas; Schindler, Roland

    2017-12-01

    The cyclic 3',5'-adenosine monophosphate (cAMP) signalling pathway constitutes an ancient signal transduction pathway present in prokaryotes and eukaryotes. Previously, it was thought that in eukaryotes three effector proteins mediate cAMP signalling, namely protein kinase A (PKA), exchange factor directly activated by cAMP (EPAC) and the cyclic-nucleotide gated channels. However, recently a novel family of cAMP effector proteins emerged and was termed the Popeye domain containing (POPDC) family, which consists of three members POPDC1, POPDC2 and POPDC3. POPDC proteins are transmembrane proteins, which are abundantly present in striated and smooth muscle cells. POPDC proteins bind cAMP with high affinity comparable to PKA. Presently, their biochemical activity is poorly understood. However, mutational analysis in animal models as well as the disease phenotype observed in patients carrying missense mutations suggests that POPDC proteins are acting by modulating membrane trafficking of interacting proteins. In this review, we will describe the current knowledge about this gene family and also outline the apparent gaps in our understanding of their role in cAMP signalling and beyond. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Adenyl cyclases and cAMP in plant signaling - Past and present

    KAUST Repository

    Gehring, Christoph A.

    2010-06-25

    In lower eukaryotes and animals 3\\'-5\\'-cyclic adenosine monophosphate (cAMP) and adenyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, have long been established as key components and second messengers in many signaling pathways. In contrast, in plants, both the presence and biological role of cAMP have been a matter of ongoing debate and some controversy. Here we shall focus firstly on the discovery of cellular cAMP in plants and evidence for a role of this second messenger in plant signal transduction. Secondly, we shall review current evidence of plant ACs, analyse aspects of their domain organisations and the biological roles of candidate molecules. In addition, we shall assess different approaches based on search motifs consisting of functionally assigned amino acids in the catalytic centre of annotated and/or experimentally tested nucleotide cyclases that can contribute to the identification of novel candidate molecules with AC activity such as F-box and TIR proteins. 2010 Gehring; licensee BioMed Central Ltd.

  1. Adenyl cyclases and cAMP in plant signaling - Past and present

    KAUST Repository

    Gehring, Christoph A

    2010-01-01

    In lower eukaryotes and animals 3'-5'-cyclic adenosine monophosphate (cAMP) and adenyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, have long been established as key components and second messengers in many signaling pathways. In contrast, in plants, both the presence and biological role of cAMP have been a matter of ongoing debate and some controversy. Here we shall focus firstly on the discovery of cellular cAMP in plants and evidence for a role of this second messenger in plant signal transduction. Secondly, we shall review current evidence of plant ACs, analyse aspects of their domain organisations and the biological roles of candidate molecules. In addition, we shall assess different approaches based on search motifs consisting of functionally assigned amino acids in the catalytic centre of annotated and/or experimentally tested nucleotide cyclases that can contribute to the identification of novel candidate molecules with AC activity such as F-box and TIR proteins. 2010 Gehring; licensee BioMed Central Ltd.

  2. An odor-specific threshold deficit implicates abnormal cAMP signaling in youths at clinical risk for psychosis.

    Science.gov (United States)

    Kamath, Vidyulata; Moberg, Paul J; Calkins, Monica E; Borgmann-Winter, Karin; Conroy, Catherine G; Gur, Raquel E; Kohler, Christian G; Turetsky, Bruce I

    2012-07-01

    While olfactory deficits have been reported in schizophrenia and youths at-risk for psychosis, few studies have linked these deficits to current pathophysiological models of the illness. There is evidence that disrupted cyclic adenosine 3',5'-monophosphate (cAMP) signaling may contribute to schizophrenia pathology. As cAMP mediates olfactory signal transduction, the degree to which this disruption could manifest in olfactory impairment was ascertained. Odor-detection thresholds to two odorants that differ in the degree to which they activate intracellular cAMP were assessed in clinical risk and low-risk participants. Birhinal assessments of odor-detection threshold sensitivity to lyral and citralva were acquired in youths experiencing prodromal symptoms (n=17) and controls at low risk for developing psychosis (n=15). Citralva and lyral are odorants that differ in cAMP activation; citralva is a strong cAMP activator and lyral is a weak cAMP activator. The overall group-by-odor interaction was statistically significant. At-risk youths showed significantly reduced odor detection thresholds for lyral, but showed intact detection thresholds for citralva. This odor-specific threshold deficit was uncorrelated with deficits in odor identification or discrimination, which were also present. ROC curve analysis revealed that olfactory performance correctly classified at-risk and low-risk youths with greater than 97% accuracy. This study extends prior findings of an odor-specific hyposmia implicating cAMP-mediated signal transduction in schizophrenia and unaffected first-degree relatives to include youths at clinical risk for developing the disorder. These results suggest that dysregulation of cAMP signaling may be present during the psychosis prodrome. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. 42 CFR 447.504 - Determination of AMP.

    Science.gov (United States)

    2010-10-01

    ... 42 Public Health 4 2010-10-01 2010-10-01 false Determination of AMP. 447.504 Section 447.504... AMP. (a) AMP means, with respect to a covered outpatient drug of a manufacturer (including those sold... wholesalers for drugs distributed to the retail pharmacy class of trade. AMP shall be determined without...

  4. S-AMP for non-linear observation models

    DEFF Research Database (Denmark)

    Cakmak, Burak; Winther, Ole; Fleury, Bernard H.

    2015-01-01

    Recently we presented the S-AMP approach, an extension of approximate message passing (AMP), to be able to handle general invariant matrix ensembles. In this contribution we extend S-AMP to non-linear observation models. We obtain generalized AMP (GAMP) as the special case when the measurement...

  5. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth; Meier, Stuart Kurt; Gehring, Christoph A

    2016-01-01

    Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms

  6. Interaction of biodegradative threonine dehydratase (TD) of Escherichia coli with 8-azido-AMP, a photoaffinity analog of AMP

    International Nuclear Information System (INIS)

    Patil, R.V.; Datta, P.

    1987-01-01

    Stimulation of TD activity by AMP (Ka = 40 μM) is known to accompany the conversion of monomeric form of the enzyme to its tetramer and a decrease in the Km for threonine. In comparison, 8-azido-AMP (N 3 AMP) simulated TD activity only partially, lowered the Km for threonine and stabilized a dimeric form of the protein. Competition experiments revealed that N 3 AMP exerted an apparent dominant effect in counteracting the AMP-mediated enzyme activation, indicating complex mutual interactions between these ligands. UV irradiation of TD with increasing concentrations of 3 H-N 3 AMP (up to 150 μM) resulted in gradual loss of enzyme activity and concomitant incorporation of N 3 AMP into protein; upon complete inactivation, 0.75 mol of N 3 AMP was bound per mol tetramer. The presence of AMP during photolysis reduced the extent of enzyme inactivation as well as the incorporation of N 3 AMP into protein. Photolabeling of TD with 20 μM 3 H-N 3 AMP revealed one labeled tryptic peptide; at higher N 3 AMP concentrations (>300 μM), two labeled peptides were found, one of which was identical to that found with low N 3 AMP concentration. The cumulative results suggest that N 3 AMP can act as an allosteric modifier and that the peptide labeled at low N 3 AMP may represent the AMP binding site on the protein molecule

  7. Cyclic Voltammograms from First Principles

    DEFF Research Database (Denmark)

    Karlberg, Gustav; Jaramillo, Thomas; Skulason, Egill

    2007-01-01

    Cyclic voltammetry is a fundamental experimental tool for characterizing electrochemical surfaces. Whereas cyclic voltammetry is widely used within the field of electrochemistry, a way to quantitatively and directly relate the cyclic voltammogram to ab initio calculations has been lacking, even f...

  8. HOST liner cyclic facilities

    Science.gov (United States)

    Schultz, D.

    1983-01-01

    The HOST Liner Cyclic Program is utilizing two types of test apparatus, rectangular box rigs and a full annular rig. To date two quartz lamp cyclic box rigs have been tested and a third is to begin testing in late October 1983. The box rigs are used to evaluate 5x8 inch rectangular linear samples. A 21 inch diameter outer liner simulator is also being built up for testing beginning in April 1984. All rigs are atmospheric rigs. The first box rig, a three 6-kVA lamp installation, was operated under adverse conditions to determine feasibility of using quartz lamps for cyclic testing. This work was done in December 1981 and looked promising. The second box rig, again using three 6-kVA lamps, was operated to obtain instrumentation durability information and initial data input to a Finite Element Model. This limited test program was conducted in August 1983. Five test plates were run. Instrumentation consisted of strain gages, thermocouples and thermal paint. The strain gages were found to fail at 1200 F as expected though plates were heated to 1700 F. The third box rig, containing four 6-kVA lamps, is in build up for testing to begin in late October 1983. In addition to 33 percent greater power input, this rig has provision for 400 F backside line cooling air and a viewing port suitable for IR camera viewing. The casing is also water cooled for extended durability.

  9. Retinal Cyclic Nucleotide-Gated Channels: From Pathophysiology to Therapy

    Directory of Open Access Journals (Sweden)

    Stylianos Michalakis

    2018-03-01

    Full Text Available The first step in vision is the absorption of photons by the photopigments in cone and rod photoreceptors. After initial amplification within the phototransduction cascade the signal is translated into an electrical signal by the action of cyclic nucleotide-gated (CNG channels. CNG channels are ligand-gated ion channels that are activated by the binding of cyclic guanosine monophosphate (cGMP or cyclic adenosine monophosphate (cAMP. Retinal CNG channels transduce changes in intracellular concentrations of cGMP into changes of the membrane potential and the Ca2+ concentration. Structurally, the CNG channels belong to the superfamily of pore-loop cation channels and share a common gross structure with hyperpolarization-activated cyclic nucleotide-gated (HCN channels and voltage-gated potassium channels (KCN. In this review, we provide an overview on the molecular properties of CNG channels and describe their physiological role in the phototransduction pathways. We also discuss insights into the pathophysiological role of CNG channel proteins that have emerged from the analysis of CNG channel-deficient animal models and human CNG channelopathies. Finally, we summarize recent gene therapy activities and provide an outlook for future clinical application.

  10. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A.

    2017-10-04

    The cyclic nucleotide monophosphates (cNMPs), and notably 3′,5′-cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are now accepted as key signaling molecules in many processes in plants including growth and differentiation, photosynthesis, and biotic and abiotic defense. At the single molecule level, we are now beginning to understand how cNMPs modify specific target molecules such as cyclic nucleotide-gated channels, while at the systems level, a recent study of the Arabidopsis cNMP interactome has identified novel target molecules with specific cNMP-binding domains. A major advance came with the discovery and characterization of a steadily increasing number of guanylate cyclases (GCs) and adenylate cyclases (ACs). Several of the GCs are receptor kinases and include the brassinosteroid receptor, the phytosulfokine receptor, the Pep receptor, the plant natriuretic peptide receptor as well as a nitric oxide sensor. We foresee that in the near future many more molecular mechanisms and biological roles of GCs and ACs and their catalytic products will be discovered and further establish cNMPs as a key component of plant responses to the environment.

  11. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A; Turek, Ilona S.

    2017-01-01

    The cyclic nucleotide monophosphates (cNMPs), and notably 3′,5′-cyclic guanosine monophosphate (cGMP) and 3′,5′-cyclic adenosine monophosphate (cAMP) are now accepted as key signaling molecules in many processes in plants including growth and differentiation, photosynthesis, and biotic and abiotic defense. At the single molecule level, we are now beginning to understand how cNMPs modify specific target molecules such as cyclic nucleotide-gated channels, while at the systems level, a recent study of the Arabidopsis cNMP interactome has identified novel target molecules with specific cNMP-binding domains. A major advance came with the discovery and characterization of a steadily increasing number of guanylate cyclases (GCs) and adenylate cyclases (ACs). Several of the GCs are receptor kinases and include the brassinosteroid receptor, the phytosulfokine receptor, the Pep receptor, the plant natriuretic peptide receptor as well as a nitric oxide sensor. We foresee that in the near future many more molecular mechanisms and biological roles of GCs and ACs and their catalytic products will be discovered and further establish cNMPs as a key component of plant responses to the environment.

  12. Regulation of melanogenesis: the role of cAMP and MITF

    Directory of Open Access Journals (Sweden)

    Michał Otręba

    2012-01-01

    Full Text Available The article presents the melanogenesis pathway and the role of cyclic adenosine monophosphate (cAMP and microphthalmia transcription factor (MITF in regulation of this process. Products of melanogenesis are eu- and/or pheomelanins synthesized in a multistage process of tyrosine oxidation and polymerization. The conversions require the presence of tyrosinase (TYR, key enzyme, tyrosine hydroxylase isoform I (THI and tyrosinase related proteins (TRP1 and TRP2. Many types of signal molecules and transcription factors participate in regulation of melanin synthesis, but the most important are cAMP and MITF. cAMP is the second messenger in the intracellular signal cascade, which is synthesized from adenosine triphosphate (ATP by adenylyl cyclase, activated among others by the melanocortin receptor and the αS subunit of G protein. The signal molecule cAMP regulates MITF, TYR, THI, GTP-cyclohydroxylase I (GTP-CHI transcription and phenylalanine hydroxylase (PAH phosphorylation at Ser16 by protein kinase A (PKA. Mutations of genes encoding proteins belonging to the cAMP signal cascade may lead to McCune-Albright and Carney syndromes. MITF is one of the most important nuclear transcription factors regulating melanogenesis. Currently 10 isoforms of human MITF are known, but in melanocytes only MITF-M, MITF-Mdel, MITF-A and MITF-H occur. MITF transcription factor regulates melanogenesis by activation of tyrosinase, TRP1 and TRP2 transcription. It also affects expression of other factors regulating melanosome maturation, biogenesis and transport. Moreover, it regulates melanocyte proliferation and protection against apoptosis. Mutations of the MITF gene may lead to hereditary diseases: Waardenburg type II and Tietz syndromes.

  13. Plasticity of regulation of mannitol phosphotransferase system operon by CRP-cAMP complex in Vibrio cholerae.

    Science.gov (United States)

    Zhou, Yan Yan; Zhang, Hong Zhi; Liang, Wei Li; Zhang, Li Juan; Zhu, Jun; Kan, Biao

    2013-10-01

    The complex of the cyclic AMP receptor protein (CRP) and cAMP is an important transcriptional regulator of numerous genes in prokaryotes. The transport of mannitol through the phosphotransferase systems (PTS) is regulated by the CRP-cAMP complex. The aim of the study is to investigate how the CRP-cAMP complex acting on the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype. The crp mutant strain was generated by homologous recombination to assess the need of CRP to activate the mannitol PTS operon of V. cholerae El Tor. Electrophoretic mobility shift assays (EMSA) and the reporter plasmid pBBRlux were used to confirm the role that the CRP-cAMP complex playing on the mannitol PTS operon mtl. In this study, we confirmed that CRP is strictly needed for the activation of the mtl operon. We further experimentally identified five CRP binding sites within the promoter region upstream of the mannitol PTS operon mtl of the Vibrio cholerae El Tor biotype and found that these sites display different affinities for CRP and provide different contributions to the activation of the operon. The five binding sites collectively confer the strong activation of mannitol transfer by CRP in V. cholerae, indicating an elaborate and subtle CRP activation mechanism. Copyright © 2013 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  14. Phosphodiesterase inhibitors suppress Lactobacillus casei cell-wall-induced NF-κB and MAPK activations and cell proliferation through protein kinase A--or exchange protein activated by cAMP-dependent signal pathway.

    Science.gov (United States)

    Saito, Takekatsu; Sugimoto, Naotoshi; Ohta, Kunio; Shimizu, Tohru; Ohtani, Kaori; Nakayama, Yuko; Nakamura, Taichi; Hitomi, Yashiaki; Nakamura, Hiroyuki; Koizumi, Shoichi; Yachie, Akihiro

    2012-01-01

    Specific strains of Lactobacillus have been found to be beneficial in treating some types of diarrhea and vaginosis. However, a high mortality rate results from underlying immunosuppressive conditions in patients with Lactobacillus casei bacteremia. Cyclic AMP (cAMP) is a small second messenger molecule that mediates signal transduction. The onset and progression of inflammatory responses are sensitive to changes in steady-state cAMP levels. L. casei cell wall extract (LCWE) develops arteritis in mice through Toll-like receptor-2 signaling. The purpose of this study was to investigate whether intracellular cAMP affects LCWE-induced pathological signaling. LCWE was shown to induce phosphorylation of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and cell proliferation in mice fibroblast cells. Theophylline and phosphodiesterase inhibitor increased intracellular cAMP and inhibited LCWE-induced cell proliferation as well as phosphorylation of NF-κB and MAPK. Protein kinase A inhibitor H89 prevented cAMP-induced MAPK inhibition, but not cAMP-induced NF-κB inhibition. An exchange protein activated by cAMP (Epac) agonist inhibited NF-κB activation but not MAPK activation. These results indicate that an increase in intracellular cAMP prevents LCWE induction of pathological signaling pathways dependent on PKA and Epac signaling.

  15. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    Energy Technology Data Exchange (ETDEWEB)

    Arana, Maite Rocío, E-mail: arana@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Tocchetti, Guillermo Nicolás, E-mail: gtocchetti@live.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Domizi, Pablo, E-mail: domizi@ibr-conicet.gov.ar [Instituto de Biología Molecular y Celular de Rosario (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Arias, Agostina, E-mail: agoarias@yahoo.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Rigalli, Juan Pablo, E-mail: jprigalli@gmail.com [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Ruiz, María Laura, E-mail: ruiz@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); and others

    2015-09-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  16. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    International Nuclear Information System (INIS)

    Arana, Maite Rocío; Tocchetti, Guillermo Nicolás; Domizi, Pablo; Arias, Agostina; Rigalli, Juan Pablo; Ruiz, María Laura

    2015-01-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  17. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Science.gov (United States)

    Pongkorpsakol, Pawin; Pathomthongtaweechai, Nutthapoom; Srimanote, Potjanee; Soodvilai, Sunhapas; Chatsudthipong, Varanuj; Muanprasat, Chatchai

    2014-09-01

    Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84) cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+)-K(+) ATPases and Na(+)-K(+)-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+) channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+)-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+)-activated basolateral K(+) channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+)-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  18. Inhibition of cAMP-activated intestinal chloride secretion by diclofenac: cellular mechanism and potential application in cholera.

    Directory of Open Access Journals (Sweden)

    Pawin Pongkorpsakol

    2014-09-01

    Full Text Available Cyclic AMP-activated intestinal Cl- secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl- secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl- secretion in human intestinal epithelial (T84 cells with IC50 of ∼ 20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl- current showed that diclofenac reversibly inhibited CFTR Cl- channel activity (IC50 ∼ 10 µM via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na(+-K(+ ATPases and Na(+-K(+-Cl- cotransporters, but inhibited cAMP-activated basolateral K(+ channels with IC50 of ∼ 3 µM. In addition, diclofenac suppressed Ca(2+-activated Cl- channels, inwardly rectifying Cl- channels, and Ca(2+-activated basolateral K(+ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT-induced Cl- secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼ 70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca(2+-activated Cl- secretion by inhibiting both apical Cl- channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal

  19. Cyclic approximation to stasis

    Directory of Open Access Journals (Sweden)

    Stewart D. Johnson

    2009-06-01

    Full Text Available Neighborhoods of points in $mathbb{R}^n$ where a positive linear combination of $C^1$ vector fields sum to zero contain, generically, cyclic trajectories that switch between the vector fields. Such points are called stasis points, and the approximating switching cycle can be chosen so that the timing of the switches exactly matches the positive linear weighting. In the case of two vector fields, the stasis points form one-dimensional $C^1$ manifolds containing nearby families of two-cycles. The generic case of two flows in $mathbb{R}^3$ can be diffeomorphed to a standard form with cubic curves as trajectories.

  20. Accelerated cyclic corrosion tests

    Directory of Open Access Journals (Sweden)

    Prošek T.

    2016-06-01

    Full Text Available Accelerated corrosion testing is indispensable for material selection, quality control and both initial and residual life time prediction for bare and painted metallic, polymeric, adhesive and other materials in atmospheric exposure conditions. The best known Neutral Salt Spray (NSS test provides unrealistic conditions and poor correlation to exposures in atmosphere. Modern cyclic accelerated corrosion tests include intermittent salt spray, wet and dry phases and eventually other technical phases. They are able to predict the material performance in service more correctly as documented on several examples. The use of NSS should thus be restricted for quality control.

  1. Isolation and characterization of cAMP-free and cAMP-bound forms of bovine heart type II cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Cobb, C.E.

    1986-01-01

    Bovine heart type II cAMP-dependent protein kinase holoenzyme (cAMP-PK) was purified to homogeneity as determined by denaturing SDS-PAGE. An HPLC-DEAE purification step resolved two distinct peaks of cAMP-dependent kinase activity, which were designated Peak 1 and Peak 2 based on their order of elution. They had the same Stoke's radii and had very similar sedimentation coefficients. As determined by densitometric scanning of SDS-PAGE brands, by their mobility on denaturing PAGE, and by the ratios of equilibrium [ 3 H] cAMP binding to maximal kinase activity, the subunit stoichiometry of the two peaks was the same. In a cAMP assay it was found that Peak 1 holoenzyme was cAMP-free, but half of the Peak 2 holoenzyme cAMP binding sites contained cAMP. Dissociation assays indicated that the cAMP was equally distributed in binding Site 1 and Site 2 of Peak 2. Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP, and Peak 2 could be partially converted to Peak 1 by aging. The interconvertibility of the two holoenzyme peaks strongly suggested that the difference between the two peaks was caused by the presence of cAMP in Peak 2

  2. The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors.

    Directory of Open Access Journals (Sweden)

    Philip D Townsend

    Full Text Available The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition.

  3. A generalized allosteric mechanism for cis-regulated cyclic nucleotide binding domains.

    Directory of Open Access Journals (Sweden)

    Alexandr P Kornev

    2008-04-01

    Full Text Available Cyclic nucleotides (cAMP and cGMP regulate multiple intracellular processes and are thus of a great general interest for molecular and structural biologists. To study the allosteric mechanism of different cyclic nucleotide binding (CNB domains, we compared cAMP-bound and cAMP-free structures (PKA, Epac, and two ionic channels using a new bioinformatics method: local spatial pattern alignment. Our analysis highlights four major conserved structural motifs: 1 the phosphate binding cassette (PBC, which binds the cAMP ribose-phosphate, 2 the "hinge," a flexible helix, which contacts the PBC, 3 the beta(2,3 loop, which provides precise positioning of an invariant arginine from the PBC, and 4 a conserved structural element consisting of an N-terminal helix, an eight residue loop and the A-helix (N3A-motif. The PBC and the hinge were included in the previously reported allosteric model, whereas the definition of the beta(2,3 loop and the N3A-motif as conserved elements is novel. The N3A-motif is found in all cis-regulated CNB domains, and we present a model for an allosteric mechanism in these domains. Catabolite gene activator protein (CAP represents a trans-regulated CNB domain family: it does not contain the N3A-motif, and its long range allosteric interactions are substantially different from the cis-regulated CNB domains.

  4. [Asthma and cyclic neutropenia].

    Science.gov (United States)

    Salazar Cabrera, A N; Berrón Pérez, R; Ortega Martell, J A; Onuma Takane, E

    1996-01-01

    We report a male with history of recurrent infections (recurrent oral aphtous disease [ROAD], middle ear infections and pharyngo amigdalitis) every 3 weeks since he was 7 months old. At the age of 3 years cyclic neutropenia was diagnosed with cyclic fall in the total neutrophil count in blood smear every 21 days and prophylactic antimicrobial therapy was indicated. Episodic events every 3 weeks of acute asthma and allergic rhinitis were detected at the age of 6 years old and specific immunotherapy to Bermuda grass was given during 3 years with markedly improvement in his allergic condition but not in the ROAD. He came back until the age of 16 with episodic acute asthma and ROAD. The total neutrophil count failed to 0 every 21 days and surprisingly the total eosinophil count increased up to 2,000 at the same time, with elevation of serum IgE (412 Ul/mL). Specific immunotherapy to D.pt. and Aller.a. and therapy with timomodulin was indicated. After 3 months we observed clinical improvement in the asthmatic condition and the ROAD disappeared, but the total neutrophil count did not improve. We present this case as a rare association between 2 diseases with probably no etiological relationship but may be physiopatological that could help to understand more the pathogenesis of asthma.

  5. Ekpyrotic and cyclic cosmology

    International Nuclear Information System (INIS)

    Lehners, Jean-Luc

    2008-01-01

    Ekpyrotic and cyclic cosmologies provide theories of the very early and of the very late universe. In these models, the big bang is described as a collision of branes - and thus the big bang is not the beginning of time. Before the big bang, there is an ekpyrotic phase with equation of state w=P/(ρ) >>1 (where P is the average pressure and ρ the average energy density) during which the universe slowly contracts. This phase resolves the standard cosmological puzzles and generates a nearly scale-invariant spectrum of cosmological perturbations containing a significant non-Gaussian component. At the same time it produces small-amplitude gravitational waves with a blue spectrum. The dark energy dominating the present-day cosmological evolution is reinterpreted as a small attractive force between our brane and a parallel one. This force eventually induces a new ekpyrotic phase and a new brane collision, leading to the idea of a cyclic universe. This review discusses the detailed properties of these models, their embedding in M-theory and their viability, with an emphasis on open issues and observational signatures

  6. Topoisomerase 1 Inhibition Promotes Cyclic GMP-AMP Synthase-Dependent Antiviral Responses

    OpenAIRE

    Pépin, Geneviève; Nejad, Charlotte; Ferrand, Jonathan; Thomas, Belinda J.; Stunden, H. James; Sanij, Elaine; Foo, Chwan-Hong; Stewart, Cameron R.; Cain, Jason E.; Bardin, Philip G.; Williams, Bryan R. G.; Gantier, Michael P.

    2017-01-01

    ABSTRACT Inflammatory responses, while essential for pathogen clearance, can also be deleterious to the host. Chemical inhibition of topoisomerase 1 (Top1) by low-dose camptothecin (CPT) can suppress transcriptional induction of antiviral and inflammatory genes and protect animals from excessive and damaging inflammatory responses. We describe the unexpected finding that minor DNA damage from topoisomerase 1 inhibition with low-dose CPT can trigger a strong antiviral immune response through c...

  7. MAST: a Mega Amp Spherical Tokamak

    International Nuclear Information System (INIS)

    Darke, A.C.; Harbar, J.R.; Hay, J.H.; Hicks, J.B.; Hill, J.W.; McKenzie, J.S.; Morris, A.W.; Nightingale, M.P.S.; Todd, T.N.; Voss, G.M.; Watkins, J.R.

    1995-01-01

    The highly successful tight aspect ratio tokamak research pioneered on the START machine at Culham, together with the attractive possibilities of the concept, suggest a larger device should be considered. The design of a Mega Amp Spherical Tokamak is described, operating at much higher currents and over longer pulses than START and compatible with strong additional heating. (orig.)

  8. NIKHEF: AmPS of electron beam

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    Now operational at the Dutch National Institute for Nuclear Physics and High Energy Physics (NIKHEF), Amsterdam, is a new tool for studying the electromagnetic properties of nuclei. Called AmPS - Amsterdam pulse stretcher - this ring provides experiments with a smoother, almost continuous supply of electrons

  9. Effect of sevoflurane on the ATPase activity of hippocampal neurons in a rat model of cerebral ischemia-reperfusion injury via the cAMP-PKA signaling pathway.

    Science.gov (United States)

    Liu, Tie-Jun; Zhang, Jin-Cun; Gao, Xiao-Zeng; Tan, Zhi-Bin; Wang, Jian-Jun; Zhang, Pan-Pan; Cheng, Ai-Bin; Zhang, Shu-Bo

    2018-01-01

    We aim to investigate the effects of sevoflurane on the ATPase activity of the hippocampal neurons in rats with cerebral ischemia-reperfusion injury (IRI) via the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) signaling pathway. Sixty rats were assigned into the normal, model and sevoflurane groups (n = 20, the latter two groups were established as focal cerebral IRI models). The ATPase activity was detected using an ultramicro Na (+)-K (+)-ATP enzyme kit. Immunohistochemical staining was used to detect the positive protein expression of cAMP and PKA. The hippocampal neurons were assigned to the normal, IRI, IRI + sevoflurane, IRI + forskolin, IRI + H89 and IRI + sevoflurane + H89 groups. qRT-PCR and Western blotting were performed for the expressions of cAMP, PKA, cAMP-responsive element-binding protein (CREB) and brain derived neurotrophic factor (BDNF). The normal and sevoflurane groups exhibited a greater positive protein expression of cAMP and PKA than the model group. Compared with the normal group, the expressions of cAMP, PKA, CREB and BDNF all reduced in the IRI, model and IRI + H89 groups. The sevoflurane group showed higher cAMP, PKA, CREB and BDNF expressions than the model group. Compared with the IRI group, ATPase activity and expressions of cAMP, PKA, CREB and BDNF all increased in the normal, IRI + sevoflurane and IRI + forskolin groups but decreased in the IRI + H89 group. It suggests that sevoflurane could enhance ATPase activity in hippocampal neurons of cerebral IRI rats through activating cAMP-PKA signaling pathway. Copyright © 2017. Published by Elsevier Taiwan.

  10. Z₂-double cyclic codes

    OpenAIRE

    Borges, J.

    2014-01-01

    A binary linear code C is a Z2-double cyclic code if the set of coordinates can be partitioned into two subsets such that any cyclic shift of the coordinates of both subsets leaves invariant the code. These codes can be identified as submodules of the Z2[x]-module Z2[x]/(x^r − 1) × Z2[x]/(x^s − 1). We determine the structure of Z2-double cyclic codes giving the generator polynomials of these codes. The related polynomial representation of Z2-double cyclic codes and its duals, and the relation...

  11. Manual for Cyclic Triaxial Test

    DEFF Research Database (Denmark)

    Shajarati, Amir; Sørensen, Kris Wessel; Nielsen, Søren Kjær

    This manual describes the different steps that is included in the procedure for conducting a cyclic triaxial test at the geotechnical Laboratory at Aalborg University. Furthermore it contains a chapter concerning some of the background theory for the static triaxial tests. The cyclic/dynamic tria......This manual describes the different steps that is included in the procedure for conducting a cyclic triaxial test at the geotechnical Laboratory at Aalborg University. Furthermore it contains a chapter concerning some of the background theory for the static triaxial tests. The cyclic...

  12. Adrenal hormones and liver cAMP in exercising rats--different modes of anesthesia.

    Science.gov (United States)

    Winder, W W; Fuller, E O; Conlee, R K

    1983-11-01

    We have compared five different modes of anesthesia (iv and ip pentobarbital sodium, ether, CO2, and cervical dislocation) with respect to their effects on liver glycogen, liver adenosine 3',5'-cyclic monophosphate (cAMP), blood glucose and lactate, plasma corticosterone, norepinephrine, and epinephrine in resting rats and in rats run on a treadmill at 26 m/min for 30 min. Ether, CO2, and cervical dislocation were found to be unsuitable due to the marked elevation in plasma catecholamines seen in both resting and exercising rats. Injection of pentobarbital sodium ip required an average of 8 min before onset of surgical anesthesia as opposed to less than 5 s for iv pentobarbital. Exercising rats anesthetized with ip pentobarbital showed markedly lower plasma catecholamines compared with rats given iv pentobarbital. Hepatic cAMP increased in response to exercise in all groups except the ip pentobarbital group. This is most likely due to the long delay between the end of the exercise and freezing of the liver in the ip pentobarbital-anesthetized animals. We conclude that iv injection of pentobarbital is the most suitable method of anesthesia for obtaining accurate measurements of plasma stress hormones, substrates, and metabolites and of hepatic cAMP and glycogen in resting and exercising rats.

  13. Cross-talk between signaling pathways can generate robust oscillations in calcium and cAMP.

    Directory of Open Access Journals (Sweden)

    Fernando Siso-Nadal

    Full Text Available BACKGROUND: To control and manipulate cellular signaling, we need to understand cellular strategies for information transfer, integration, and decision-making. A key feature of signal transduction is the generation of only a few intracellular messengers by many extracellular stimuli. METHODOLOGY/PRINCIPAL FINDINGS: Here we model molecular cross-talk between two classic second messengers, cyclic AMP (cAMP and calcium, and show that the dynamical complexity of the response of both messengers increases substantially through their interaction. In our model of a non-excitable cell, both cAMP and calcium concentrations can oscillate. If mutually inhibitory, cross-talk between the two second messengers can increase the range of agonist concentrations for which oscillations occur. If mutually activating, cross-talk decreases the oscillation range, but can generate 'bursting' oscillations of calcium and may enable better filtering of noise. CONCLUSION: We postulate that this increased dynamical complexity allows the cell to encode more information, particularly if both second messengers encode signals. In their native environments, it is unlikely that cells are exposed to one stimulus at a time, and cross-talk may help generate sufficiently complex responses to allow the cell to discriminate between different combinations and concentrations of extracellular agonists.

  14. AMP is an adenosine A1 receptor agonist.

    Science.gov (United States)

    Rittiner, Joseph E; Korboukh, Ilia; Hull-Ryde, Emily A; Jin, Jian; Janzen, William P; Frye, Stephen V; Zylka, Mark J

    2012-02-17

    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5'-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5'-monophosphonate, ACP) directly activated the adenosine A(1) receptor (A(1)R). In contrast, AMP only activated the adenosine A(2B) receptor (A(2B)R) after hydrolysis to adenosine by ecto-5'-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A(1)R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A(1)R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A(1)R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A(1)R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine.

  15. 7 CFR 772.14 - Reamortization of AMP loans.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Reamortization of AMP loans. 772.14 Section 772.14... AGRICULTURE SPECIAL PROGRAMS SERVICING MINOR PROGRAM LOANS § 772.14 Reamortization of AMP loans. The Agency may approve reamortization of AMP loans provided: (a) There is no extension of the final maturity date...

  16. AMP Deaminase 3 Deficiency Enhanced 5′-AMP Induction of Hypometabolism

    Science.gov (United States)

    Daniels, Isadora Susan; O′Brien, William G.; Nath, Vinay; Zhao, Zhaoyang; Lee, Cheng Chi

    2013-01-01

    A hypometabolic state can be induced in mice by 5′-AMP administration. Previously we proposed that an underlying mechanism for this hypometabolism is linked to reduced erythrocyte oxygen transport function due to 5′-AMP uptake altering the cellular adenylate equilibrium. To test this hypothesis, we generated mice deficient in adenosine monophosphate deaminase 3 (AMPD3), the key catabolic enzyme for 5′-AMP in erythrocytes. Mice deficient in AMPD3 maintained AMPD activities in all tissues except erythrocytes. Developmentally and morphologically, the Ampd3−/− mice were indistinguishable from their wild type siblings. The levels of ATP, ADP but not 5′-AMP in erythrocytes of Ampd3−/− mice were significantly elevated. Fasting blood glucose levels of the Ampd3−/− mice were comparable to wild type siblings. In comparison to wild type mice, the Ampd3−/− mice displayed a deeper hypometabolism with a significantly delayed average arousal time in response to 5′-AMP administration. Together, these findings demonstrate a central role of AMPD3 in the regulation of 5′-AMP mediated hypometabolism and further implicate erythrocytes in this behavioral response. PMID:24066180

  17. AMPS Supporting Research and Technology (SR and T) report. Atmospheric, Magnetospheric and Plasmas in Space (AMPS) definition study

    Science.gov (United States)

    1976-01-01

    A listing of candidate technology areas that require additional study is presented. These candidate tasks, identified during the AMPS Phase B studies, are requisites to the design, development, and operation of the AMPS concept selected for preliminary design.

  18. The clinical correlation of regulatory T cells and cyclic adenosine monophosphate in enterovirus 71 infection.

    Directory of Open Access Journals (Sweden)

    Shih-Min Wang

    Full Text Available Brainstem encephalitis (BE and pulmonary edema (PE are notable complications of enterovirus 71 (EV71 infection.This study investigated the immunoregulatory characterizations of EV71 neurological complications by disease severity and milrinone treatment.Patients <18 years with virologically confirmed EV71 infections were enrolled and divided into 2 groups: the hand, foot, and mouth disease (HFMD or BE group, and the autonomic nervous system (ANS dysregulation or PE group. Cytokine and cyclic adenosine monophosphate (cAMP levels, and the regulatory T cell (Tregs profiles of the patients were determined.Patients with ANS dysregulation or PE exhibited significantly low frequency of CD4(+CD25(+Foxp3+ and CD4(+Foxp3(+ T cells compared with patients with HFMD or BE. The expression frequency of CD4-CD8- was also significantly decreased in patients with ANS dysregulation or PE. Among patients with ANS dysregulation or PE, the expression frequency of CD4+Foxp3+ increased markedly after milrinone treatment, and was associated with reduction of plasma levels IL-6, IL-8 and IL-10. Plasma concentrations of cAMP were significantly decreased in patients with ANS dysregulation or PE compared with patients with HFMD or BE; however, cAMP levels increased after milrinone treatment.These findings suggested decreased different regulatory T populations and cAMP expression correlate with increased EV71 disease severity. Improved outcome after milrinone treatment may associate with increased regulatory T populations, cAMP expression and modulation of cytokines levels.

  19. The arabidopsis cyclic nucleotide interactome

    KAUST Repository

    Donaldson, Lara Elizabeth

    2016-05-11

    Background Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals. Methods An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling. Results A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment. Conclusions We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

  20. Osthole Enhances Osteogenesis in Osteoblasts by Elevating Transcription Factor Osterix via cAMP/CREB Signaling In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Zhong-Rong Zhang

    2017-06-01

    Full Text Available Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L. Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB. Blockage of cAMP/CREB downstream signals with protein kinase A (PKA inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression.

  1. Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model.

    Science.gov (United States)

    Shao, Xiao-Mei; Sun, Jing; Jiang, Yong-Liang; Liu, Bo-Yi; Shen, Zui; Fang, Fang; Du, Jun-Ying; Wu, Yuan-Yuan; Wang, Jia-Ling; Fang, Jian-Qiao

    2016-01-01

    Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA) can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs) in a rat pain memory model. To explore the critical role of protein kinase A (PKA) in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC) in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP), PKA, cAMP response element-binding protein (CREB), and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA.

  2. S-AMP: Approximate Message Passing for General Matrix Ensembles

    DEFF Research Database (Denmark)

    Cakmak, Burak; Winther, Ole; Fleury, Bernard H.

    2014-01-01

    the approximate message-passing (AMP) algorithm to general matrix ensembles with a well-defined large system size limit. The generalization is based on the S-transform (in free probability) of the spectrum of the measurement matrix. Furthermore, we show that the optimality of S-AMP follows directly from its......We propose a novel iterative estimation algorithm for linear observation models called S-AMP. The fixed points of S-AMP are the stationary points of the exact Gibbs free energy under a set of (first- and second-) moment consistency constraints in the large system limit. S-AMP extends...

  3. cAMP-induced activation of protein kinase A and p190B RhoGAP mediates down-regulation of TC10 activity at the plasma membrane and neurite outgrowth.

    Science.gov (United States)

    Koinuma, Shingo; Takeuchi, Kohei; Wada, Naoyuki; Nakamura, Takeshi

    2017-11-01

    Cyclic AMP plays a pivotal role in neurite growth. During outgrowth, a trafficking system supplies membrane at growth cones. However, the cAMP-induced signaling leading to the regulation of membrane trafficking remains unknown. TC10 is a Rho family GTPase that is essential for specific types of vesicular trafficking. Recent studies have shown a role of TC10 in neurite growth in NGF-treated PC12 cells. Here, we investigated a mechanical linkage between cAMP and TC10 in neuritogenesis. Plasmalemmal TC10 activity decreased abruptly after cAMP addition in neuronal cells. TC10 was locally inactivated at extending neurite tips in cAMP-treated PC12 cells. TC10 depletion led to a decrease in cAMP-induced neurite outgrowth. Constitutively active TC10 could not rescue this growth reduction, supporting our model for a role of GTP hydrolysis of TC10 in neuritogenesis by accelerating vesicle fusion. The cAMP-induced TC10 inactivation was mediated by PKA. Considering cAMP-induced RhoA inactivation, we found that p190B, but not p190A, mediated inactivation of TC10 and RhoA. Upon cAMP treatment, p190B was recruited to the plasma membrane. STEF depletion and Rac1-N17 expression reduced cAMP-induced TC10 inactivation. Together, the PKA-STEF-Rac1-p190B pathway leading to inactivation of TC10 and RhoA at the plasma membrane plays an important role in cAMP-induced neurite outgrowth. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

  4. 2′,3′-cAMP, 3′-AMP, and 2′-AMP inhibit human aortic and coronary vascular smooth muscle cell proliferation via A2B receptors

    Science.gov (United States)

    Ren, Jin; Gillespie, Delbert G.

    2011-01-01

    Rat vascular smooth muscle cells (VSMCs) from renal microvessels metabolize 2′,3′-cAMP to 2′-AMP and 3′-AMP, and these AMPs are converted to adenosine that inhibits microvascular VSMC proliferation via A2B receptors. The goal of this study was to test whether this mechanism also exists in VSMCs from conduit arteries and whether it is similarly expressed in human vs. rat VSMCs. Incubation of rat and human aortic VSMCs with 2′,3′-cAMP concentration-dependently increased levels of 2′-AMP and 3′-AMP in the medium, with a similar absolute increase in 2′-AMP vs. 3′-AMP. In contrast, in human coronary VSMCs, 2′,3′-cAMP increased 2′-AMP levels yet had little effect on 3′-AMP levels. In all cell types, 2′,3′-cAMP increased levels of adenosine, but not 5′-AMP, and 2′,3′-AMP inhibited cell proliferation. Antagonism of A2B receptors (MRS-1754), but not A1 (1,3-dipropyl-8-cyclopentylxanthine), A2A (SCH-58261), or A3 (VUF-5574) receptors, attenuated the antiproliferative effects of 2′,3′-cAMP. In all cell types, 2′-AMP, 3′-AMP, and 5′-AMP increased adenosine levels, and inhibition of ecto-5′-nucleotidase blocked this effect of 5′-AMP but not that of 2′-AMP nor 3′-AMP. Also, 2′-AMP, 3′-AMP, and 5′-AMP, like 2′,3′-cAMP, exerted antiproliferative effects that were abolished by antagonism of A2B receptors with MRS-1754. In conclusion, VSMCs from conduit arteries metabolize 2′,3′-cAMP to AMPs, which are metabolized to adenosine. In rat and human aortic VSMCs, both 2′-AMP and 3′-AMP are involved in this process, whereas, in human coronary VSMCs, 2′,3′-cAMP is mainly converted to 2′-AMP. Because adenosine inhibits VSMC proliferation via A2B receptors, local vascular production of 2′,3′-cAMP may protect conduit arteries from atherosclerosis. PMID:21622827

  5. The Monge-Ampère equation

    CERN Document Server

    Gutiérrez, Cristian E

    2016-01-01

    Now in its second edition, this monograph explores the Monge-Ampère equation and the latest advances in its study and applications. It provides an essentially self-contained systematic exposition of the theory of weak solutions, including regularity results by L. A. Caffarelli. The geometric aspects of this theory are stressed using techniques from harmonic analysis, such as covering lemmas and set decompositions. An effort is made to present complete proofs of all theorems, and examples and exercises are offered to further illustrate important concepts. Some of the topics considered include generalized solutions, non-divergence equations, cross sections, and convex solutions. New to this edition is a chapter on the linearized Monge-Ampère equation and a chapter on interior Hölder estimates for second derivatives. Bibliographic notes, updated and expanded from the first edition, are included at the end of every chapter for further reading on Monge-Ampère-type equations and their diverse applications in th...

  6. The Applied Mathematics for Power Systems (AMPS)

    Energy Technology Data Exchange (ETDEWEB)

    Chertkov, Michael [Los Alamos National Laboratory

    2012-07-24

    Increased deployment of new technologies, e.g., renewable generation and electric vehicles, is rapidly transforming electrical power networks by crossing previously distinct spatiotemporal scales and invalidating many traditional approaches for designing, analyzing, and operating power grids. This trend is expected to accelerate over the coming years, bringing the disruptive challenge of complexity, but also opportunities to deliver unprecedented efficiency and reliability. Our Applied Mathematics for Power Systems (AMPS) Center will discover, enable, and solve emerging mathematics challenges arising in power systems and, more generally, in complex engineered networks. We will develop foundational applied mathematics resulting in rigorous algorithms and simulation toolboxes for modern and future engineered networks. The AMPS Center deconstruction/reconstruction approach 'deconstructs' complex networks into sub-problems within non-separable spatiotemporal scales, a missing step in 20th century modeling of engineered networks. These sub-problems are addressed within the appropriate AMPS foundational pillar - complex systems, control theory, and optimization theory - and merged or 'reconstructed' at their boundaries into more general mathematical descriptions of complex engineered networks where important new questions are formulated and attacked. These two steps, iterated multiple times, will bridge the growing chasm between the legacy power grid and its future as a complex engineered network.

  7. Optimal Design for Reactivity Ratio Estimation: A Comparison of Techniques for AMPS/Acrylamide and AMPS/Acrylic Acid Copolymerizations

    Directory of Open Access Journals (Sweden)

    Alison J. Scott

    2015-11-01

    Full Text Available Water-soluble polymers of acrylamide (AAm and acrylic acid (AAc have significant potential in enhanced oil recovery, as well as in other specialty applications. To improve the shear strength of the polymer, a third comonomer, 2-acrylamido-2-methylpropane sulfonic acid (AMPS, can be added to the pre-polymerization mixture. Copolymerization kinetics of AAm/AAc are well studied, but little is known about the other comonomer pairs (AMPS/AAm and AMPS/AAc. Hence, reactivity ratios for AMPS/AAm and AMPS/AAc copolymerization must be established first. A key aspect in the estimation of reliable reactivity ratios is design of experiments, which minimizes the number of experiments and provides increased information content (resulting in more precise parameter estimates. However, design of experiments is hardly ever used during copolymerization parameter estimation schemes. In the current work, copolymerization experiments for both AMPS/AAm and AMPS/AAc are designed using two optimal techniques (Tidwell-Mortimer and the error-in-variables-model (EVM. From these optimally designed experiments, accurate reactivity ratio estimates are determined for AMPS/AAm (rAMPS = 0.18, rAAm = 0.85 and AMPS/AAc (rAMPS = 0.19, rAAc = 0.86.

  8. Dendritic diameter influences the rate and magnitude of hippocampal cAMP and PKA transients during β-adrenergic receptor activation.

    Science.gov (United States)

    Luczak, Vincent; Blackwell, Kim T; Abel, Ted; Girault, Jean-Antoine; Gervasi, Nicolas

    2017-02-01

    In the hippocampus, cyclic-adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) form a critical signaling cascade required for long-lasting synaptic plasticity, learning and memory. Plasticity and memory are known to occur following pathway-specific changes in synaptic strength that are thought to result from spatially and temporally coordinated intracellular signaling events. To better understand how cAMP and PKA dynamically operate within the structural complexity of hippocampal neurons, we used live two-photon imaging and genetically-encoded fluorescent biosensors to monitor cAMP levels or PKA activity in CA1 neurons of acute hippocampal slices. Stimulation of β-adrenergic receptors (isoproterenol) or combined activation of adenylyl cyclase (forskolin) and inhibition of phosphodiesterase (IBMX) produced cAMP transients with greater amplitude and rapid on-rates in intermediate and distal dendrites compared to somata and proximal dendrites. In contrast, isoproterenol produced greater PKA activity in somata and proximal dendrites compared to intermediate and distal dendrites, and the on-rate of PKA activity did not differ between compartments. Computational models show that our observed compartmental difference in cAMP can be reproduced by a uniform distribution of PDE4 and a variable density of adenylyl cyclase that scales with compartment size to compensate for changes in surface to volume ratios. However, reproducing our observed compartmental difference in PKA activity required enrichment of protein phosphatase in small compartments; neither reduced PKA subunits nor increased PKA substrates were sufficient. Together, our imaging and computational results show that compartment diameter interacts with rate-limiting components like adenylyl cyclase, phosphodiesterase and protein phosphatase to shape the spatial and temporal components of cAMP and PKA signaling in CA1 neurons and suggests that small neuronal compartments are most sensitive to cAMP

  9. Linderane Suppresses Hepatic Gluconeogenesis by Inhibiting the cAMP/PKA/CREB Pathway Through Indirect Activation of PDE 3 via ERK/STAT3

    Directory of Open Access Journals (Sweden)

    Wei Xie

    2018-05-01

    Full Text Available The role of phosphodiesterase 3 (PDE3, a cyclic AMP (cAMP-degrading enzyme, in modulating gluconeogenesis remains unknown. Here, linderane, a natural compound, was found to inhibit gluconeogenesis by activating hepatic PDE3 in rat primary hepatocytes. The underlying molecular mechanism and its effects on whole-body glucose and lipid metabolism were investigated. The effect of linderane on gluconeogenesis, cAMP content, phosphorylation of cAMP-response element-binding protein (CREB and PDE activity were examined in cultured primary hepatocytes and C57BL/6J mice. The precise mechanism by which linderane activates PDE3 and inhibits the cAMP pathway was explored using pharmacological inhibitors. The amelioration of metabolic disorders was observed in ob/ob mice. Linderane inhibited gluconeogenesis, reduced phosphoenolpyruvate carboxykinase (Pck1 and glucose-6-phosphatase (G6pc gene expression, and decreased intracellular cAMP concentration and CREB phosphorylation in rat primary hepatocytes under both basal and forskolin-stimulated conditions. In rat primary hepatocytes, it also increased total PDE and PDE3 activity but not PDE4 activity. The suppressive effect of linderane on the cAMP pathway and gluconeogenesis was abolished by the non-specific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX and the specific PDE3 inhibitor cilostazol. Linderane indirectly activated PDE3 through extracellular regulated protein kinase 1/2 (ERK1/2 and signal transducer and activator of transcription 3 (STAT3 activation. Linderane improved glucose and lipid metabolism after chronic oral administration in ob/ob mice. Our findings revealed linderane as an indirect PDE3 activator that suppresses gluconeogenesis through cAMP pathway inhibition and has beneficial effects on metabolic syndromes in ob/ob mice. This investigation highlighted the potential for PDE3 activation in the treatment of type 2 diabetes.

  10. The influence of dibutyryl adenosine cyclic monophosphate on cell proliferation in the epithelium of the jejunal crypts, the colonic crypts and in colonic carcinomata of rat.

    Science.gov (United States)

    Tutton, P J; Barkla, D H

    1980-01-01

    1. Cell proliferation in the jejunal crypts, the colonic crypts and in dimethylhydrazine (DMH)-induced adenocarcinomata of rat colon was measured using a stathmokinetic technique. 2. Dibutryl cyclic adneosine monophosphate (dibutyryl cAMP) was found to inhibit cell proliferation in colonic crypts and in colonic adenocarcinomata. 3. Dibutryl cAMP at very high doses was found to inhibit jejunal crypt cell proliferation but at lower doses was found to accelerate jejunal crypt cell proliferation. 4. Neither bilateral adrenalectomy nor chemical sympathectomy was found to abolish the ability of dibutryl cAMP to stimulate jejunal crypt cell proliferation. 5. The present results are difficult to interpret in terms of known hormonal influences on cell proliferation in the tissues examined and of established actions, of these hormones on cyclic nucleotide metabolism in other tissues.

  11. Changes in the Arabidopsis thaliana Proteome Implicate cAMP in Biotic and Abiotic Stress Responses and Changes in Energy Metabolism

    KAUST Repository

    Alquraishi, May Majed; Gehring, Christoph A; Marondedze, Claudius

    2016-01-01

    The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is increasingly recognized as having many different roles in plant responses to environmental stimuli. To gain further insights into these roles, Arabidopsis thaliana cell suspension culture was treated with 100 nM of cell permeant 8-bromo-cAMP for 5 or 10 min. Here, applying mass spectrometry and comparative proteomics, 20 proteins were identified as differentially expressed and we noted a specific bias in proteins with a role in abiotic stress, particularly cold and salinity, biotic stress as well as proteins with a role in glycolysis. These findings suggest that cAMP is sufficient to elicit specific stress responses that may in turn induce complex changes to cellular energy homeostasis.

  12. Changes in the Arabidopsis thaliana Proteome Implicate cAMP in Biotic and Abiotic Stress Responses and Changes in Energy Metabolism

    KAUST Repository

    Alquraishi, May Majed

    2016-06-01

    The second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) is increasingly recognized as having many different roles in plant responses to environmental stimuli. To gain further insights into these roles, Arabidopsis thaliana cell suspension culture was treated with 100 nM of cell permeant 8-bromo-cAMP for 5 or 10 min. Here, applying mass spectrometry and comparative proteomics, 20 proteins were identified as differentially expressed and we noted a specific bias in proteins with a role in abiotic stress, particularly cold and salinity, biotic stress as well as proteins with a role in glycolysis. These findings suggest that cAMP is sufficient to elicit specific stress responses that may in turn induce complex changes to cellular energy homeostasis.

  13. Prognosis of Cyclic Vomiting Syndrome

    Directory of Open Access Journals (Sweden)

    J. Gordon Millichap

    2016-03-01

    Full Text Available Investigators from Teikyo University School of Medicine, Tokyo, Japan, evaluated the clinical features, prognosis, and prophylaxis of cyclic vomiting syndrome and the relationship between the syndrome and levels of adrenocorticotropic/antidiuretic hormones (ACTH/ADH.

  14. Novel Radioligands for Cyclic Nucleotide Phosphodiesterase Imaging with Positron Emission Tomography: An Update on Developments Since 2012

    Directory of Open Access Journals (Sweden)

    Susann Schröder

    2016-05-01

    Full Text Available Cyclic nucleotide phosphodiesterases (PDEs are a class of intracellular enzymes that inactivate the secondary messenger molecules, cyclic adenosine monophosphate (cAMP and cyclic guanosine monophosphate (cGMP. Thus, PDEs regulate the signaling cascades mediated by these cyclic nucleotides and affect fundamental intracellular processes. Pharmacological inhibition of PDE activity is a promising strategy for treatment of several diseases. However, the role of the different PDEs in related pathologies is not completely clarified yet. PDE-specific radioligands enable non-invasive visualization and quantification of these enzymes by positron emission tomography (PET in vivo and provide an important translational tool for elucidation of the relationship between altered expression of PDEs and pathophysiological effects as well as (pre-clinical evaluation of novel PDE inhibitors developed as therapeutics. Herein we present an overview of novel PDE radioligands for PET published since 2012.

  15. Functionalized linear and cyclic polyolefins

    Energy Technology Data Exchange (ETDEWEB)

    Tuba, Robert; Grubbs, Robert H.

    2018-02-13

    This invention relates to methods and compositions for preparing linear and cyclic polyolefins. More particularly, the invention relates to methods and compositions for preparing functionalized linear and cyclic polyolefins via olefin metathesis reactions. Polymer products produced via the olefin metathesis reactions of the invention may be utilized for a wide range of materials applications. The invention has utility in the fields of polymer and materials chemistry and manufacture.

  16. Cyclic Processing for Context Fusion

    DEFF Research Database (Denmark)

    Kjærgaard, Mikkel Baun

    2007-01-01

    Many machine-learning techniques use feedback information. However, current context fusion systems do not support this because they constrain processing to be structured as acyclic processing. This paper proposes a generalization which enables the use of cyclic processing in context fusion systems....... A solution is proposed to the inherent problem of how to avoid uncontrollable looping during cyclic processing. The solution is based on finding cycles using graph-coloring and breaking cycles using time constraints....

  17. PdeH, a high-affinity cAMP phosphodiesterase, is a key regulator of asexual and pathogenic differentiation in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    2010-05-01

    Full Text Available Cyclic AMP-dependent pathways mediate the communication between external stimuli and the intracellular signaling machinery, thereby influencing important aspects of cellular growth, morphogenesis and differentiation. Crucial to proper function and robustness of these signaling cascades is the strict regulation and maintenance of intracellular levels of cAMP through a fine balance between biosynthesis (by adenylate cyclases and hydrolysis (by cAMP phosphodiesterases. We functionally characterized gene-deletion mutants of a high-affinity (PdeH and a low-affinity (PdeL cAMP phosphodiesterase in order to gain insights into the spatial and temporal regulation of cAMP signaling in the rice-blast fungus Magnaporthe oryzae. In contrast to the expendable PdeL function, the PdeH activity was found to be a key regulator of asexual and pathogenic development in M. oryzae. Loss of PdeH led to increased accumulation of intracellular cAMP during vegetative and infectious growth. Furthermore, the pdeHDelta showed enhanced conidiation (2-3 fold, precocious appressorial development, loss of surface dependency during pathogenesis, and highly reduced in planta growth and host colonization. A pdeHDelta pdeLDelta mutant showed reduced conidiation, exhibited dramatically increased (approximately 10 fold cAMP levels relative to the wild type, and was completely defective in virulence. Exogenous addition of 8-Br-cAMP to the wild type simulated the pdeHDelta defects in conidiation as well as in planta growth and development. While a fully functional GFP-PdeH was cytosolic but associated dynamically with the plasma membrane and vesicular compartments, the GFP-PdeL localized predominantly to the nucleus. Based on data from cAMP measurements and Real-Time RTPCR, we uncover a PdeH-dependent biphasic regulation of cAMP levels during early and late stages of appressorial development in M. oryzae. We propose that PdeH-mediated sustenance and dynamic regulation of cAMP signaling

  18. AMP Is an Adenosine A1 Receptor Agonist*

    Science.gov (United States)

    Rittiner, Joseph E.; Korboukh, Ilia; Hull-Ryde, Emily A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

    2012-01-01

    Numerous receptors for ATP, ADP, and adenosine exist; however, it is currently unknown whether a receptor for the related nucleotide adenosine 5′-monophosphate (AMP) exists. Using a novel cell-based assay to visualize adenosine receptor activation in real time, we found that AMP and a non-hydrolyzable AMP analog (deoxyadenosine 5′-monophosphonate, ACP) directly activated the adenosine A1 receptor (A1R). In contrast, AMP only activated the adenosine A2B receptor (A2BR) after hydrolysis to adenosine by ecto-5′-nucleotidase (NT5E, CD73) or prostatic acid phosphatase (PAP, ACPP). Adenosine and AMP were equipotent human A1R agonists in our real-time assay and in a cAMP accumulation assay. ACP also depressed cAMP levels in mouse cortical neurons through activation of endogenous A1R. Non-selective purinergic receptor antagonists (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid and suramin) did not block adenosine- or AMP-evoked activation. Moreover, mutation of His-251 in the human A1R ligand binding pocket reduced AMP potency without affecting adenosine potency. In contrast, mutation of a different binding pocket residue (His-278) eliminated responses to AMP and to adenosine. Taken together, our study indicates that the physiologically relevant nucleotide AMP is a full agonist of A1R. In addition, our study suggests that some of the physiological effects of AMP may be direct, and not indirect through ectonucleotidases that hydrolyze this nucleotide to adenosine. PMID:22215671

  19. Radioprotection of the rat parotid gland by cAMP

    International Nuclear Information System (INIS)

    Sodicoff, M.; Conger, A.D.

    1983-01-01

    Most earlier studies showing a radioprotective effect by cAMP show only slight degrees of protection. The present study demonstrates a substantial protective effect (DMF, 1.63) of exogenously administered cAMP on the rat parotid gland and supports the mechanism suggested previously for protection afforded the parotid glands by the β-adrenergic agonist isoproterenol, which is known to elevate endogenous intracellular cAMP

  20. Didactical formulation of the Ampère law

    International Nuclear Information System (INIS)

    Barchiesi, Dominique

    2014-01-01

    The Ampère law is useful to calculate the magnetostatic field in the cases of distributions of current with high degree of symmetry. Nevertheless the magnetic field produced by a thin straight wire carrying a current I requires the Biot–Savart law and the use of the Ampère law leads to a mistake. A didactical formulation of the Ampère law is proposed to prevent misinterpretations. (letters and comments)

  1. The cAMP-PKA Signaling Pathway Regulates Pathogenicity, Hyphal Growth, Appressorial Formation, Conidiation, and Stress Tolerance in Colletotrichum higginsianum.

    Science.gov (United States)

    Zhu, Wenjun; Zhou, Man; Xiong, Zeyang; Peng, Fang; Wei, Wei

    2017-01-01

    Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. Understanding the mechanisms of the cruciferous plant- C. higginsianum interactions will be important in facilitating efficient control of anthracnose diseases. The cAMP-PKA signaling pathway plays important roles in diverse physiological processes of multiple pathogens. C. higginsianum contains two genes, ChPKA1 and ChPKA2 , that encode the catalytic subunits of cyclic AMP (cAMP)-dependent protein kinase A (PKA). To analyze the role of cAMP signaling pathway in pathogenicity and development in C. higginsianum , we characterized ChPKA1 and ChPKA2 genes, and adenylate cyclase ( ChAC ) gene. The ChPKA1 and ChAC deletion mutants were unable to cause disease and significantly reduced in hyphal growth, tolerance to cell wall inhibitors, conidiation, and appressorial formation with abnormal germ tubes, but they had an increased tolerance to elevated temperatures and exogenous H 2 O 2 . In contrast, the ChPKA2 mutant had no detectable alteration of phenotypes, suggesting that ChPKA1 contributes mainly to PKA activities in C. higginsianum . Moreover, we failed to generate Δ ChPKA1ChPKA2 double mutant, indicating that deletion of both PKA catalytic subunits is lethal in C. higginsianum and the two catalytic subunits possibly have overlapping functions. These results indicated that ChPKA1 is the major PKA catalytic subunit in cAMP-PKA signaling pathway and plays significant roles in hyphal growth, pathogenicity, appressorial formation, conidiation, and stress tolerance in C. higginsianum .

  2. The cAMP-PKA Signaling Pathway Regulates Pathogenicity, Hyphal Growth, Appressorial Formation, Conidiation, and Stress Tolerance in Colletotrichum higginsianum

    Directory of Open Access Journals (Sweden)

    Wenjun Zhu

    2017-07-01

    Full Text Available Colletotrichum higginsianum is an economically important pathogen that causes anthracnose disease in a wide range of cruciferous crops. Understanding the mechanisms of the cruciferous plant–C. higginsianum interactions will be important in facilitating efficient control of anthracnose diseases. The cAMP-PKA signaling pathway plays important roles in diverse physiological processes of multiple pathogens. C. higginsianum contains two genes, ChPKA1 and ChPKA2, that encode the catalytic subunits of cyclic AMP (cAMP-dependent protein kinase A (PKA. To analyze the role of cAMP signaling pathway in pathogenicity and development in C. higginsianum, we characterized ChPKA1 and ChPKA2 genes, and adenylate cyclase (ChAC gene. The ChPKA1 and ChAC deletion mutants were unable to cause disease and significantly reduced in hyphal growth, tolerance to cell wall inhibitors, conidiation, and appressorial formation with abnormal germ tubes, but they had an increased tolerance to elevated temperatures and exogenous H2O2. In contrast, the ChPKA2 mutant had no detectable alteration of phenotypes, suggesting that ChPKA1 contributes mainly to PKA activities in C. higginsianum. Moreover, we failed to generate ΔChPKA1ChPKA2 double mutant, indicating that deletion of both PKA catalytic subunits is lethal in C. higginsianum and the two catalytic subunits possibly have overlapping functions. These results indicated that ChPKA1 is the major PKA catalytic subunit in cAMP-PKA signaling pathway and plays significant roles in hyphal growth, pathogenicity, appressorial formation, conidiation, and stress tolerance in C. higginsianum.

  3. Duodenal activation of cAMP-dependent protein kinase induces vagal afferent firing and lowers glucose production in rats.

    Science.gov (United States)

    Rasmussen, Brittany A; Breen, Danna M; Luo, Ping; Cheung, Grace W C; Yang, Clair S; Sun, Biying; Kokorovic, Andrea; Rong, Weifang; Lam, Tony K T

    2012-04-01

    The duodenum senses nutrients to maintain energy and glucose homeostasis, but little is known about the signaling and neuronal mechanisms involved. We tested whether duodenal activation of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) is sufficient and necessary for cholecystokinin (CCK) signaling to trigger vagal afferent firing and regulate glucose production. In rats, we selectively activated duodenal PKA and evaluated changes in glucose kinetics during the pancreatic (basal insulin) pancreatic clamps and vagal afferent firing. The requirement of duodenal PKA signaling in glucose regulation was evaluated by inhibiting duodenal activation of PKA in the presence of infusion of the intraduodenal PKA agonist (Sp-cAMPS) or CCK1 receptor agonist (CCK-8). We also assessed the involvement of a neuronal network and the metabolic impact of duodenal PKA activation in rats placed on high-fat diets. Intraduodenal infusion of Sp-cAMPS activated duodenal PKA and lowered glucose production, in association with increased vagal afferent firing in control rats. The metabolic and neuronal effects of duodenal Sp-cAMPS were negated by coinfusion with either the PKA inhibitor H89 or Rp-CAMPS. The metabolic effect was also negated by coinfusion with tetracaine, molecular and pharmacologic inhibition of NR1-containing N-methyl-d-aspartate (NMDA) receptors within the dorsal vagal complex, or hepatic vagotomy in rats. Inhibition of duodenal PKA blocked the ability of duodenal CCK-8 to reduce glucose production in control rats, whereas duodenal Sp-cAMPS bypassed duodenal CCK resistance and activated duodenal PKA and lowered glucose production in rats on high-fat diets. We identified a neural glucoregulatory function of duodenal PKA signaling. Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.

  4. PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome

    Science.gov (United States)

    Terrin, Anna; Monterisi, Stefania; Stangherlin, Alessandra; Zoccarato, Anna; Koschinski, Andreas; Surdo, Nicoletta C.; Mongillo, Marco; Sawa, Akira; Jordanides, Niove E.; Mountford, Joanne C.

    2012-01-01

    Previous work has shown that the protein kinase A (PKA)–regulated phosphodiesterase (PDE) 4D3 binds to A kinase–anchoring proteins (AKAPs). One such protein, AKAP9, localizes to the centrosome. In this paper, we investigate whether a PKA–PDE4D3–AKAP9 complex can generate spatial compartmentalization of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. Real-time imaging of fluorescence resonance energy transfer reporters shows that centrosomal PDE4D3 modulated a dynamic microdomain within which cAMP concentration selectively changed over the cell cycle. AKAP9-anchored, centrosomal PKA showed a reduced activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally, disruption of the centrosomal cAMP microdomain by local displacement of PDE4D3 impaired cell cycle progression as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent modulation of the enzyme activation threshold rather than on overall changes in cAMP levels. Further, we provide for the first time direct evidence that control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals. PMID:22908311

  5. CRIS-a novel cAMP-binding protein controlling spermiogenesis and the development of flagellar bending.

    Directory of Open Access Journals (Sweden)

    Anke Miriam Krähling

    Full Text Available The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD. Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca(2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca(2+-regulated proteins that--in mature sperm--are involved in flagellar bending.

  6. Inotropic responses of the frog ventricle to adenosine triphosphate and related changes in endogenous cyclic nucleotides.

    Science.gov (United States)

    Flitney, F W; Singh, J

    1980-07-01

    1. A study has been made of a well documented but poorly understood response of the isolated frog ventricle to treatment with exogenous adenosine 5' triphosphate (ATP). Measurements of membrane potential, isometric twitch tension and levels of endogenous 3',5'-cyclic nucleotides have been made at various times during the ATP-induced response. 2. ATP elicits a characteristic triphasic response, which comprises an initial, abrupt increase in contractility, rising to a maximum within a few beats (first phase); followed by a period when the twitch amplitude falls, sometimes to below the control level (second phase); and superceded by a more slowly developing and longer-lasting increase in contractile force (third phase). The response is unaffected by atropine, propranolol or phentolamine. However, the prostaglandin synthetase inhibitor indomethacin depresses the first phase and entirely suppresses the third phase. 3. The inotropic effects of ATP are accompanied by changes in the shape of the action potential. These effects are dose-related. The duration of the action potential (D-30mV) and its positive overshoot (O) are increased during all phases of the response, for [ATP]o's up to 10(-5) M. However, at higher [ATP]o's, D-30mV and O ar both reduced during the second phase (but not the first or third phase), when isometric twitch tension is also depressed. The relationship between action potential duration and twitch tension (P) for different [ATP]o's is linear for all three phases of the response, but the slopes of the curves (delta P/delta D) are markedly different, indicating that the sensitivity of the contractile system to membrane depolarization is not constant, but varies continuously throughout the response. 4. ATP has a potent stimulatory effect on the metabolism of endogenous 3',5'-cyclic nucleotides. The time courses of the changes in adenosine 3','5-cyclic monophosphate (3',5'-cyclic AMP) and guanosine 3',5'-cyclic monophosphate (3',5'-cyclic GMP) are

  7. On Improvements of Cyclic MUSIC

    Directory of Open Access Journals (Sweden)

    H. Howard Fan

    2005-01-01

    Full Text Available Many man-made signals encountered in communications exhibit cyclostationarity. By exploiting cyclostationarity, cyclic MUSIC has been shown to be able to separate signals with different cycle frequencies, thus, to be able to perform signal selective direction of-arrival (DOA estimation. However, as will be shown in this paper, the DOA estimation of cyclic MUSIC is actually biased. We show in this paper that by properly choosing the frequency for evaluating the steering vector, the bias of DOA estimation can be substantially reduced and the performance can be improved. Furthermore, we propose another algorithm exploiting cyclic conjugate correlation to further improve the performance of DOA estimation. Simulation results show the effectiveness of both of our methods.

  8. Design of a cyclic multiverse

    Energy Technology Data Exchange (ETDEWEB)

    Piao Yunsong, E-mail: yspiao@gucas.ac.c [College of Physical Sciences, Graduate School of Chinese Academy of Sciences, Beijing 100049 (China)

    2010-08-09

    Recently, it has been noticed that the amplification of the amplitude of curvature perturbation cycle by cycle can lead to a cyclic multiverse scenario, in which the number of universes increases cycle by cycle. However, this amplification will also inevitably induce either the ultimate end of corresponding cycle, or the resulting spectrum of perturbations inside corresponding universe is not scale invariant, which baffles the existence of observable universes. In this Letter, we propose a design of a cyclic multiverse, in which the observable universe can emerges naturally. The significance of a long period of dark energy before the turnaround of each cycle for this implementing is shown.

  9. Design of a cyclic multiverse

    International Nuclear Information System (INIS)

    Piao Yunsong

    2010-01-01

    Recently, it has been noticed that the amplification of the amplitude of curvature perturbation cycle by cycle can lead to a cyclic multiverse scenario, in which the number of universes increases cycle by cycle. However, this amplification will also inevitably induce either the ultimate end of corresponding cycle, or the resulting spectrum of perturbations inside corresponding universe is not scale invariant, which baffles the existence of observable universes. In this Letter, we propose a design of a cyclic multiverse, in which the observable universe can emerges naturally. The significance of a long period of dark energy before the turnaround of each cycle for this implementing is shown.

  10. Nature of a solar cyclicity

    International Nuclear Information System (INIS)

    Romanchuk, P.R.

    1981-01-01

    The paper contains a critical review of works on studying a cyclic character of solar activity. An introduction of cyclic curves with a frequency spectrum is established to be insolvent. The Wolf, Newcomb and Waldmeier approach seems to be useful. Some evidence is given in favour of the author's conception of solar activity ciclicity of a tide nature. It is accounted for a continuous double and single effect of planets, a resonant character of this effect due to which a 10-year period of Jupiter and Saturn is transformed into an 11-year cycle of activity [ru

  11. Modulation of phenotype and function of human CD4+CD25+ T regulatory lymphocytes mediated by cAMP elevating agents

    Directory of Open Access Journals (Sweden)

    Antonella Riccomi

    2016-09-01

    Full Text Available We have shown that Cholera Toxin (CT and other cyclic AMP (cAMP elevating agents induce up-regulation of the inhibitory molecule CTLA-4 in human resting CD4+ T lymphocytes, which following the treatment acquired suppressive functions. In this study, we evaluated the effect of cAMP elevating agents on human CD4+CD25+ T cells, which include the T regulatory (Treg cells that play a pivotal role in the maintenance of immunological tolerance. We found that cAMP elevating agents induce up-regulation of CTLA-4 in CD4+CD25- and further enhance its expression in CD4+CD25+ T cells. We observed an increase of two isoforms of mRNA coding for the membrane and the soluble CTLA-4 molecules, suggesting that the regulation of CTLA-4 expression by cAMP is at the transcriptional level. In addition, we found that the increase of cAMP in CD4+CD25+ T cells converts the CD4+CD25+Foxp3- T cells in CD4+CD25+Foxp3+ T cells, whereas the increase of cAMP in CD4+CD25- T cells did not up-regulate Foxp3 in the absence of activation stimuli. To investigate the function of these cells, we performed an in vitro suppression assay by culturing CD4+CD25+ T cells untreated or pre-treated with CT with anti-CD3 mAbs-stimulated autologous PBMC. We found that CT enhances the inhibitory function of CD4+CD25+ T cells, CD4+ and CD8+ T cell proliferation and IFNγ production are strongly inhibited by CD4+CD25+ T cells pre-treated with cAMP elevating agents. Furthermore, we found that CD4+CD25+ T lymphocytes pre-treated with cAMP elevating agents induce the up-regulation of CD80 and CD86 co-stimulatory molecules on immature dendritic cells (DCs in the absence of antigenic stimulation, however without leading to full DC maturation. These data show that the increase of intracellular cAMP modulates the phenotype and function of human CD4+CD25+ T cells.

  12. Regularizing properties of Complex Monge-Amp\\`ere flows

    OpenAIRE

    Tô, Tat Dat

    2016-01-01

    We study the regularizing properties of complex Monge-Amp\\`ere flows on a K\\"ahler manifold $(X,\\omega)$ when the initial data are $\\omega$-psh functions with zero Lelong number at all points. We prove that the general Monge-Amp\\`ere flow has a solution which is immediately smooth. We also prove the uniqueness and stability of solution.

  13. Effects of cholesterol and cAMP on progesterone production in cultured luteal cells isolated from pseudopregnant cat ovaries.

    Science.gov (United States)

    Arikan, Sevket; Yigit, Ayse Arzu

    2009-10-01

    The present study was designed to incubate luteal cells isolated from pseudopregnant cats and to investigate the effects of cholesterol and cAMP on luteal progesterone production. Corpora lutea were collected from the cats on days 10 and 15 of pseudopregnancy. Luteal cells were isolated from the ovaries by collagenase digestion. Steroidogenic luteal cells were stained for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity. Cells (2 x 10(4)) staining positive for 3beta-HSD were cultured for up to 7 days. The cells were treated with 22(R)-hydroxycholesterol (22R-HC) and dibutyryl cyclic AMP (dbcAMP) on days 1, 3 and 7. Treatment of cells with 22R-HC resulted in a dose-dependent increase (pprogesterone production. When 22R-HC was used at a concentration of 10 microg/ml, it resulted in 2.7- and 5.1-fold increases in progesterone production on days 3 and 5, respectively. When the dose was doubled (20 microg/ml), treated cells produced four times more progesterone on days 3 and 7, and three times more on day 5. By day 7, progesterone production increased up to 9.1 times more than the control. Incubation of cells with both concentrations of dbcAMP (0.1 mM and 1 mM) resulted in significant stimulations of progesterone on days 5 and 7 (pProgesterone production was increased up to 2- and 2.9-fold of the control when cells were treated with lower concentration of dbcAMP (0.1 mM) on days 5 and 7, respectively. Incubation of cells with 1 mM concentrations of dbcAMP induced a 3.2-fold increase on day 5 and a 5-fold increase on day 7. In conclusion, a successful incubation was performed for long-life culturing of luteal cells collected from pseudopregnant cats. The method works well and allows for optimal growth and development of cells in the culture. The present study also demonstrated that incubating cat luteal cells with 22R-HC and dbcAMP induces a significant increase in luteal progesterone synthesis.

  14. Endogenous Parathyroid Hormone Promotes Fracture Healing by Increasing Expression of BMPR2 through cAMP/PKA/CREB Pathway in Mice

    Directory of Open Access Journals (Sweden)

    Wei Zhou

    2017-06-01

    Full Text Available Background/Aims: Endogenous parathyroid hormone (PTH plays an important role in fracture healing. This study investigated whether endogenous PTH regulates fracture healing by bone morphogenetic protein (BMP and/or the transforming growth factor-β (TGF-β signaling pathway. Methods: Eight-week-old wild-type (WT and PTH-knockout (PTH KO male mice were selected, and models of open right-femoral fracture were constructed. Fracture healing and callus characteristics of mice in the two groups were compared by X-ray, micro-computed tomography, histological, and immunohistochemical examinations. Bone marrow mesenchymal stem cells (BMMSCs of 8-week-old WT and PTHKO male mice were obtained and induced into osteoblasts and chondrocytes. Results: We found that expression levels of Runt-related transcription factor (RUNX2, bone morphogenetic protein-receptor-type Ⅱ (BMPR2, phosphorylated Smad 1/5/8, and phosphorylated cyclic adenosine monophosphate-responsive element binding protein (CREB in the callus of PTHKO mice were significantly decreased, whereas no significant difference in expression of SOX9, TGF-βR2,or pSMAD2/3 was observed between PTHKO and WT mice. Additionally, the activity of osteoblast alkaline phosphatase was low at 7 days post-induction, and was upregulated by addition of PTH or dibutyryl cyclic adenosine monophosphate (dbcAMP to the cell culture. Furthermore, H89 (protein kinase A inhibitoreliminated the simulating effects of PTH and dbcAMP, and a low concentration of cyclic adenosine monophosphate (cAMP was observed in PTHKO mouse BMMSCs. Conclusion: These results suggested that endogenous PTH enhanced BMPR2 expression by a cAMP/PKA/CREB pathway in osteoblasts, and increased RUNX2 expression through transduction of the BMP/pSMAD1/5/8 signaling pathway.

  15. Resveratrol stimulates AMP kinase activity in neurons.

    Science.gov (United States)

    Dasgupta, Biplab; Milbrandt, Jeffrey

    2007-04-24

    Resveratrol is a polyphenol produced by plants that has multiple beneficial activities similar to those associated with caloric restriction (CR), such as increased life span and delay in the onset of diseases associated with aging. CR improves neuronal health, and the global beneficial effects of CR have been postulated to be mediated by the nervous system. One key enzyme thought to be activated during CR is the AMP-activated kinase (AMPK), a sensor of cellular energy levels. AMPK is activated by increases in the cellular AMP:ATP ratio, whereupon it functions to help preserve cellular energy. In this regard, the regulation of dietary food intake by hypothalamic neurons is mediated by AMPK. The suppression of nonessential energy expenditure by activated AMPK along with the CR mimetic and neuroprotective properties of resveratrol led us to hypothesize that neuronal activation of AMPK could be an important component of resveratrol activity. Here, we show that resveratrol activated AMPK in Neuro2a cells and primary neurons in vitro as well as in the brain. Resveratrol and the AMPK-activating compound 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) promoted robust neurite outgrowth in Neuro2a cells, which was blocked by genetic and pharmacologic inhibition of AMPK. Resveratrol also stimulated mitochondrial biogenesis in an AMPK-dependent manner. Resveratrol-stimulated AMPK activity in neurons depended on LKB1 activity but did not require the NAD-dependent protein deacetylase SIRT1 during this time frame. These findings suggest that neuronal activation of AMPK by resveratrol could affect neuronal energy homeostasis and contribute to the neuroprotective effects of resveratrol.

  16. The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

    Science.gov (United States)

    Tariqul Islam, A F M; Yue, Haicen; Scavello, Margarethakay; Haldeman, Pearce; Rappel, Wouter-Jan; Charest, Pascale G

    2018-08-01

    To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Effects of sodium ions on rat thyrocyte (FRTL-5 cells) swelling- and thyrotropin-activated taurine efflux dependent on cAMP and Epac.

    Science.gov (United States)

    Fugelli, Kjell

    2016-03-01

    Cellular osmolyte release is important in preventing water accumulation and swelling. However, the signaling pathways that detect volume increase and activate solute efflux are still not fully understood. We investigated efflux activation of the osmolyte taurine which is actively accumulated in rat thyrocytes (FRTL-5). Efflux of accumulated [(3)H]taurine was stimulated by cellular swelling and thyrotropin (TSH). These effects were significantly diminished in cells having reduced TSH receptor concentrations. Phosphodiesterase inhibitors (IBMX, Rolipram) enhanced both responses. An analog of forskolin (FSK; 7-deacetyl-7-[O-(N-methylpiperazino)-γ-butyryl] dihydrochloride) and an analog of cAMP, specific for activating exchange protein activated directly by cAMP (Epac; 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, acetoxymethyl ester), significantly stimulated [(3)H]taurine efflux. A cAMP analog specific for activating protein kinase A (PKA; N6-benzoyladenosine-3',5'-cyclic monophosphate, acetoxymethyl ester) had no significant stimulatory effect on [(3)H]taurine efflux rate. The amiloride analog, 5-(N-ethyl-N-isopropyl)-amiloride, which inhibits a TSH-stimulated Na(+)/H(+) exchanger, enhanced (100 %) and ouabain inhibited (50 %) the TSH-stimulated [(3)H]taurine efflux rate. The effect of FSK on efflux was strongly potentiated by Na(+)-free iso-osmotic conditions and by osmolality/cell volume that affected also the db-cAMP-stimulated efflux. The TSH receptors and downstream elements of the signaling pathway comprising adenylyl cyclase, cAMP and Epac appeared to mediate the hormone-induced signal for [(3)H]taurine efflux from FRTL-5 cells. With less evidence, the cell volume/osmolality-induced [(3)H]taurine efflux cascade appeared to share some of the hormone signaling elements and to modulate the hormone signaling pathway at two levels through cellular Na(+).

  18. Inhibitory action of certain cyclophosphate derivatives of cAMP on cAMP-dependent protein kinases

    NARCIS (Netherlands)

    Wit, René J.W. de; Hekstra, Doeke; Jastorff, Bernd; Stec, Wojciech J.; Baraniak, Janina; Driel, Roel van; Haastert, Peter J.M. van

    1984-01-01

    A series cAMP derivatives with modifications in the adenine, ribose and cyclophosphate moiety were screened for their binding affinity for the two types of cAMP-binding sites in mammalian protein kinase type I. In addition, the activation of the kinase by these analogs was monitored. The binding

  19. Alteration of sodium, potassium-adenosine triphosphatase activity in rabbit ciliary processes by cyclic adenosine monophosphate-dependent protein kinase

    International Nuclear Information System (INIS)

    Delamere, N.A.; Socci, R.R.; King, K.L.

    1990-01-01

    The response of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) to cyclic adenosine monophosphate (cAMP)-dependent protein kinase was examined in membranes obtained from rabbit iris-ciliary body. In the presence of the protein kinase together with 10(-5) M cAMP, Na,K-ATPase activity was reduced. No change in Na,K-ATPase activity was detected in response to the protein kinase without added cAMP. Likewise cAMP alone did not alter Na,K-ATPase activity. Reduction of Na,K-ATPase activity was also observed in the presence of the cAMP-dependent protein kinase catalytic subunit. The response of the enzyme to the kinase catalytic subunit was also examined in membranes obtained from rabbit ciliary processes. In the presence of 8 micrograms/ml of the catalytic subunit, ciliary process Na,K-ATPase activity was reduced by more than 50%. To examine whether other ATPases were suppressed by the protein kinase, calcium-stimulated ATPase activity was examined; its activity was stimulated by the catalytic subunit. To test whether the response of the ciliary process Na,K-ATPase is unique, experiments were also performed using membrane preparations from rabbit lens epithelium or rabbit kidney; the catalytic subunit significantly reduced the activity of Na,K-ATPase from the kidney but not the lens. These Na,K-ATPase studies suggest that in the iris-ciliary body, cAMP may alter sodium pump activity. In parallel 86Rb uptake studies, we observed that ouabain-inhibitable potassium uptake by intact pieces of iris-ciliary body was reduced by exogenous dibutryl cAMP or by forskolin

  20. A cAMP/PKA/Kinesin-1 Axis Promotes the Axonal Transport of Mitochondria in Aging Drosophila Neurons.

    Science.gov (United States)

    Vagnoni, Alessio; Bullock, Simon L

    2018-04-23

    Mitochondria play fundamental roles within cells, including energy provision, calcium homeostasis, and the regulation of apoptosis. The transport of mitochondria by microtubule-based motors is critical for neuronal structure and function. This process allows local requirements for mitochondrial functions to be met and also facilitates recycling of these organelles [1, 2]. An age-related reduction in mitochondrial transport has been observed in neurons of mammalian and non-mammalian organisms [3-6], and has been proposed to contribute to the broader decline in neuronal function that occurs during aging [3, 5-7]. However, the factors that influence mitochondrial transport in aging neurons are poorly understood. Here we provide evidence using the tractable Drosophila wing nerve system that the cyclic AMP/protein kinase A (cAMP/PKA) pathway promotes the axonal transport of mitochondria in adult neurons. The level of the catalytic subunit of PKA decreases during aging, and acute activation of the cAMP/PKA pathway in aged flies strongly stimulates mitochondrial motility. Thus, the age-related impairment of transport is reversible. The expression of many genes is increased by PKA activation in aged flies. However, our results indicate that elevated mitochondrial transport is due in part to upregulation of the heavy chain of the kinesin-1 motor, the level of which declines during aging. Our study identifies evolutionarily conserved factors that can strongly influence mitochondrial motility in aging neurons. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  1. Treatments for diabetes mellitus type II: New perspectives regarding the possible role of calcium and cAMP interaction.

    Science.gov (United States)

    Carvalho, Diego Soares; de Almeida, Alexandre Aparecido; Borges, Aurélio Ferreira; Campos, Vannucci

    2018-07-05

    Diabetes mellitus (DM) is among the top ten causes of death worldwide. It is considered to be one of the major global epidemics of the 21st century, with a significant impact on public health budgets. DM is a metabolic disorder with multiple etiologies. Its pathophysiology is marked by dysfunction of pancreatic β-cells which compromises the synthesis and secretion of insulin along with resistance to insulin action in peripheral tissues (muscle and adipose). Subjects presenting insulin resistance in DM type 2 often also exhibit increased insulin secretion and hyperinsulinemia. Insulin secretion is controlled by several factors such as nutrients, hormones, and neural factors. Exocytosis of insulin granules has, as its main stimulus, increased intracellular calcium ([Ca +2 ]i) and it is further amplified by cyclic AMP (cAMP). In the event of this hyperfunction, it is very common for β-cells to go into exhaustion leading to failure or death. Several animal studies have demonstrated pleiotropic effects of L-type Ca 2+ channel blockers (CCBs). In animal models of obesity and diabetes, treatment with CCBs promoted restoration of insulin secretion, glycemic control, and reduction of pancreatic β-cell apoptosis. In addition, hypertensive individuals treated with CCBs presented a lower incidence of DM when compared with other antihypertensive agents. In this review, we propose that pharmacological manipulation of the Ca 2+ /cAMP interaction system could lead to important targets for pharmacological improvement of insulin secretion in DM type 2. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Extracellular 2′,3′-cAMP Is a Source of Adenosine*

    OpenAIRE

    Jackson, Edwin K.; Ren, Jin; Mi, Zaichuan

    2009-01-01

    We discovered that renal injury releases 2′,3′-cAMP (positional isomer of 3′,5′-cAMP) into the interstitium. This finding motivated a novel hypothesis: renal injury leads to activation of an extracellular 2′,3′-cAMP-adenosine pathway (i.e. metabolism of extracellular 2′,3′-cAMP to 3′-AMP and 2′-AMP, which are metabolized to adenosine, a retaliatory metabolite). In isolated rat kidneys, arterial infusions of 2′,3′-cAMP (30 μmol/liter) increased the mean venous secretion of 3′-AMP (3,400-fold),...

  3. Deformation mechanisms in cyclic creep and fatigue

    International Nuclear Information System (INIS)

    Laird, C.

    1979-01-01

    Service conditions in which static and cyclic loading occur in conjunction are numerous. It is argued that an understanding of cyclic creep and cyclic deformation are necessary both for design and for understanding creep-fatigue fracture. Accordingly a brief, and selective, review of cyclic creep and cyclic deformation at both low and high strain amplitudes is provided. Cyclic loading in conjunction with static loading can lead to creep retardation if cyclic hardening occurs, or creep acceleration if softening occurs. Low strain amplitude cyclic deformation is understood in terms of dislocation loop patch and persistent slip band behavior, high strain deformation in terms of dislocation cell-shuttling models. While interesting advances in these fields have been made in the last few years, the deformation mechanisms are generally poorly understood

  4. Sequencing Cyclic Peptides by Multistage Mass Spectrometry

    Science.gov (United States)

    Mohimani, Hosein; Yang, Yu-Liang; Liu, Wei-Ting; Hsieh, Pei-Wen; Dorrestein, Pieter C.; Pevzner, Pavel A.

    2012-01-01

    Some of the most effective antibiotics (e.g., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of mass spectrometry based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage mass spectrometry, and show its advantages over single stage mass spectrometry. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria. PMID:21751357

  5. Cyclic peptide therapeutics: past, present and future.

    Science.gov (United States)

    Zorzi, Alessandro; Deyle, Kaycie; Heinis, Christian

    2017-06-01

    Cyclic peptides combine several favorable properties such as good binding affinity, target selectivity and low toxicity that make them an attractive modality for the development of therapeutics. Over 40 cyclic peptide drugs are currently in clinical use and around one new cyclic peptide drug enters the market every year on average. The vast majority of clinically approved cyclic peptides are derived from natural products, such as antimicrobials or human peptide hormones. New powerful techniques based on rational design and in vitro evolution have enabled the de novo development of cyclic peptide ligands to targets for which nature does not offer solutions. A look at the cyclic peptides currently under clinical evaluation shows that several have been developed using such techniques. This new source for cyclic peptide ligands introduces a freshness to the field, and it is likely that de novo developed cyclic peptides will be in clinical use in the near future. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

    Science.gov (United States)

    Lee, S; Parent, C A; Insall, R; Firtel, R A

    1999-09-01

    We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras

  7. Applications of AMPS-1D for solar cell simulation

    Science.gov (United States)

    Zhu, Hong; Kalkan, Ali Kaan; Hou, Jingya; Fonash, Stephen J.

    1999-03-01

    The AMPS-1D PC computer program is now used by over 70 groups world-wide for detector and solar cell analysis. It has proved to be a very powerful tool in understanding device operation and physics for single crystal, poly-crystalline and amorphous structures. For example, AMPS-1D has been successful in explaining the "red kink" [1] and the "transient effect" in CdS/CIGS poly-crystalline solar cells. It has been used to show that thin film poly-Si structures, with reasonable light trapping, are capable of competitive solar cell conversion efficiencies. In the case of a-Si:H structures, it has been used, for example, to settle the discrepancies in bandgap measurement, to predict the effective QE>1 phenomenon later seen in these materials [2], to determine the relative roles of interface and bulk properties, and to point the direction toward 16% triple junction structures. In general AMPS-1D is used for cell and detector design, material parameter sensitivity studies, and parameter extraction. Recently we have shown that it can be used to determine optimum structure and light and voltage biasing conditions in the material parameter extraction function. Information on AMPS can be found at www.psu.edu/dept/AMPS/amps_web/AMPS.html and at other web sites set up by user groups.

  8. Dysfunctional Hyperpolarization-Activated Cyclic Nucleotide-gated Ion Channels in Cardiac Diseases

    Directory of Open Access Journals (Sweden)

    Xiaoqi Zhao

    Full Text Available Abstract Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are reverse voltage-dependent, and their activation depends on the hyperpolarization of the membrane and may be directly or indirectly regulated by the cyclic adenosine monophosphate (cAMP or other signal-transduction cascades. The distribution, quantity and activation states of HCN channels differ in tissues throughout the body. Evidence exhibits that HCN channels play critical roles in the generation and conduction of the electrical impulse and the physiopathological process of some cardiac diseases. They may constitute promising drug targets in the treatment of these cardiac diseases. Pharmacological treatment targeting HCN channels is of benefit to these cardiac conditions.

  9. Cyclic mononucleotides modulate potassium and calcium flux responses to H2O2 in Arabidopsis roots

    KAUST Repository

    Ordoñez, Natalia Maria

    2014-02-13

    Cyclic mononucleotides are messengers in plant stress responses. Here we show that hydrogen peroxide (H2O2) induces rapid net K+-efflux and Ca2+-influx in Arabidopsis roots. Pre-treatment with either 10 μM cAMP or cGMP for 1 or 24 h does significantly reduce net K+-leakage and Ca2+-influx, and in the case of the K+-fluxes, the cell permeant cyclic mononucleotides are more effective. We also examined the effect of 10 μM of the cell permeant 8-Br-cGMP on the Arabidopsis microsomal proteome and noted a specific increase in proteins with a role in stress responses and ion transport, suggesting that cGMP is sufficient to directly and/or indirectly induce complex adaptive changes to cellular stresses induced by H2O2. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. AMP-18 Targets p21 to Maintain Epithelial Homeostasis.

    Science.gov (United States)

    Chen, Peili; Li, Yan Chun; Toback, F Gary

    2015-01-01

    Dysregulated homeostasis of epithelial cells resulting in disruption of mucosal barrier function is an important pathogenic mechanism in inflammatory bowel diseases (IBD). We have characterized a novel gastric protein, Antrum Mucosal Protein (AMP)-18, that has pleiotropic properties; it is mitogenic, anti-apoptotic and can stimulate formation of tight junctions. A 21-mer synthetic peptide derived from AMP-18 exhibits the same biological functions as the full-length protein and is an effective therapeutic agent in mouse models of IBD. In this study we set out to characterize therapeutic mechanisms and identify molecular targets by which AMP-18 maintains and restores disrupted epithelial homeostasis in cultured intestinal epithelial cells and a mouse model of IBD. Tumor necrosis factor (TNF)-α, a pro-inflammatory cytokine known to mediate gastrointestinal (GI) mucosal injury in IBD, was used to induce intestinal epithelial cell injury, and study the effects of AMP-18 on apoptosis and the cell cycle. An apoptosis array used to search for targets of AMP-18 in cells exposed to TNF-α identified the cyclin-dependent kinase inhibitor p21 WAF1/CIP1. Treatment with AMP-18 blunted increases in p21 expression and apoptosis, while reversing disturbed cell cycle kinetics induced by TNF-α. AMP-18 appears to act through PI3K/AKT pathways to increase p21 phosphorylation, thereby reducing its nuclear accumulation to overcome the antiproliferative effects of TNF-α. In vitamin D receptor-deficient mice with TNBS-induced IBD, the observed increase in p21 expression in colonic epithelial cells was suppressed by treatment with AMP peptide. The results indicate that AMP-18 can maintain and/or restore the homeostatic balance between proliferation and apoptosis in intestinal epithelial cells to protect and repair mucosal barrier homeostasis and function, suggesting a therapeutic role in IBD.

  11. AMP-18 Targets p21 to Maintain Epithelial Homeostasis.

    Directory of Open Access Journals (Sweden)

    Peili Chen

    Full Text Available Dysregulated homeostasis of epithelial cells resulting in disruption of mucosal barrier function is an important pathogenic mechanism in inflammatory bowel diseases (IBD. We have characterized a novel gastric protein, Antrum Mucosal Protein (AMP-18, that has pleiotropic properties; it is mitogenic, anti-apoptotic and can stimulate formation of tight junctions. A 21-mer synthetic peptide derived from AMP-18 exhibits the same biological functions as the full-length protein and is an effective therapeutic agent in mouse models of IBD. In this study we set out to characterize therapeutic mechanisms and identify molecular targets by which AMP-18 maintains and restores disrupted epithelial homeostasis in cultured intestinal epithelial cells and a mouse model of IBD. Tumor necrosis factor (TNF-α, a pro-inflammatory cytokine known to mediate gastrointestinal (GI mucosal injury in IBD, was used to induce intestinal epithelial cell injury, and study the effects of AMP-18 on apoptosis and the cell cycle. An apoptosis array used to search for targets of AMP-18 in cells exposed to TNF-α identified the cyclin-dependent kinase inhibitor p21 WAF1/CIP1. Treatment with AMP-18 blunted increases in p21 expression and apoptosis, while reversing disturbed cell cycle kinetics induced by TNF-α. AMP-18 appears to act through PI3K/AKT pathways to increase p21 phosphorylation, thereby reducing its nuclear accumulation to overcome the antiproliferative effects of TNF-α. In vitamin D receptor-deficient mice with TNBS-induced IBD, the observed increase in p21 expression in colonic epithelial cells was suppressed by treatment with AMP peptide. The results indicate that AMP-18 can maintain and/or restore the homeostatic balance between proliferation and apoptosis in intestinal epithelial cells to protect and repair mucosal barrier homeostasis and function, suggesting a therapeutic role in IBD.

  12. Monopod bucket foundations under cyclic lateral loading

    DEFF Research Database (Denmark)

    Foglia, Aligi; Ibsen, Lars Bo

    on bucket foundations under lateral cyclic loading. The test setup is described in detail and a comprehensive experimental campaign is presented. The foundation is subjected to cyclic overturning moment, cyclic horizontal loading and constant vertical loading, acting on the same plane for thousands...

  13. 40 CFR 721.2120 - Cyclic amide.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Cyclic amide. 721.2120 Section 721... Cyclic amide. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as a cyclic amide (PMN P-92-131) is subject to reporting under this section for the...

  14. Physiological desensitization of carbohydrate permeases and adenylate cyclase to regulation by the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium. Involvement of adenosine cyclic 3',5'-phosphate and inducer.

    Science.gov (United States)

    Saier, M H; Keeler, D K; Feucht, B U

    1982-03-10

    Adenylate cyclase and a number of carbohydrate transport systems are subject to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. These sensitive carbohydrate transport systems are desensitized to regulation by the phosphotransferase system, and adenylate cyclase is deactivated when cells are grown in medium containing cyclic AMP. These effects are specific for cyclic AMP and are potentiated by the genetic loss of cyclic AMP phosphodiesterase. Inclusion in the growth medium of an inducer of a sensitive transport system also promotes desensitization of that particular transport system. Inducer-promoted desensitization is specific for the particular target transport system, while cyclic AMP-promoted desensitization is general and affects several systems. Desensitization of the permeases to regulation, and inactivation of adenylate cyclase, are slow processes which are blocked by chloramphenicol and are therefore presumably dependent on protein synthesis. Several sugar substrates of the phosphotransferase system are capable of regulating the sensitive carbohydrate transport systems. The evidence suggests that desensitization to this regulation does not result from a direct effect on the functioning of Enzyme I, a small heat-stable protein of the phosphotransferase system, HPr, or an Enzyme II of the phosphotransferase system, but specifically uncouples the permease systems from regulation.

  15. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP-eNOS/NO-cyclic

  16. Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1 Expression by Orphan Nuclear Receptor Nr4a1

    Directory of Open Access Journals (Sweden)

    Michael P. Greenwood

    2017-12-01

    Full Text Available Cyclic AMP (cAMP inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1 is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH. We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5′UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.

  17. DisA and c-di-AMP act at the intersection between DNA-damage response and stress homeostasis in exponentially growing Bacillus subtilis cells.

    Science.gov (United States)

    Gándara, Carolina; Alonso, Juan C

    2015-03-01

    Bacillus subtilis contains two vegetative diadenylate cyclases, DisA and CdaA, which produce cyclic di-AMP (c-di-AMP), and one phosphodiesterase, GdpP, that degrades it into a linear di-AMP. We report here that DisA and CdaA contribute to elicit repair of DNA damage generated by alkyl groups and H2O2, respectively, during vegetative growth. disA forms an operon with radA (also termed sms) that encodes a protein distantly related to RecA. Among different DNA damage agents tested, only methyl methane sulfonate (MMS) affected disA null strain viability, while radA showed sensitivity to all of them. A strain lacking both disA and radA was as sensitive to MMS as the most sensitive single parent (epistasis). Low c-di-AMP levels (e.g. by over-expressing GdpP) decreased the ability of cells to repair DNA damage caused by MMS and in less extent by H2O2, while high levels of c-di-AMP (absence of GdpP or expression of sporulation-specific diadenylate cyclase, CdaS) increased cell survival. Taken together, our results support the idea that c-di-AMP is a crucial signalling molecule involved in DNA repair with DisA and CdaA contributing to modulate different DNA damage responses during exponential growth. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Activity of cAMP-dependent protein kinases and cAMP-binding proteins of rat kidney cytosol during dehydration

    International Nuclear Information System (INIS)

    Zelenina, M.N.; Solenov, E.I.; Ivanova, L.N.

    1985-01-01

    The activity of cAMP-dependent protein kinases, the binding of cAMP, and the spectrum of cAMP-binding proteins in the cytosol of the renal papilla was studied in intact rats and in rats after 24 h on a water-deprived diet. It was found that the activation of protein kinases by 10 -6 M cAMP is significantly higher in the experimental animals than in the intact animals. In chromatography on DEAE-cellulose, the positions of the peaks of specific reception of cAMP corresponded to the peaks of the regulatory subunits of cAMP-dependent protein kinases of types I and II. In this case, in intact animals more than 80% of the binding activity was detected in peaks II, whereas in rats subjected to water deprivation, more than 60% of the binding was observed in peak I. The general regulatory activity of the cytosol was unchanged in the experimental animals in comparison with intact animals. It is suggested that during dehydration there is an induction of the synthesis of the regulatory subunit of type I cAMP-dependent protein kinase in the renal papilla

  19. The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING.

    Science.gov (United States)

    Diner, Elie J; Burdette, Dara L; Wilson, Stephen C; Monroe, Kathryn M; Kellenberger, Colleen A; Hyodo, Mamoru; Hayakawa, Yoshihiro; Hammond, Ming C; Vance, Russell E

    2013-05-30

    The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic GMP-AMP (cGAMP) upon binding to an enzyme called cGAMP synthase (cGAS). cGAMP activates an interferon response by binding to a downstream receptor called STING. Here, we identify natural variants of human STING (hSTING) that are poorly responsive to cGAMP yet, unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a noncanonical cyclic dinucleotide, cyclic [G(2'-5')pA(3'-5')p], which contains a single 2'-5' phosphodiester bond. Cyclic [G(2'-5')pA(3'-5')p] potently activates diverse hSTING receptors and, therefore, may be a useful adjuvant or immunotherapeutic. Our results indicate that hSTING variants have evolved to distinguish conventional (3'-5') cyclic dinucleotides, known to be produced mainly by bacteria, from the noncanonical cyclic dinucleotide produced by mammalian cGAS. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  20. The Innate Immune DNA Sensor cGAS Produces a Noncanonical Cyclic Dinucleotide that Activates Human STING

    Directory of Open Access Journals (Sweden)

    Elie J. Diner

    2013-05-01

    Full Text Available The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic GMP-AMP (cGAMP upon binding to an enzyme called cGAMP synthase (cGAS. cGAMP activates an interferon response by binding to a downstream receptor called STING. Here, we identify natural variants of human STING (hSTING that are poorly responsive to cGAMP yet, unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a noncanonical cyclic dinucleotide, cyclic [G(2′-5′pA(3′-5′p], which contains a single 2′-5′ phosphodiester bond. Cyclic [G(2′-5′pA(3′-5′p] potently activates diverse hSTING receptors and, therefore, may be a useful adjuvant or immunotherapeutic. Our results indicate that hSTING variants have evolved to distinguish conventional (3′-5′ cyclic dinucleotides, known to be produced mainly by bacteria, from the noncanonical cyclic dinucleotide produced by mammalian cGAS.

  1. Amped Up! - Volume 1, No. 3, May/June 2015

    Energy Technology Data Exchange (ETDEWEB)

    None

    2015-05-01

    Welcome to the latest issue of our bimonthly newsletter, Amped Up!, highlighting the initiatives, events and technologies in the Office of Energy Efficiency and Renewable Energy that influence change.

  2. Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

    Science.gov (United States)

    Simko, Veronika; Iuliano, Filippo; Sevcikova, Andrea; Labudova, Martina; Barathova, Monika; Radvak, Peter; Pastorekova, Silvia; Pastorek, Jaromir; Csaderova, Lucia

    2017-08-31

    Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

  3. Interrogating the Spatiotemporal Landscape of Neuromodulatory GPCR Signaling by Real-Time Imaging of cAMP in Intact Neurons and Circuits

    Directory of Open Access Journals (Sweden)

    Brian S. Muntean

    2018-01-01

    Full Text Available Summary: Modulation of neuronal circuits is key to information processing in the brain. The majority of neuromodulators exert their effects by activating G-protein-coupled receptors (GPCRs that control the production of second messengers directly impacting cellular physiology. How numerous GPCRs integrate neuromodulatory inputs while accommodating diversity of incoming signals is poorly understood. In this study, we develop an in vivo tool and analytical suite for analyzing GPCR responses by monitoring the dynamics of a key second messenger, cyclic AMP (cAMP, with excellent quantitative and spatiotemporal resolution in various neurons. Using this imaging approach in combination with CRISPR/Cas9 editing and optogenetics, we interrogate neuromodulatory mechanisms of defined populations of neurons in an intact mesolimbic reward circuit and describe how individual inputs generate discrete second-messenger signatures in a cell- and receptor-specific fashion. This offers a resource for studying native neuronal GPCR signaling in real time. : Muntean et al. develop an in vivo reagent to study processing of neurotransmitter GPCR signals by monitoring real-time dynamics of cAMP responses. They demonstrate application of this approach, in combination with CRISPR/Cas9 gene editing and optogenetics, to interrogate the functional organization of a striatal circuit. Keywords: cAMP, GPCR, neuromodulation, dopamine, striatum, imaging, optogenetics

  4. [Effect of baicalin on ATPase and LDH and its regulatory effect on the AC/cAMP/PKA signaling pathway in rats with attention deficit hyperactivity disorder].

    Science.gov (United States)

    Zhou, Rong-Yi; Wang, Jiao-Jiao; You, Yue; Sun, Ji-Chao; Song, Yu-Chen; Yuan, Hai-Xia; Han, Xin-Min

    2017-05-01

    To study the effect of baicalin on synaptosomal adenosine triphosphatase (ATPase) and lactate dehydrogenase (LDH) and its regulatory effect on the adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway in rats with attention deficit hyperactivity disorder (ADHD). A total of 40 SHR rats were randomly divided into five groups: ADHD model, methylphenidate hydrochloride treatment (0.07 mg/mL), and low-dose (3.33 mg/mL), medium-dose (6.67 mg/mL), and high-dose (10 mg/mL) baicalin treatment (n=8 each). Eight WKY rats were selected as normal control group. Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structure. Colorimetry was used to measure the activities of ATPase and LDH in synaptosomes. ELISA was used to measure the content of AC, cAMP, and PKA. Compared with the normal control group, the ADHD model group had a significant reduction in the ATPase activity, a significant increase in the LDH activity, and significant reductions in the content of AC, cAMP, and PKA (PATPase activity (PATPase activity (PATPase activity (PATPase and LDH activities in rats with ADHD. The effect of baicalin is dose-dependent, and high-dose baicalin has a significantly greater effect than methylphenidate hydrochloride. Baicalin exerts its therapeutic effect possibly by upregulating the AC/cAMP/PKA signaling pathway.

  5. The reciprocal interaction of sympathetic nervous system and cAMP-PKA-NF-kB pathway in immune suppression after experimental stroke.

    Science.gov (United States)

    Zuo, Lei; Shi, Luhang; Yan, Fuling

    2016-08-03

    Sympathetic nervous system(SNS) is involved in the mechanism of immune suppression after stroke. Furthermore, as the pro-inflammatory effect of nuclear factor kappa B(NF-kB) is inhibited after stroke, which is regulated by cyclic adenosine monophosphate(cAMP) and proteinkinase A(PKA). The cAMP-PKA-NF-kB pathway might play an important role in noradrenergic-mediated immune dysfunction. The purpose of our research is to analyze how SNS interfere with the immune system after acute stroke and the underlying mechanism of cAMP-PKA-NF-kB pathway in regulating the inflammation. 32 healthy male Sprague-Dawley rats were divided into 4 groups equally and randomly (1) Sham operation group; (2) middle cerebral artery occlusion; (MCAO) control group; (3) propranolol MCAO group; (4) isopropylarterenol sham group. 72h later after MCAO or sham operation, tumor necrosis factor-α(TNF-α)and interleukine-10(IL-10) in serum as well as cAMP, PKA and NF-kB in spleen cells were tested. TNF-α decreased while IL-10 increased in serum after acute ischemia stroke (pkB was inhibited (pkB is down-regulated. Since the pro-inflammatory effect of NF-kB slacked, the immune system may be inhibited after stroke. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Airborne Multisensor Pod System (AMPS) data management overview

    Energy Technology Data Exchange (ETDEWEB)

    Wiberg, J.D.; Blough, D.K.; Daugherty, W.R.; Hucks, J.A.; Gerhardstein, L.H.; Meitzler, W.D.; Melton, R.B.; Shoemaker, S.V.

    1994-09-01

    An overview of the Data Management Plan for the Airborne Multisensor Pod System (AMPS) pro-grain is provided in this document. The Pacific Northwest Laboratory (PNL) has been assigned the responsibility of data management for the program, which includes defining procedures for data management and data quality assessment. Data management is defined as the process of planning, acquiring, organizing, qualifying and disseminating data. The AMPS program was established by the U.S. Department of Energy (DOE), Office of Arms Control and Non-Proliferation (DOE/AN) and is integrated into the overall DOE AN-10.1 technology development program. Sensors used for collecting the data were developed under the on-site inspection, effluence analysis, and standoff sensor program, the AMPS program interacts with other technology programs of DOE/NN-20. This research will be conducted by both government and private industry. AMPS is a research and development program, and it is not intended for operational deployment, although the sensors and techniques developed could be used in follow-on operational systems. For a complete description of the AMPS program, see {open_quotes}Airborne Multisensor Pod System (AMPS) Program Plan{close_quotes}. The primary purpose of the AMPS is to collect high-quality multisensor data to be used in data fusion research to reduce interpretation problems associated with data overload and to derive better information than can be derived from any single sensor. To collect the data for the program, three wing-mounted pods containing instruments with sensors for collecting data will be flight certified on a U.S. Navy RP-3A aircraft. Secondary objectives of the AMPS program are sensor development and technology demonstration. Pod system integrators and instrument developers will be interested in the performance of their deployed sensors and their supporting data acquisition equipment.

  7. Effects of electron beam irradiation on inorganic exchanger AMP

    International Nuclear Information System (INIS)

    Rao, K.L.N.; Mathew, C.; Deshpande, R.S.; Jadhav, A.V.; Pande, B.M.; Shukla, J.P.

    1996-01-01

    The heteropolyacid salt inorganic exchanger ammonium molybdophosphate (AMP) was subjected to an electron dose upto 2 MGy to assess any possible radiation damage. The breakthrough and total exchange capacity of AMP for Cs + from simulated fission product solutions were determined for both control and irradiated samples. The scanning electron microscopy (SEM) and energy dispersive x-ray analysis (EDX) were deployed to examine any marked microscopic changes taking place in this exchanger. (author). 3 refs., 3 figs

  8. The glutamate carboxypeptidase AMP1 mediates abscisic acid and abiotic stress responses in Arabidopsis.

    Science.gov (United States)

    Shi, Yiting; Wang, Zheng; Meng, Pei; Tian, Siqi; Zhang, Xiaoyan; Yang, Shuhua

    2013-07-01

    ALTERED MERISTEM PROGRAM1 (AMP1) encodes a glutamate carboxypeptidase that plays an important role in shoot apical meristem development and phytohormone homeostasis. We isolated a new mutant allele of AMP1, amp1-20, from a screen for abscisic acid (ABA) hypersensitive mutants and characterized the function of AMP1 in plant stress responses. amp1 mutants displayed ABA hypersensitivity, while overexpression of AMP1 caused ABA insensitivity. Moreover, endogenous ABA concentration was increased in amp1-20- and decreased in AMP1-overexpressing plants under stress conditions. Application of ABA reduced the AMP1 protein level in plants. Interestingly, amp1 mutants accumulated excess superoxide and displayed hypersensitivity to oxidative stress. The hypersensitivity of amp1 to ABA and oxidative stress was partially rescued by reactive oxygen species (ROS) scavenging agent. Furthermore, amp1 was tolerant to freezing and drought stress. The ABA hypersensitivity and freezing tolerance of amp1 was dependent on ABA signaling. Moreover, amp1 had elevated soluble sugar content and showed hypersensitivity to high concentrations of sugar. By contrast, the contents of amino acids were changed in amp1 mutant compared to the wild-type. This study suggests that AMP1 modulates ABA, oxidative and abotic stress responses, and is involved in carbon and amino acid metabolism in Arabidopsis. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  9. Database for the ampC alleles in Acinetobacter baumannii.

    Directory of Open Access Journals (Sweden)

    Nabil Karah

    Full Text Available Acinetobacter baumannii is a troublesome opportunistic pathogen with a high capacity for clonal dissemination. We announce the establishment of a database for the ampC locus in A. baumannii, in which novel ampC alleles are differentiated based on the occurrence of ≥ 1 nucleotide change, regardless of whether it is silent or missense. The database is openly accessible at the pubmlst platform for A. baumannii (http://pubmlst.org/abaumannii/. Forty-eight distinctive alleles of the ampC locus have so far been identified and deposited in the database. Isolates from clonal complex 1 (CC1, according to the Pasteur multilocus sequence typing scheme, had a variety of the ampC locus alleles, including alleles 1, 3, 4, 5, 6, 7, 8, 13, 14, 17, and 18. On the other hand, isolates from CC2 had the ampC alleles 2, 3, 19, 20, 21, 22, 23, 24, 26, 27, 28, and 46. Allele 3 was characteristic for sequence types ST3 or ST32. The ampC alleles 10, 16, and 25 were characteristic for CC10, ST16, and CC25, respectively. Our study points out that novel gene databases, in which alleles are numbered based on differences in their nucleotide identities, should replace traditional records that use amino acid substitutions to define new alleles.

  10. Dynamic Changes of Endothelium Derived Factors and Cyclic Nucleotides in Ascites Broilers and Control of L-arginine

    Institute of Scientific and Technical Information of China (English)

    HAN Bo; WANG Xiao-long

    2003-01-01

    A flock of AA breed chickens were reared in peterstme brood-vait chamber using high energypelleted feed, at 14 days of age, 400 birds were separated into 3 groups randomly as follows: 100 birds wereexposed to normal ambient temperature (20℃) as control group, 150 birds were exposed to low ambient tem-perature (11℃) in order to induce ascites as treatment Ⅰ group, another 100 birds were also exposed to lowambient temperature (11℃) and fed the diets containing 1% L-arginine for ascitic prophylactic treatment astreatment Ⅱ group. The blood samples were collected on 3, 4, 5, 6, 7 wk, respectively, to measure the con-tents of plasma endothelin (ET-1), angiotensin Ⅱ (Ang Ⅱ ) and cyclic adenosine monophosphate (cAMP) andcyclic guanosine monophosphate (cGMP). The results indicated that the contents of cAMP, cGMP, Ang Ⅱand the ratio of cAMP/cGMP in treatment Ⅰ and ascitic broilers were higher than that of correspondent con-trol group(P<0.01, P<0.05), ET-1 of preascitic broilers were higher than that of control group(P<0.05),while there were insignificant differences with later ascitic broilers, the contents of cAMP and cGMP in treat-ment Ⅱ were higher than treatment Ⅰ and control group(P<0.01, P<0.05), whereas, the ratio of cAMP/cGMP and the contents of Ang Ⅱ were gradually decreased than that of control group(P<0.05), the contentsof ET-1 were not changed. By further analysis, the increased plasma Ang Ⅱ at low ambient temperature condi-tion in broilers made endothelium cell secretion of increased ET-1, cAMP, cGMP and decreased NO. So lowtemperature accelarated ascites syndrome in broilers. Supplemented L-arginine can decrease ET-1, cAMP andcAMP/cGMP. It is concluded that cAMP mediated pulmonary hypertension syndrome in broilers.

  11. 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine inhibit TNF-α and CXCL10 production from activated primary murine microglia via A2A receptors.

    Science.gov (United States)

    Newell, Elizabeth A; Exo, Jennifer L; Verrier, Jonathan D; Jackson, Travis C; Gillespie, Delbert G; Janesko-Feldman, Keri; Kochanek, Patrick M; Jackson, Edwin K

    2015-01-12

    Some cells, tissues and organs release 2',3'-cAMP (a positional isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'-AMP plus 3'-AMP and convert these AMPs to adenosine (called the extracellular 2',3'-cAMP-adenosine pathway). Recent studies show that microglia have an extracellular 2',3'-cAMP-adenosine pathway. The goal of the present study was to investigate whether the extracellular 2',3'-cAMP-adenosine pathway could have functional consequences on the production of cytokines/chemokines by activated microglia. Experiments were conducted in cultures of primary murine microglia. In the first experiment, the effect of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production was determined. In the next experiment, the first protocol was replicated but with the addition of 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (0.1 μM; antagonist of adenosine receptors). The last experiment compared the ability of 2-chloro-N(6)-cyclopentyladenosine (CCPA) (10 μM; selective A1 agonist), 5'-N-ethylcarboxamide adenosine (NECA) (10 μM; agonist for all adenosine receptor subtypes) and CGS21680 (10 μM; selective A2A agonist) to inhibit LPS-induced TNF-α and CXCL10 production. (1) 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine similarly inhibited LPS-induced TNF-α and CXCL10 production; (2) DPSPX nearly eliminated the inhibitory effects of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production; (3) CCPA did not affect LPS-induced TNF-α and CXCL10; (4) NECA and CGS21680 similarly inhibited LPS-induced TNF-α and CXCL10 production. 2',3'-cAMP and its metabolites (3'-AMP, 2'-AMP and adenosine) inhibit LPS-induced TNF-α and CXCL10 production via A2A-receptor activation. Adenosine and its precursors, via A2A receptors, likely suppress TNF-α and CXCL10 production by activated microglia in brain diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. 2’,3’-cAMP, 3’-AMP, 2’-AMP and Adenosine Inhibit TNF-α and CXCL10 Production From Activated Primary Murine Microglia via A2A Receptors

    Science.gov (United States)

    Newell, Elizabeth A.; Exo, Jennifer L.; Verrier, Jonathan D.; Jackson, Travis C.; Gillespie, Delbert G.; Janesko-Feldman, Keri; Kochanek, Patrick M.

    2014-01-01

    Background Some cells, tissues and organs release 2’,3’-cAMP (a positional isomer of 3’,5’-cAMP) and convert extracellular 2’,3’-cAMP to 2’-AMP plus 3’-AMP and convert these AMPs to adenosine (called the extracellular 2’,3’-cAMP-adenosine pathway). Recent studies show that microglia have an extracellular 2’,3’-cAMP-adenosine pathway. The goal of the present study was to investigate whether the extracellular 2’,3’-cAMP-adenosine pathway could have functional consequences on the production of cytokines/chemokines by activated microglia. Methods Experiments were conducted in cultures of primary murine microglia. In the first experiment, the effect of 2’,3’-cAMP, 3’-AMP, 2’-AMP and adenosine on LPS-induced TNF-α and CXCL10 production was determined. In the next experiment, the first protocol was replicated but with the addition of 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (0.1 µM; antagonist of adenosine receptors). The last experiment compared the ability of 2-chloro-N6-cyclopentyladenosine (CCPA) (10 µM; selective A1 agonist), 5’-N-ethylcarboxamide adenosine (NECA) (10 µM; agonist for all adenosine receptor subtypes) and CGS21680 (10 µM; selective A2A agonist) to inhibit LPS-induced TNF-α and CXCL10 production. Results 1) 2’,3’-cAMP, 3’-AMP, 2’-AMP and adenosine similarly inhibited LPS-induced TNF-α and CXCL10 production; 2) DPSPX nearly eliminated the inhibitory effects of 2’,3’-cAMP, 3’-AMP, 2’-AMP and adenosine on LPS-induced TNF-α and CXCL10 production; 3) CCPA did not affect LPS-induced TNF-α and CXCL10; 4) NECA and CGS21680 similarly inhibited LPS-induced TNF-α and CXCL10 production. Conclusions 2’,3’-cAMP and its metabolites (3’-AMP, 2’-AMP and adenosine) inhibit LPS-induced TNF-α and CXCL10 production via A2A-receptor activation. Adenosine and its precursors, via A2A receptors, likely suppress TNF-α and CXCL10 production by activated microglia in brain diseases. PMID:25451117

  13. Genetic variation in Hyperpolarization-activated cyclic nucleotide-gated (HCN channels and its relationship with neuroticism, cognition and risk of depression

    Directory of Open Access Journals (Sweden)

    Andrew Mark Mcintosh

    2012-07-01

    Full Text Available Hyperpolarization-activated cyclic nucleotide-gated (HCN channels are encoded by four genes (HCN1-4 and, through activation by cyclic AMP (cAMP, represent a point of convergence for several psychosis risk genes. On the basis of positive preliminary data, we sought to test whether genetic variation in HCN1-4 conferred risk of depression or cognitive impairment in the Generation Scotland: Scottish Family Health Study. HCN1, HCN2, HCN3 and HCN4 were genotyped for 43 haplotype-tagging SNPs and tested for association with DSM-IV depression, neuroticism and a battery of cognitive tests assessing cognitive ability, memory, verbal fluency and psychomotor performance. No association was found between any HCN channel gene SNP and risk of depression, neuroticism or on any cognitive measure. The current study does not support a genetic role for HCN channels in conferring risk of depression or cognitive impairment in human subjects within the Scottish population.

  14. On charge-3 cyclic monopoles

    International Nuclear Information System (INIS)

    Braden, H W; D'Avanzo, Antonella; Enolski, V Z

    2011-01-01

    We determine the spectral curve of charge-3 BPS su(2) monopoles with C 3 cyclic symmetry. The symmetry means that the genus 4 spectral curve covers a (Toda) spectral curve of genus 2. A well adapted homology basis is presented enabling the theta functions and monopole data of the genus 4 curve to be given in terms of genus 2 data. The Richelot correspondence, a generalization of the arithmetic mean, is used to solve for this genus 2 curve. Results of other approaches are compared

  15. On numerically pluricanonical cyclic coverings

    International Nuclear Information System (INIS)

    Kulikov, V S; Kharlamov, V M

    2014-01-01

    We investigate some properties of cyclic coverings f:Y→X (where X is a complex surface of general type) branched along smooth curves B⊂X that are numerically equivalent to a multiple of the canonical class of X. Our main results concern coverings of surfaces of general type with p g =0 and Miyaoka-Yau surfaces. In particular, such coverings provide new examples of multi-component moduli spaces of surfaces with given Chern numbers and new examples of surfaces that are not deformation equivalent to their complex conjugates

  16. Cyclic graphs and Apery's theorem

    International Nuclear Information System (INIS)

    Sorokin, V N

    2002-01-01

    This is a survey of results about the behaviour of Hermite-Pade approximants for graphs of Markov functions, and a survey of interpolation problems leading to Apery's result about the irrationality of the value ζ(3) of the Riemann zeta function. The first example is given of a cyclic graph for which the Hermite-Pade problem leads to Apery's theorem. Explicit formulae for solutions are obtained, namely, Rodrigues' formulae and integral representations. The asymptotic behaviour of the approximants is studied, and recurrence formulae are found

  17. A system for cyclical voltametry

    International Nuclear Information System (INIS)

    Silva, R.P. da; Chierice, G.O.

    1974-01-01

    The constrution of a system composed by two instruments, voltametric circuit and potenciostate is depicted. Both instruments junction joined so that the voltametric circuit works as a triangular pulse generator, capable of operating with independent ascendant and descendant slope change, with unique pulse of continuous regime. The circuit of the potenciostate is composed of an amplifier with high entrance impedance and capable of supplying relatively high currents at the exit. The equipment was tested to study the aqueous Pb 2+ system in mercury electrode. this system depicted for the cyclical-voltometry technique set in use at I.E.A., Sao Paulo (Brazil), has very good linearity

  18. Plasma-focused cyclic accelerators

    International Nuclear Information System (INIS)

    Mondelli, A.A.; Chernin, D.P.

    1985-01-01

    The use of ambient plasma to neutralize the transverse forces of an intense particle beam has been known for many years. Most recently, the so-called ion-focused regime (IFR) for beam propagation has been used as a means of focusing intense electron beams in linear accelerators and suggested for injecting an electron beam across magnetic field lines into a high-current cyclic accelerator. One technique for generating the required background plasma for IFR propagation is to use a laser to ionize ambient gas in the accelerator chamber. For cyclic accelerators a technique is required for carrying the plasma channel and the beam around a bend. Multiple laser-generated channels with dipole magnetic fields to switch the beam from one channel to the next have been tested at Sandia. This paper discusses an alternative means of plasma production for IFR, viz. by using rf breakdown. For this approach the accelerator chamber acts as a waveguide. With a suitable driving frequency, a waveguide mode can be driven which has its peak field intensity on the axis with negligible fields at the chamber walls. The plasma production and hence the beam propagation is thereby isolated from the walls. This technique is not limited to toroidal accelerators. It may be applied to any accelerator or recirculator geometry as well as for beam steering and for injection or extraction of beams in closed accelerator configurations

  19. Cyclic Vomiting Syndrome in Children

    Directory of Open Access Journals (Sweden)

    T.V. Sorokman

    2016-08-01

    Full Text Available Introduction. Cyclic vomiting syndrome (CVS — is a fairly common disease of unknown etiology that affects children of all age groups and sometimes adult population and refers to the functional disorders of the gastrointestinal tract. Objective: to evaluate the effectiveness of the usage of Rehydron Optim for oral rehydration therapy in children. Materials and methods. The treatment of 40 children aged 3 to 11 years with CVS (15 persons and primary acetonemic syndrome (25 persons in the period of acetonemic crisis, including 15 boys and 25 girls, was analyzed. All children were observed in the outpatient department of the Regional children’s hospital of Chernivtsi. Diagnosis was established based on anamnesis, clinical and laboratory data. Patients underwent required clinico-biological tests and instrumental examinations. The dynamics of the following syndromes was investigated: pain, vomiting, dehydration and intoxication. Rehydration therapy in all cases was oral with the usage of Rehydron Optim. Results of the study and their discussion. A cyclical vomiting was observed in children with primary acetonemic syndrome with satisfactory condition in attack-free period. Migraine-like headaches prevailed in 36 patients (80 %, and the age of these patients was older than 7 years. Same children had episodes of paroxysmal autonomic failure. Almost all surveyed children had in their family history the risk factors for CVS development. All children had positive dynamics of the main basic clinical manifestations on the background of oral rehydration therapy using Rehydron Optim. Within the 1st day of oral rehydration therapy with Rehydron Optim in children, we have noted a significant decrease in the incidence of lethargy, vomiting, spastic abdominal pain, smell of acetone in the exhaled air (p < 0.05. In children with the I degree of dehydration, clinical signs of dehydration were not seen before the treatment, and children with the II degree had an

  20. [Cyclic Cushing's Syndrome - rare or rarely recognized].

    Science.gov (United States)

    Kiałka, Marta; Doroszewska, Katarzyna; Mrozińska, Sandra; Milewicz, Tomasz; Stochmal, Ewa

    2015-01-01

    Cyclic Cushing's syndrome is a type of Cushing's disease which is characterized by alternating periods of increasing and decreasing levels of cortisol in the blood. The diagnostic criteria for cyclic Cushing's syndrome are at least three periods of hypercortisolism alternating with at least two episodes of normal levels of serum cortisol concentration. The epidemiology, signs, symptoms, pathogenesis and treatment of cyclic Cushing's syndrome have been discussed.

  1. Regulation of cessation of respiration and killing by cyclic 3',5'-adenosine monophosphate and its receptor protein after far-ultraviolet irradiation of Escherichia coli

    International Nuclear Information System (INIS)

    Swenson, P.A.; Schenley, R.L.; Joshi, J.G.

    1978-01-01

    When Escherichia coli B/r cultures are irradiated with ultraviolet light (UV) (254 nm), those cells that are killed stop respiring by 60 min after irradiation. Post-UV treatment with cyclic adenosine 3',5'-adenosine monophosphate (cAMP) causes more cells to stop respiring and to die. We have studied these effects at a UV fluence of 52 I/m 2 in a a wild-type E. coli K 12 strain and in mutants defective in cAMP metabolism. Strain CA 8,000 has crp + and cya + genes for the cAMP receptor protein (CRP) (required for transcription of operons regulated by cAMP) and for adenylate cyclase, respectively; CA 7901 is crp - ; and CA 8306 is a cya deletion (Δ). The wild-type culture showed a small transient cessation of respiration, and addition of cAMP caused cessation to be nearly complete. The crp - culture showed no evidence of cessation of respiration, and cAMP had no effect. The Δ cya mutant also showed no cessation of respiration, but cAMP (5 mM) caused as complete inhibition as in the wild type. cAMP caused a 10-fold loss in viability of UV-irradiated wild-type and Δ cya liquid cultures but had no effect on the cpr - culture. Respiration and viability changes were also studied in a double mutant, CA8404 Δ cya crp*, which has an altered CRP that is, with respect to the lac operon, independent of cAMP. The respiration response to UV was similar to that of the wild-type culture, and both respiration and viability of cells in liquid culture were sensitive to cAMP. The survival data, obtained by plating immediately after irradiation, show the wild type, Δ cya strains, and Δ cya crp* to be equally sensitive and the crp - strain to be more resistant. We conclude that cessation of respiration and cell killing after UV irradiation are regulated by cAMP and the CRP. (orig.) [de

  2. Modeling Cyclic Variation of Intracranial Pressure

    National Research Council Canada - National Science Library

    Daley, M

    2001-01-01

    ...) recording during mechanical ventilation are due to cyclic extravascular compressional modulation primarily of the cerebral venous bed, an established isovolumetric model of cerebrospinal fluid...

  3. Behaviour of Cohesionless Soils During Cyclic Loading

    DEFF Research Database (Denmark)

    Shajarati, Amir; Sørensen, Kris Wessel; Nielsen, Søren Kjær

    Offshore wind turbine foundations are typically subjected to cyclic loading from both wind and waves, which can lead to unacceptable deformations in the soil. However, no generally accepted standardised method is currently available, when accounting for cyclic loading during the design of offshore...... wind turbine foundations. Therefore a literature study is performed in order to investigate existing research treating the behaviour of cohesionless soils, when subjected to cyclic loading. The behaviour of a soil subjected to cyclic loading is found to be dependent on; the relative density, mean...

  4. Cyclical subnormal separation in A-groups

    International Nuclear Information System (INIS)

    Makarfi, M.U.

    1995-12-01

    Three main results, concerning A-groups in respect of cyclical subnormal separation as defined in, are presented. It is shown in theorem A that any A-group that is generated by elements of prime order and satisfying the cyclical subnormal separation conditions is metabelian. The two other main results give necessary and sufficient conditions for A-groups, that are split extensions of certain abelian p-groups by a metabelian p'-group, to satisfy the cyclical subnormal separation condition. There is also a result which shows that A-groups with elementary abelian Sylow subgroups are cyclically separated as defined. (author). 7 refs

  5. [Studies on the relation between glucose metabolism and c-AMP formation in dental pulps in the presence of inflammatory chemical mediators in vitro].

    Science.gov (United States)

    Kiyohara, H

    1989-01-01

    The relationship between glucose metabolism and cyclic-AMP production in dental pulp in the presence of chemical mediators was investigated in vitro. It is generally accepted that oxidation of glucose-6-14C is indicative of metabolism by the glycolytic pathway whereas that of glucose-1-14C occurs by the hexose monophosphate shunt. The 14CO2 productions from both routes were compared in dental pulp from cattle and rats in the presence of each of several chemical mediators: bradykinin (1.7-3.3 micrograms/ml), prostaglandin E1 (0.3 micrograms/ml), prostaglandin E2 (0.3 micrograms/ml), histamine (33 micrograms/ml), and 5-hydroxytryptamine (33 micrograms/ml). The effects of dental filling materials on glucose oxidation, and cyclic-AMP production by chemical mediators in pulp tissues were also investigated. The results obtained were as follows: 1) Glucose oxidation in dental pulp was stimulated by chemical mediators generally by way of the Embden-Meyerhof Parnas pathway, and was further stimulated by the medium containing bradykinin. However, it was depressed in the presence of higher concentrations of chemical mediators, especially depressed in the HMS pathway. 2) The oxidation ratio of glucose-1-14C to glucose-6-14C (G1/G6) in dental pulp was 4 to 8 in the cattle and 0.6 in the rat, showing clear differences in glucose oxidation between the two animals. 3) Moreover, glucose oxidation in rat dental pulp was 60 to 80 times higher in the EMP pathway and 5 to 10 times higher in the HMS pathway than those in the cattle. 4) Dental filling materials such as silicate cement, zinc phosphate cement, calcium hydroxide, and eugenol cement severely depressed glucose-6-14C oxidation in bovine dental pulp when used at high concentrations, but not at low concentrations. 5) The chemical mediators tested in this experiment (PGE1, PGE2, histamine, 5-HT, bradykinin, and substance P) stimulated cyclic AMP production in rat dental pulp. The production was highest with PGE1 and PGE2. The

  6. Persönlichkeit der Fussballer AMP Futbol = Personality footballers AMP Futbol

    Directory of Open Access Journals (Sweden)

    Paweł Kalinowski

    2016-08-01

    Full Text Available Kalinowski, Paweł, Lischka Ołpińska, Marta . Persönlichkeit der Fussballer AMP Futbol = Personality footballers AMP Futbol. Journal of Education, Health and Sport. 2016;6(8:227-235. eISSN 2391-8306. DOI http://dx.doi.org/10.5281/zenodo.60260 http://ojs.ukw.edu.pl/index.php/johs/article/view/3751       The journal has had 7 points in Ministry of Science and Higher Education parametric evaluation. Part B item 755 (23.12.2015. 755 Journal of Education, Health and Sport eISSN 2391-8306 7 © The Author (s 2016; This article is published with open access at Licensee Open Journal Systems of Kazimierz Wielki University in Bydgoszcz, Poland Open Access. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s and source are credited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/4.0/ which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. This is an open access article licensed under the terms of the Creative Commons Attribution Non Commercial License (http://creativecommons.org/licenses/by-nc/4.0/ which permits unrestricted, non commercial use, distribution and reproduction in any medium, provided the work is properly cited. The authors declare that there is no conflict of interests regarding the publication of this paper. Received: 15.07.2016. Revised 25.07.2016. Accepted: 12.08.2016.   QUALITY IN SPORT 3 (2 2016, s. 65-72, e-ISSN 2450-3118 Praca wpłynęła do redakcji 13 III 2016, praca została przyjęta do druku 23 VI 2016 DOI: http://dx.doi.org/10.5281/zenodo.57217 PBN: https://pbn.nauka.gov.pl/sedno-webapp/works/735282     Paweł Kalinowski[1], Marta Lischka Ołpińska Doktoranden AWF Pozna

  7. cAMP/PKA signaling pathway contributes to neuronal apoptosis via regulating IDE expression in a mixed model of type 2 diabetes and Alzheimer's disease.

    Science.gov (United States)

    Li, Huajie; Yang, Song; Wu, Jian; Ji, Lei; Zhu, Linfeng; Cao, Liping; Huang, Jinzhong; Jiang, Qingqing; Wei, Jiang; Liu, Meng; Mao, Keshi; Wei, Ning; Xie, Wei; Yang, Zhilong

    2018-02-01

    Type 2 diabetes (T2D) may play a relevant role in the development of Alzheimer's disease (AD), however, the underlying mechanism was not clear yet. We developed an animal model presenting both AD and T2D, morris water maze (MWM) test and recognition task were performed to trace the cognitive function. Fasting plasma glucose (FPG) and oral glucose tolerance test (OGTT) were determined to trace the metabolism evolution. TUNEL assay and apoptosis-related protein levels were analyzed for the detection of neuronal apoptosis. Cyclic adenosine monophosphate (cAMP) agonist bucladesine or protein kinase (PKA) inhibitor H-89 were used to determine the effects of cAMP/PKA signaling pathway on IDE expression and neuronal apoptosis. The results showed that T2D contributes to the AD progress by accelerating and worsening spatial memory and recognition dysfunctions. Metabolic parameters and glucose tolerance were significantly changed in the presence of the AD and T2D. The significantly induced neuronal apoptosis and increased pro-apoptotic proteins in mice with AD and T2D were also observed. We showed the decreased expression level of IDE and the activating of cAMP/PKA signaling pathway in AD and T2D mice. Further studies indicated that cAMP agonist decreased the expression level of IDE and induced the neuronal apoptosis in mice with AD and T2D; whereas PKA inhibitor H-89 treatment showed the completely opposite results. Our study indicated that, in the T2D and AD mice, cAMP/PKA signaling pathway and IDE may participate in the contribute role of T2D in accelerating the pathological process of AD via causing the accumulation of Aβ and neuronal apoptosis. © 2017 Wiley Periodicals, Inc.

  8. Exposure to a specific time-varying electromagnetic field inhibits cell proliferation via cAMP and ERK signaling in cancer cells.

    Science.gov (United States)

    Buckner, Carly A; Buckner, Alison L; Koren, Stan A; Persinger, Michael A; Lafrenie, Robert M

    2018-04-01

    Exposure to specific electromagnetic field (EMF) patterns can affect a variety of biological systems. We have shown that exposure to Thomas-EMF, a low-intensity, frequency-modulated (25-6 Hz) EMF pattern, inhibited growth and altered cell signaling in malignant cells. Exposure to Thomas-EMF for 1 h/day inhibited the growth of malignant cells including B16-BL6 mouse melanoma cells, MDA-MB-231, MDA-MB-468, BT-20, and MCF-7 human breast cancer and HeLa cervical cancer cells but did not affect non-malignant cells. The Thomas-EMF-dependent changes in cell proliferation were mediated by adenosine 3',5'-cyclic monophosphate (cAMP) and extracellular-signal-regulated kinase (ERK) signaling pathways. Exposure of malignant cells to Thomas-EMF transiently changed the level of cellular cAMP and promoted ERK phosphorylation. Pharmacologic inhibitors (SQ22536) and activators (forskolin) of cAMP production both blocked the ability of Thomas-EMF to inhibit cell proliferation, and an inhibitor of the MAP kinase pathway (PD98059) was able to partially block Thomas-EMF-dependent inhibition of cell proliferation. Genetic modulation of protein kinase A (PKA) in B16-BL6 cells also altered the effect of Thomas-EMF on cell proliferation. Cells transfected with the constitutively active form of PKA (PKA-CA), which interfered with ERK phosphorylation, also interfered with the Thomas-EMF effect on cell proliferation. The non-malignant cells did not show any EMF-dependent changes in cAMP levels, ERK phosphorylation, or cell growth. These data indicate that exposure to the specific Thomas-EMF pattern can inhibit the growth of malignant cells in a manner dependent on contributions from the cAMP and MAP kinase pathways. Bioelectromagnetics. 39;217-230, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Linoleic acid enhance the production of moncolin K and red pigments in Monascus ruber by activating mokH and mokA, and by accelerating cAMP-PkA pathway.

    Science.gov (United States)

    Huang, Jing; Liao, NanQing; Li, HaoMing

    2018-04-01

    Monacolin K, an inhibitor of HMG-CoA reductase, is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus ruber. The mokH gene encoding Zn(II)2Cys6 binding protein and mokA gene encoding polyketide synthase are presumed to activate monacolin K production. In this study, linoleic acid could be a quorum sensing signaling molecule to increase monacolin K production in the cyclic AMP(cAMP)-protein kinase A(PKA) signaling pathway. Analysis of the PKA activity and the cAMP concentration shows that linoleic acid could increase cAMP concentration and activate PKA. Analysis of the RT-qPCR products demonstrates that 256μM and 512μM linoleic acid can up-regulate mokH and mokA gene transcript levels. Especially with 512μM linoleic acid addition, linoleic acid increase 1.35 folds of monacolin K production, but 64μM linoleic acid increase 1.94 folds of red pigment production in Monascus ruber. These results show the cAMP-PkA pathway activity can up-regulate mokA and mokH gene, which enhance the yield of Monacolin K. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Plasma-focused cyclic accelerators

    International Nuclear Information System (INIS)

    Mondelli, A.A.; Chernin, D.P.

    1985-01-01

    The use of ambient plasma to neutralize the transverse forces of an intense particle beam has been known for many years. Most recently, the so-called ion-focused regime (IFR) for beam propagation has been used as a means of focusing intense electron beams in linear accelerators and suggested for injecting an electron beam across magnetic field lines into a high-current cyclic accelerator. One technique for generating the required background plasma for IFR propagation is to use a laser to ionize ambient gas in the accelerator chamber. This paper discusses an alternative means of plasma production for IFR, viz. by using RF breakdown. For this approach the accelerator chamber acts as a waveguide. This technique is not limited to toroidal accelerators. It may be applied to any accelerator or recirculator geometry as well as for beam steering and for injection or extraction of beams in closed accelerator configurations

  11. Modeling of CO2 absorber using an AMP solution

    DEFF Research Database (Denmark)

    Gabrielsen, Jostein; Michelsen, Michael Locht; Stenby, Erling Halfdan

    2006-01-01

    Abstract: An explicit model for carbon dioxide (CO2) solubility in an aqueous solution of 2-amino-2-methyl-1-propanol (AMP) has been proposed and an expression for the heat of absorption of CO2 has been developed as a function of loading and temperature. A rate-based steady-state model for CO2...... to absorption of CO2 into an AMP solution in a packed tower and validated against pilot-plant data from the literature. (c) 2006 American Institute of Chemical Engineers....... absorption into an AMP solution has been proposed, using both the proposed expression for the CO2 solubility and the expression for the heat of absorption along with an expression for the enhancement factor and physicochemical data from the literature. The proposed model has successfully been applied...

  12. Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation.

    Directory of Open Access Journals (Sweden)

    Kenji Ezoe

    Full Text Available Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK or 3-isobutyl-1-methylxanthine (IBMX to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.

  13. Physiological and Molecular Effects of the Cyclic Nucleotides cAMP and cGMP on Arabidopsis thaliana

    KAUST Repository

    Herrera, Natalia M.

    2012-01-01

    transport in Arabidopsis thaliana leaves and, that these changes at the molecular level can have functional biological consequences. For this reason we tested if CNs modulate the photosynthetic rate, responses to high light and root ion transport. Real time

  14. Activation of Cyclic AMP Synthesis by Full and Partial Beta-Adrenergic Receptor Agonists in Chicken Skeletal Muscle Cells

    Science.gov (United States)

    Young, R. B.; Bridge, K. Y.

    2003-01-01

    Several beta-adrenergic receptor (bAR) agonists are known to cause hypertrophy of skeletal muscle tissue. Accordingly, five bAR agonists encompassing a range in activity from strong to weak were evaluated for their ability to stimulate CAMP accumulation in embryonic chicken skeletal muscle cells in culture. Two strong agonists (epinephrine and isoproterenol), one moderate agonist (albuterol), and two weak agonists known to cause hypertrophy in animals (clenbuterol and cimaterol) were studied. Dose response curves were determined over six orders of magnitude in concentration for each agonist, and values were determined for their maximum stimulation of CAMP synthesis rate (Bmax) and the agonist concentration at which 50% stimulation of CAMP synthesis (EC50) occurred. Bmax values decreased in the following order: isoproterenol, epinephrine, albuterol, cimaterol, clenbuterol. Cimaterol and clenbuterol at their Bmax concentrations were approximately 15-fold weaker than isoproterenol in stimulating the rate of CAMP synthesis. When cimaterol and clenbuterol were added to culture media at concentrations known to cause significant muscle hypertrophy in animals, there was no detectable effect on stimulation of CAMP synthesis. Finally, these same levels of cimaterol and clenbuterol did not antagonize the stimulation of CAMP by either epinephrine or isoproterenol.

  15. Cyclic AMP signalling in Dictyostelium : G-proteins activate separate Ras pathways using specific RasGEFs

    NARCIS (Netherlands)

    Kae, Helmut; Kortholt, Arjan; Rehmann, Holger; Insall, RobertH.; Van Haastert, Peter J. M.; Spiegelman, George B.; Weeks, Gerald

    In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium

  16. Properties of the luminal membrane of isolated perfused rat pancreatic ducts. Effect of cyclic AMP and blockers of chloride transport

    DEFF Research Database (Denmark)

    Novak, I; Greger, R

    1988-01-01

    - and interlobular ducts of rat pancreas was used. Responses of the epithelium to inhibitors and agonists were monitored by electrophysiological techniques. Addition of HCO-3/CO2 to the bath side of nonstimulated ducts depolarized the PD across the basolateral membrane (PDbl) by about 9 mV, as also observed...

  17. The Role of Cyclic AMP and Its Relationship to Parathyroid Hormone Response in an In Vitro Model of Chondrogenesis.

    Science.gov (United States)

    1992-06-01

    factors on bone and cartilage cell equilibria and differentiation will enhance our understanding of bone metabolism in health and disease . A. Parathyroid...1984. Difference between 1-84 parathyroid hormone and the 1-34 fragment on renal tubular calcium transport in the dog . Miner. Electrolyte Metab., ]A...general dentist during a four-year tour. In June 1989, Dr. Semba entered the Postdoctoral Periodontics program at the University of Texas Health Science Center in San Antonio in conjunction with Wilford Hall USAF Medical Center.

  18. Prevalence and molecular characterization of clinical isolates of Escherichia coli expressing an AmpC phenotype

    DEFF Research Database (Denmark)

    Jørgensen, Rikke Lind; Nielsen, Jesper Boye; Friis-Møller, Alice

    2010-01-01

    . Hyperproduction of AmpC beta-lactamase was confirmed by isoelectric focusing (IEF). The presence of a plasmid-mediated ampC gene (pAmpC) was detected by multiplex PCR. The promoter and the entire reading frame of the chromosomal ampC gene were sequenced to identify promoter mutations associated...... by multilocus sequence typing (MLST). The remaining isolates all had mutations or insertions in the promoter region, which could explain increased expression of the chromosomal AmpC enzyme. Mutations in the ampC gene associated with extended activity were rare and did not cause resistance to cefepime...

  19. Gamma and neutron irradiation tests on commercial IC op amps

    International Nuclear Information System (INIS)

    Kennedy, E.J.; Morris, A.C. Jr.; Su, D.K.

    1985-01-01

    Experimental results of gamma and neutron irradiation tests on 30 types of integrated-circuit operational amplifiers from 11 manufacturers are presented. All units were low-cost, commercial-grade devices. Op amps were evaluated for changes in offset voltage, input bias current, power supply current, open-loop gain, gain-bandwidth product, slew rate, power-supply and common-mode rejection ratios. Bipolar transistor op amps with resistive collector load resistors for the input stage indicated the best radiation hardness

  20. Analisis Derau dalam Perancangan Rangkaian Terintegrasi Op-Amp

    Directory of Open Access Journals (Sweden)

    Siti Sendari

    2009-02-01

    Full Text Available Perancangan IC Op-Amp, memerlukan spesifikasi-spesifikasi yang meliputi parameter proses dan parameter perancangan. Dalam menentukan parameter perancangan, derau merupakan suatu hal yang tidak bisa ditinggalkan, karena derau dapat menentukan keandalan perancangan. Untuk menganalisis derau pada IC Op-Amp langkah pertama yang harus diketahui adalah sumber-sumber derau, setelah itu dilakukan analisis derau baik secara teoritis maupun secara simulasi untuk mengetahui derau yang mungkin terjadi dalam perancangan IC, apakah sudah sesuai dengan spesifikasi yang ditentukan atau tidak. Untuk simulasi derau dapat dilakukan dengan program Spice.

  1. Cyclic completion of the anamorphic universe

    Science.gov (United States)

    Ijjas, Anna

    2018-04-01

    Cyclic models of the universe have the advantage of avoiding initial conditions problems related to postulating any sort of beginning in time. To date, the best known viable examples of cyclic models have been ekpyrotic. In this paper, we show that the recently proposed anamorphic scenario can also be made cyclic. The key to the cyclic completion is a classically stable, non-singular bounce. Remarkably, even though the bounce construction was originally developed to connect a period of contraction with a period of expansion both described by Einstein gravity, we show here that it can naturally be modified to connect an ordinary contracting phase described by Einstein gravity with a phase of anamorphic smoothing. The paper will present the basic principles and steps in constructing cyclic anamorphic models.

  2. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    LENUS (Irish Health Repository)

    Gegenbauer, Kristina

    2013-11-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

  3. A common signaling pathway is activated in erythroid cells expressing high levels of fetal hemoglobin: a potential role for cAMP-elevating agents in β-globin disorders

    Directory of Open Access Journals (Sweden)

    Ikuta T

    2013-12-01

    Full Text Available Tohru Ikuta,1 Yuichi Kuroyanagi,1 Nadine Odo,1 Siyang Liu21Department of Anesthesiology and Perioperative Medicine, 2Department of Physiology, Medical College of Georgia, Georgia Regents University, Augusta, GA, USABackground: Although erythroid cells prepared from fetal liver, cord blood, or blood from β-thalassemia patients are known to express fetal hemoglobin at high levels, the underlying mechanisms remain elusive. We previously showed that cyclic nucleotides such as cAMP and cGMP induce fetal hemoglobin expression in primary erythroid cells. Here we report that cAMP signaling contributes to high-level fetal hemoglobin expression in erythroid cells prepared from cord blood and β-thalassemia.Methods: The status of the cAMP signaling pathway was investigated using primary erythroid cells prepared from cord blood and the mononuclear cells of patients with β-thalassemia; erythroid cells from adult bone marrow mononuclear cells served as the control.Results: We found that intracellular cAMP levels were higher in erythroid cells from cord blood and β-thalassemia than from adult bone marrow. Protein kinase A activity levels and cAMP-response element binding protein phosphorylation were higher in erythroid cells from cord blood or β-thalassemia than in adult bone marrow progenitors. Mitogen-activated protein kinase pathways, which play a role in fetal hemoglobin expression, were not consistently activated in cord blood or β-thalassemia erythroid cells. When cAMP signaling was activated in adult erythroid cells, fetal hemoglobin was induced at high levels and associated with reduced expression of BCL11A, a silencer of the β-globin gene.Conclusion: These results suggest that activated cAMP signaling may be a common mechanism among erythroid cells with high fetal hemoglobin levels, in part because of downregulation of BCL11A. Activation of the cAMP signaling pathway with cAMP-elevating agents may prove to be an important signaling mechanism to

  4. Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

    Science.gov (United States)

    Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

    2007-01-01

    Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

  5. Specificity of the Cyclic GMP-Binding Activity and of a Cyclic GMP-Dependent Cyclic GMP Phosphodiesterase in Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Walsum, Hans van; Meer, Rob C. van der; Bulgakov, Roman; Konijn, Theo M.

    1982-01-01

    The nucleotide specificity of the cyclic GMP-binding activity in a homogenate of Dictyostelium discoideum was determined by competition of cyclic GMP derivatives with [8-3H] cyclic GMP for the binding sites. The results indicate that cyclic GMP is bound to the binding proteins by hydrogen bonds at

  6. Evolution of the cAMP-dependent protein kinase (PKA catalytic subunit isoforms.

    Directory of Open Access Journals (Sweden)

    Kristoffer Søberg

    Full Text Available The 3',5'-cyclic adenosine monophosphate (cAMP-dependent protein kinase, or protein kinase A (PKA, pathway is one of the most versatile and best studied signaling pathways in eukaryotic cells. The two paralogous PKA catalytic subunits Cα and Cβ, encoded by the genes PRKACA and PRKACB, respectively, are among the best understood model kinases in signal transduction research. In this work, we explore and elucidate the evolution of the alternative 5' exons and the splicing pattern giving rise to the numerous PKA catalytic subunit isoforms. In addition to the universally conserved Cα1/Cβ1 isoforms, we find kinase variants with short N-termini in all main vertebrate classes, including the sperm-specific Cα2 isoform found to be conserved in all mammals. We also describe, for the first time, a PKA Cα isoform with a long N-terminus, paralogous to the PKA Cβ2 N-terminus. An analysis of isoform-specific variation highlights residues and motifs that are likely to be of functional importance.

  7. Evolutionary Paths of the cAMP-Dependent Protein Kinase (PKA) Catalytic Subunits

    Science.gov (United States)

    Søberg, Kristoffer; Jahnsen, Tore; Rognes, Torbjørn; Skålhegg, Bjørn S.; Laerdahl, Jon K.

    2013-01-01

    3′,5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods. PMID:23593352

  8. Ohmyungsamycins promote antimicrobial responses through autophagy activation via AMP-activated protein kinase pathway.

    Science.gov (United States)

    Kim, Tae Sung; Shin, Yern-Hyerk; Lee, Hye-Mi; Kim, Jin Kyung; Choe, Jin Ho; Jang, Ji-Chan; Um, Soohyun; Jin, Hyo Sun; Komatsu, Masaaki; Cha, Guang-Ho; Chae, Han-Jung; Oh, Dong-Chan; Jo, Eun-Kyeong

    2017-06-13

    The induction of host cell autophagy by various autophagy inducers contributes to the antimicrobial host defense against Mycobacterium tuberculosis (Mtb), a major pathogenic strain that causes human tuberculosis. In this study, we present a role for the newly identified cyclic peptides ohmyungsamycins (OMS) A and B in the antimicrobial responses against Mtb infections by activating autophagy in murine bone marrow-derived macrophages (BMDMs). OMS robustly activated autophagy, which was essentially required for the colocalization of LC3 autophagosomes with bacterial phagosomes and antimicrobial responses against Mtb in BMDMs. Using a Drosophila melanogaster-Mycobacterium marinum infection model, we showed that OMS-A-induced autophagy contributed to the increased survival of infected flies and the limitation of bacterial load. We further showed that OMS triggered AMP-activated protein kinase (AMPK) activation, which was required for OMS-mediated phagosome maturation and antimicrobial responses against Mtb. Moreover, treating BMDMs with OMS led to dose-dependent inhibition of macrophage inflammatory responses, which was also dependent on AMPK activation. Collectively, these data show that OMS is a promising candidate for new anti-mycobacterial therapeutics by activating antibacterial autophagy via AMPK-dependent signaling and suppressing excessive inflammation during Mtb infections.

  9. Metabolic benefits of inhibiting cAMP-PDEs with resveratrol.

    Science.gov (United States)

    Chung, Jay H

    2012-10-01

    Calorie restriction (CR) extends lifespan in species ranging from yeast to mammals. There is evidence that CR also protects against aging-related diseases in non-human primates. This has led to an intense interest in the development of CR-mimetics to harness the beneficial effects of CR to treat aging-related diseases. One CR-mimetic that has received a great deal of attention is resveratrol. Resveratrol extends the lifespan of obese mice and protects against obesity-related diseases such as type 2 diabetes. The specific mechanism of resveratrol action has been difficult to elucidate because resveratrol has a promiscuous target profile. A recent finding indicates that the metabolic effects of resveratrol may result from competitive inhibition of cAMP-degrading phosphodiesterases (PDEs), which increases cAMP levels. The cAMP-dependent pathways activate AMP-activated protein kinase (AMPK), which is essential for the metabolic effects of resveratrol. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including protection against diet-induced obesity and an increase in mitochondrial function, physical stamina and glucose tolerance in mice. This discovery suggests that PDE inhibitors may be useful for treating metabolic diseases associated with aging.

  10. CMOS technology and current-feedback op-amps

    DEFF Research Database (Denmark)

    Bruun, Erik

    1993-01-01

    Some of the problems related to the application of CMOS technology to current-feedback operational amplifiers (CFB op-amps) are identified. Problems caused by the low device transconductance and by the absence of matching between p-channel and n-channel transistors are examined, and circuit...

  11. Detection of Amp C Beta Lactamases in Clinical Isolates of ...

    African Journals Online (AJOL)

    A total of 81 consecutive non repetitive clinical isolates of Escherichia coli (n=40) and Klebsiella spp. (n=41) were screened for AmpC production by disc diffusion method using cefoxitin (30 Zg) disc and confirmed by inhibitor based test using boronic acid as inhibitor. A total of 16 E.coli isolates (40%) and 16 Klebsiella ...

  12. 0011-0030.Data Representation amp Computer Arithmetic6 IEEE ...

    Indian Academy of Sciences (India)

    Home; public; Volumes; reso; 021; 01; 0011-0030.Data Representation amp Computer Arithmetic6 IEEE Standard Double Precision FormatIn.pdf. 404! error. The page your are looking for can not be found! Please check the link or use the navigation bar at the top. YouTube; Twitter; Facebook; Blog. Academy News.

  13. Occurrence and detection of AmpC β-lactamases among ...

    African Journals Online (AJOL)

    Soha A. El-Hady

    2015-04-06

    Apr 6, 2015 ... Molecular detection of plasmid mediated AmpC by multiplex PCR was conducted on them. Results: The .... b-lactamases into the external environment. AmpC .... prove satisfactory) and should not display significant homol-.

  14. Purification, characterization, and sequencing of novel antimicrobial peptides, Tu-AMP 1 and Tu-AMP 2, from bulbs of tulip (Tulipa gesneriana L.).

    Science.gov (United States)

    Fujimura, Masatoshi; Ideguchi, Mineo; Minami, Yuji; Watanabe, Keiichi; Tadera, Kenjiro

    2004-03-01

    Novel antimicrobial peptides (AMP), designated Tu-AMP 1 and Tu-AMP 2, were purified from the bulbs of tulip (Tulipa gesneriana L.) by chitin affinity chromatography and reverse-phase high-performance liquid chromatography (HPLC). They bind to chitin in a reversible way. They were basic peptides having isoelectric points of over 12. Tu-AMP 1 and Tu-AMP 2 had molecular masses of 4,988 Da and 5,006 Da on MALDI-TOF MS analysis, and their extinction coefficients of 1% aqueous solutions at 280 nm were 3.3 and 3.4, respectively. Half of all amino acid residues of Tu-AMP 1 and Tu-AMP 2 were occupied by cysteine, arginine, lysine, and proline. The concentrations of peptides required for 50% inhibition (IC(50)) of the growth of plant pathogenic bacteria and fungi were 2 to 20 microg/ml. The structural characteristics of Tu-AMP 1 and Tu-AMP 2 indicated that they were novel thionin-like antimicrobial peptides, though Tu-AMP 2 was a heterodimer composes of two short peptides joined with disulfide bonds.

  15. Cyclic characteristics of earthquake time histories

    International Nuclear Information System (INIS)

    Hall, J.R. Jr; Shukla, D.K.; Kissenpfennig, J.F.

    1977-01-01

    From an engineering standpoint, an earthquake record may be characterized by a number of parameters, one of which is its 'cyclic characteristics'. The cyclic characteristics are most significant in fatigue analysis of structures and liquefaction analysis of soils where, in addition to the peak motion, cyclic buildup is significant. Whereas duration peak amplitude and response spectra for earthquakes have been studied extensively, the cyclic characteristics of earthquake records have not received an equivalent attention. Present procedures to define the cyclic characteristics are generally based upon counting the number of peaks at various amplitude ranges on a record. This paper presents a computer approach which describes a time history by an amplitude envelope and a phase curve. Using Fast Fourier Transform Techniques, an earthquake time history is represented as a projection along the x-axis of a rotating vector-the length the vector is given by the amplitude spectra-and the angle between the vector and x-axis is given by the phase curve. Thus one cycle is completed when the vector makes a full rotation. Based upon Miner's cumulative damage concept, the computer code automatically combines the cycles of various amplitudes to obtain the equivalent number of cycles of a given amplitude. To illustrate the overall results, the cyclic characteristics of several real and synthetic earthquake time histories have been studied and are presented in the paper, with the conclusion that this procedure provides a physical interpretation of the cyclic characteristics of earthquakes. (Auth.)

  16. Immunization with Tc52 or its amino terminal domain adjuvanted with c-di-AMP induces Th17+Th1 specific immune responses and confers protection against Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Marina N Matos

    2017-02-01

    Full Text Available The development of new adjuvants enables fine modulation of the elicited immune responses. Ideally, the use of one or more adjuvants should result in the induction of a protective immune response against the specific pathogen. We have evaluated the immune response and protection against Trypanosoma cruzi infection in mice vaccinated with recombinant Tc52 or its N- and C-terminal domains (NTc52 and CTc52 adjuvanted either with the STING (Stimulator of Interferon Genes agonist cyclic di-AMP (c-di-AMP, a pegylated derivative of α-galactosylceramide (αGC-PEG, or oligodeoxynucleotides containing unmethylated CpG motifs (ODN-CpG. All groups immunized with the recombinant proteins plus adjuvant: Tc52+c-di-AMP, NTc52+c-di-AMP, CTc52+c-di-AMP, NTc52+c-di-AMP+αGC-PEG, NTc52+CpG, developed significantly higher anti-Tc52 IgG titers than controls. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 showed the highest Tc52-specific IgA titers in nasal lavages. All groups immunized with the recombinant proteins plus adjuvant developed a strong specific cellular immune response in splenocytes and lymph node cells with significant differences for groups immunized with c-di-AMP and Tc52, NTc52 or CTc52. These groups also showed high levels of Tc52-specific IL-17 and IFN-γ producing cells, while NTc52+CpG group only showed significant difference with control in IFN-γ producing cells. Groups immunized with c-di-AMP and Tc52, NTc52 or CTc52 developed predominantly a Th17 and Th1immune response, whereas for NTc52+CpG it was a dominant Th1 response. It was previously described that αGC-PEG inhibits Th17 differentiation by activating NKT cells. Thus, in this work we have also included a group immunized with both adjuvants (NTc52+c-di-AMP+αGC-PEG with the aim to modulate the Th17 response induced by c-di-AMP. This group showed a significant reduction in the number of Tc52-specific IL-17 producing splenocytes, as compared to the group NTc52+c-di-AMP, which has

  17. cAMP/PKA signalling reinforces the LATS–YAP pathway to fully suppress YAP in response to actin cytoskeletal changes

    Science.gov (United States)

    Kim, Minchul; Kim, Miju; Lee, Seunghee; Kuninaka, Shinji; Saya, Hideyuki; Lee, Ho; Lee, Sookyung; Lim, Dae-Sik

    2013-01-01

    Actin cytoskeletal damage induces inactivation of the oncoprotein YAP (Yes-associated protein). It is known that the serine/threonine kinase LATS (large tumour suppressor) inactivates YAP by phosphorylating its Ser127 and Ser381 residues. However, the events downstream of actin cytoskeletal changes that are involved in the regulation of the LATS–YAP pathway and the mechanism by which LATS differentially phosphorylates YAP on Ser127 and Ser381 in vivo have remained elusive. Here, we show that cyclic AMP (cAMP)-dependent protein kinase (PKA) phosphorylates LATS and thereby enhances its activity sufficiently to phosphorylate YAP on Ser381. We also found that PKA activity is involved in all contexts previously reported to trigger the LATS–YAP pathway, including actin cytoskeletal damage, G-protein-coupled receptor activation, and engagement of the Hippo pathway. Inhibition of PKA and overexpression of YAP cooperate to transform normal cells and amplify neural progenitor pools in developing chick embryos. We also implicate neurofibromin 2 as an AKAP (A-kinase-anchoring protein) scaffold protein that facilitates the function of the cAMP/PKA–LATS–YAP pathway. Our study thus incorporates PKA as novel component of the Hippo pathway. PMID:23644383

  18. Possible involvement of G-proteins and cAMP in the induction of progesterone hydroxylating enzyme system in the vascular wilt fungus Fusarium oxysporum.

    Science.gov (United States)

    Poli, Anna; Di Pietro, Antonio; Zigon, Dusan; Lenasi, Helena

    2009-02-01

    Fungi present the ability to hydroxylate steroids. In some filamentous fungi, progesterone induces an enzyme system which converts the compound into a less toxic hydroxylated product. We investigated the progesterone response in the vascular wilt pathogen Fusarium oxysporum, using mass spectrometry and high performance liquid chromatography (HPLC). Progesterone was mainly transformed into 15alpha-hydroxyprogesterone, which was found predominantly in the extracellular medium. The role of two conserved fungal signaling cascades in the induction of the progesterone-transforming enzyme system was studied, using knockout mutants lacking the mitogen-activated protein kinase Fmk1 or the heterotrimeric G-protein beta subunit Fgb1 functioning upstream of the cyclic adenosine monophosphate (cAMP) pathway. No steroid hydroxylation was induced in the Deltafgb1 strain, suggesting a role for the G-protein beta subunit in progesterone signaling. Exogenous cAMP restored the induction of progesterone-transforming activity in the Deltafgb1 strain, suggesting that steroid signaling in F. oxysporum is mediated by the cAMP-PKA pathway.

  19. Effects of prostaglandin E2 and cAMP elevating drugs on GM-CSF release by cultured human airway smooth muscle cells. Relevance to asthma therapy.

    Science.gov (United States)

    Lazzeri, N; Belvisi, M G; Patel, H J; Yacoub, M H; Chung, K F; Mitchell, J A

    2001-01-01

    Human airway smooth muscle (HASM) cells release granulocyte macrophage-colony stimulating factor (GM-CSF) and express cyclooxygenase (COX)-2 (resulting in the release of prostaglandin [PG] E2) after stimulation with cytokines. Because COX-2 activity can regulate a number of inflammatory processes, we have assessed its effects, as well as those of agents that modulate cyclic adenosine monophosphate (cAMP), on GM-CSF release by HASM cells. Cells stimulated with a combination of proinflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha each at 10 ng/ml) for 24 h released significant amounts of PGE2 (measured by radioimmunoassay) and GM-CSF (measured by enzyme-linked immunosorbent assay). Indomethacin and other COX-1/COX-2 inhibitors caused concentration-dependent inhibitions of PGE2 concomitantly with increases in GM-CSF formation. Addition of exogenous PGE2 or the beta2-agonist fenoterol, which increase cAMP, to cytokine-treated HASM cells had no effect on GM-CSF release unless COX activity was first blocked with indomethacin. The type 4 phosphodiesterase inhibitors rolipram and SB 207499 both caused concentration-dependent reductions in GM-CSF production. Thus, when HASM cells are activated with cytokines they release PGE2, which acts as a "braking mechanism" to limit the coproduction of GM-CSF. Moreover, agents that elevate cAMP also reduce GM-CSF formation by these cells.

  20. 7 CFR 772.10 - Transfer and assumption-AMP loans.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 7 2010-01-01 2010-01-01 false Transfer and assumption-AMP loans. 772.10 Section 772..., DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS SERVICING MINOR PROGRAM LOANS § 772.10 Transfer and assumption—AMP loans. (a) Eligibility. The Agency may approve transfers and assumptions of AMP loans when: (1) The...

  1. Occurrence and detection of AmpC β-lactamases among ...

    African Journals Online (AJOL)

    Aim of the study: We aimed to evaluate the presence of AmpC β-lactamase among Enterobacteriaceae isolates separated from patients with nosocomial infections, and to detect the genetic basis for AmpC production in these strains. Subjects and methods: 50 AmpC β-lactamase Enterobacteriaceae were analyzed for the ...

  2. Bacterial subversion of cAMP signalling inhibits cathelicidin expression, which is required for innate resistance to Mycobacterium tuberculosis

    Science.gov (United States)

    Gupta, Shashank; Winglee, Kathryn; Gallo, Richard; Bishai, William R

    2017-01-01

    Antimicrobial peptides such as cathelicidins are an important component of innate immune defence against inhaled microorganisms and have demonstrated antimicrobial activity against Mycobacterium tuberculosis with in vitro models. Despite this, little is known about the regulation and expression of cathelicidin during tuberculosis in vivo. We sought to determine whether the cathelicidin-related antimicrobial peptide (Cramp) gene, the murine functional homologue of the human cathelicidin gene (CAMP or LL-37), is required for regulating protective immunity during M. tuberculosis infection in vivo. We used Cramp−/− mice in a validated model of pulmonary tuberculosis and conducted cell-based assays with macrophages from these mice. We evaluated the in vivo susceptibility of Cramp−/− mice to infection and further dissected various pro-inflammatory immune responses against M. tuberculosis. We observed increased susceptibility of Cramp−/− mice to M. tuberculosis compared to wild type mice. Macrophages from Cramp−/− mice were unable to control M. tuberculosis growth in an in vitro infection model, were deficient in intracellular calcium influx and were defective in stimulating T-cells. Additionally, CD4 and CD8 T-cells from Cramp−/− mice produced less IFNβ upon stimulation. Furthermore, bacterial-derived cyclic-AMP modulated cathelicidin expression in macrophages. Our results demonstrate that cathelicidin is required for innate resistance to M. tuberculosis in a relevant animal model and is a key mediator in regulating the levels of pro-inflammatory cytokines by calcium and cyclic nucleotides. PMID:28097645

  3. Cyclic voltammetry and reduction mechanistic studies of ...

    African Journals Online (AJOL)

    styrylpyrylium perchlorates have been evaluated using cyclic voltammetry, in comparison to their non-methylated derivatives values. The reduction peak of all studied compounds remained chemically irreversible. The presence of the ...

  4. A cyclically actuated electrolytic drug delivery device

    KAUST Repository

    Yi, Ying; Buttner, Ulrich; Foulds, Ian G.

    2015-01-01

    This work, focusing on an implantable drug delivery system, presents the first prototype electrolytic pump that combines a catalytic reformer and a cyclically actuated mode. These features improve the release performance and extend the lifetime

  5. Introduction of a cyclic-fermentation method

    Energy Technology Data Exchange (ETDEWEB)

    Makarova, C P

    1958-01-01

    Equipment is described, consisting of 8 kettles, which permits a cyclic fermentation process and continuous ethanol production; 100% yields of ethanol are obtained, based on the starch content in grain.

  6. Results on Cyclic Signal Processing Systems

    National Research Council Canada - National Science Library

    Vaidyanathan, P

    1998-01-01

    .... A number of related problems such as the paraunitary interpolation problem and the cyclic paraunitary factorizability problem can be understood in a unified way by using the realization matrix...

  7. Cyclical Variability of Prominences, CMEs and Flares

    Indian Academy of Sciences (India)

    tribpo

    For many years, qualitative studies were made about the cyclical ... plan to review the more recent research concerning all these topics. Key words. ... are distributed in three narrow zones, which show different types of time-latitude behaviour.

  8. Anodic selective functionalization of cyclic amine derivatives

    OpenAIRE

    Onomura, Osamu

    2012-01-01

    Anodic reactions are desirable methods from the viewpoint of Green Chemistry, since no toxic oxidants are necessary for the oxidation of organic molecules. This review introduces usefulness of anodic oxidation and successive reaction for selective functionalization of cyclic amine derivatives.

  9. Macromolecular Networks Containing Fluorinated Cyclic Moieties

    Science.gov (United States)

    2015-12-12

    Briefing Charts 3. DATES COVERED (From - To) 17 Nov 2015 – 12 Dec 2015 4. TITLE AND SUBTITLE Macromolecular Networks Containing Fluorinated Cyclic... FLUORINATED CYCLIC MOIETIES 12 December 2015 Andrew J. Guenthner,1 Scott T. Iacono,2 Cynthia A. Corley,2 Christopher M. Sahagun,3 Kevin R. Lamison,4...Reinforcements Good Flame, Smoke, & Toxicity Characteristics Low Water Uptake with Near Zero Coefficient of Hygroscopic Expansion ∆ DISTRIBUTION A

  10. The Cyclicality of New Product Introductions

    OpenAIRE

    Kostas Axarloglou

    2003-01-01

    This study analyzes empirically the cyclical nature of the timing of new product introductions in U.S. manufacturing. New product introductions vary more in nonseasonal frequencies than in seasonal frequencies. However, the seasons alone account for only a small part of their total variability with demand factors being much more important. Demand fluctuations account for 35%80% and 17%43%, respectively, of the seasonal and cyclical variability of new product introductions in various industrie...

  11. The calculation of dissipated work, elastoplastic cyclic stress and cyclic strain in a structure

    International Nuclear Information System (INIS)

    Wang Xucheng; Xie Yihuan.

    1986-01-01

    With the development of the reactor technique, there is being an increasing interest in the calculation of elastoplastic response of a structure to its complex loading. This paper introduces a constitutive relation of a material for discribing unloading property, and uses it in an analysis of a real structure under a cyclic loading. The results, which include cyclic stress, cyclic strain and dissipated work, are meaningful in the researches of the structure behavior under complex loading and of the structural safety

  12. Activation of PKA in cell requires higher concentration of cAMP than in vitro: implications for compartmentalization of cAMP signalling.

    Science.gov (United States)

    Koschinski, Andreas; Zaccolo, Manuela

    2017-10-26

    cAMP is a ubiquitous second messenger responsible for the cellular effects of multiple hormones and neurotransmitters via activation of its main effector, protein kinase A (PKA). Multiple studies have shown that the basal concentration of cAMP in several cell types is about 1 μM. This value is well above the reported concentration of cAMP required to half-maximally activate PKA, which measures in the 100-300 nM range. Several hypotheses have been suggested to explain this apparent discrepancy including inaccurate measurements of intracellular free cAMP, inaccurate measurement of the apparent activation constant of PKA or shielding of PKA from bulk cytosolic cAMP via localization of the enzyme to microdomains with lower basal cAMP concentration. However, direct experimental evidence in support of any of these models is limited and a firm conclusion is missing. In this study we use multiple FRET-based reporters for the detection of cAMP and PKA activity in intact cells and we establish that the sensitivity of PKA to cAMP is almost twenty times lower when measured in cell than when measured in vitro. Our findings have important implications for the understanding of compartmentalized cAMP signalling.

  13. Inhibition of Cyclic Adenosine Monophosphate-Specific Phosphodiesterase by Various Food Plant-Derived Phytotherapeutic Agents.

    Science.gov (United States)

    Röhrig, Teresa; Pacjuk, Olga; Hernández-Huguet, Silvia; Körner, Johanna; Scherer, Katharina; Richling, Elke

    2017-11-04

    Background: Phosphodiesterases (PDEs) play a major role in the regulation of cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-mediated pathways. Their inhibitors exhibit anti-inflammatory, vasodilatory and antithrombotic effects. Therefore, consumption of foods with PDE-inhibiting potential may possess beneficial influence on the risk of cardiovascular diseases. Methods: Four plant extracts ( Arbutus unedo , Camellia sinensis , Cynara scolymus , Zingiber officinale ) with promising ingredient profiles and physiological effects were tested for their ability to inhibit cAMP-specific PDE in vitro in a radioactive assay. Results: Strawberry tree fruit ( Arbutus unedo ) and tea ( Camellia sinensis ) extracts did not inhibit PDE markedly. Alternatively, artichoke ( Cynara scolymus ) extract had a significant inhibitory influence on PDE activity (IC 50 = 0.9 ± 0.1 mg/mL) as well as its flavone luteolin (IC 50 = 41 ± 10 μM) and 3,4-dicaffeoylquinic acid (IC 50 > 1.0 mM). Additionally, the ginger ( Zingiber officinale ) extract and one of its constituents, [6]-gingerol, significantly inhibited PDE (IC 50 = 1.7 ± 0.2 mg/mL and IC 50 > 1.7 mM, respectively). Crude fractionation of ginger extract showed that substances responsible for PDE inhibition were in the lipoid fraction (IC 50 = 455 ± 19 μg/mL). Conclusions: A PDE-inhibitory effect was shown for artichoke and ginger extract. Whether PDE inhibition in vivo can be achieved through ingestion of artichoke or ginger extracts leading to physiological effects concerning cardiovascular health should be addressed in future research.

  14. Inhibition of Cyclic Adenosine Monophosphate-Specific Phosphodiesterase by Various Food Plant-Derived Phytotherapeutic Agents

    Directory of Open Access Journals (Sweden)

    Teresa Röhrig

    2017-11-01

    Full Text Available Background: Phosphodiesterases (PDEs play a major role in the regulation of cyclic adenosine monophosphate (cAMP- and cyclic guanosine monophosphate (cGMP-mediated pathways. Their inhibitors exhibit anti-inflammatory, vasodilatory and antithrombotic effects. Therefore, consumption of foods with PDE-inhibiting potential may possess beneficial influence on the risk of cardiovascular diseases. Methods: Four plant extracts (Arbutus unedo, Camellia sinensis, Cynara scolymus, Zingiber officinale with promising ingredient profiles and physiological effects were tested for their ability to inhibit cAMP-specific PDE in vitro in a radioactive assay. Results: Strawberry tree fruit (Arbutus unedo and tea (Camellia sinensis extracts did not inhibit PDE markedly. Alternatively, artichoke (Cynara scolymus extract had a significant inhibitory influence on PDE activity (IC50 = 0.9 ± 0.1 mg/mL as well as its flavone luteolin (IC50 = 41 ± 10 μM and 3,4-dicaffeoylquinic acid (IC50 > 1.0 mM. Additionally, the ginger (Zingiber officinale extract and one of its constituents, [6]-gingerol, significantly inhibited PDE (IC50 = 1.7 ± 0.2 mg/mL and IC50 > 1.7 mM, respectively. Crude fractionation of ginger extract showed that substances responsible for PDE inhibition were in the lipoid fraction (IC50 = 455 ± 19 μg/mL. Conclusions: A PDE-inhibitory effect was shown for artichoke and ginger extract. Whether PDE inhibition in vivo can be achieved through ingestion of artichoke or ginger extracts leading to physiological effects concerning cardiovascular health should be addressed in future research.

  15. cAMP/PKA-CREB-BDNF signaling pathway in hippocampus mediates cyclooxygenase 2-induced learning/memory deficits of rats subjected to chronic unpredictable mild stress.

    Science.gov (United States)

    Luo, Ying; Kuang, Shengnan; Li, Huan; Ran, Dongzhi; Yang, Junqing

    2017-05-30

    To investigate the mechanism of cyclooxygenase 2 (COX2) in learning and memory impairments in rats subjected to chronic unpredictable mild stress (CUMS), meloxicam was used intragastrically to inhibit the activity of cyclooxygenase 2. Moreover, cyclooxygenase 2 over-expressing or RNA interfere lentivirus was injected intraventricularly to increase or decrease the enzyme's expression, respectively. The body weights and sucrose consumption were used to analyze depressive behaviors, while the Morris water maze and step-down-type passive avoidance tests were carried out to evaluate the learning-memory functions. The levels of inflammatory cytokines were measured to estimate inflammation and the contents of cyclic adenosine monophosphate (cAMP) were used to measure the levels of the second messenger. Changes in cyclooxygenase 2 mRNA levels were analyzed using reverse transcription polymerase chain reaction. Moreover, the expression of cyclooxygenase 2, brain-derived neurotrophic factor (BDNF), prostaglandins receptor 3 (EP3), protein kinase A (PKA), cAMP response element binding protein (CREB), and phosphorylated CREB were estimated using immunohistochemical staining or western blotting. The results showed that CUMS led to significant depressive-like behaviors and learning and memory dysfunctions. Also, the cAMP levels decreased significantly, while levels of inflammatory cytokines and prostaglandins E2 increased significantly. The expressions of PKA, BDNF, phosphorylated CREB/CREB declined and cyclooxygenase 2 was increased. Meloxicam and cyclooxygenase 2 RNA interfere lentivirus reversed the changes caused by CUMS while cyclooxygenase 2-overexpressing lentivirus worsened these abnormalities. The findings also showed that CUMS increased cyclooxygenase 2 expression, which can cause learning and memory impairments, mainly through activating the hippocampal neuronal cAMP/PKA-CREB-BDNF signaling pathways.

  16. Decreased hepatic response to glucagon, adrenergic agonists, and cAMP in glycogenolysis, gluconeogenesis, and glycolysis in tumor-bearing rats.

    Science.gov (United States)

    Biazi, Giuliana R; Frasson, Isabele G; Miksza, Daniele R; de Morais, Hely; de Fatima Silva, Flaviane; Bertolini, Gisele L; de Souza, Helenir M

    2018-05-15

    The response to glucagon and adrenaline in cancer cachexia is poorly known. The aim of this study was to investigate the response to glucagon, adrenergic agonists (α and β) and cyclic adenosine monophosphate (cAMP) on glycogenolysis, gluconeogenesis, and glycolysis in liver perfusion of Walker-256 tumor-bearing rats with advanced cachexia. Liver ATP content was also investigated. Rats without tumor (healthy) were used as controls. Agonists α (phenylephrine) and β (isoproterenol) adrenergic, instead of adrenaline, and cAMP, the second messenger of glucagon and isoproterenol, were used in an attempt to identify mechanisms involved in the responses. Glucagon (1 nM) stimulated glycogenolysis and gluconeogenesis and inhibited glycolysis in the liver of healthy and tumor-bearing rats, but their effects were lower in tumor-bearing rats. Isoproterenol (20 µM) stimulated glycogenolysis, gluconeogenesis, and glycolysis in healthy rats and had virtually no effect in tumor-bearing rats. cAMP (9 µM) also stimulated glycogenolysis and gluconeogenesis and inhibited glycolysis in healthy rats but had practically no effect in tumor-bearing rats. Phenylephrine (2 µM) stimulated glycogenolysis and gluconeogenesis and inhibited glycolysis and these effects were also lower in tumor-bearing rats than in healthy. Liver ATP content was lower in tumor-bearing rats. In conclusion, tumor-bearing rats with advanced cachexia showed a decreased hepatic response to glucagon, adrenergic agonists (α and β), and cAMP in glycogenolysis, gluconeogenesis, and glycolysis, which may be due to a reduced rate of regulatory enzyme phosphorylation caused by the low ATP levels in the liver. © 2018 Wiley Periodicals, Inc.

  17. Effect of radioprotectant WR 2721 on cyclic nucleotides, prostaglandins, and lysosomes

    International Nuclear Information System (INIS)

    Trocha, P.J.; Catravas, G.N.

    1983-01-01

    Within 1 hr after ip injection of the radioprotectant WR 2721 into rats, splenic cGMP levels dropped and remained suppressed for 6 hr before returning to normal. However, if rats were exposed to ionizing radiation 30-40 min after WR 2721 treatment, they had higher cGMP levels at 3 hr postirradiation than the nonirradiUted, drug-treated controls, but the cGMP content was still found to be lower than that of the irradiated nondrug-treated controls. Radiation exposure of animals pretreated with WR 2721 also resulted in higher liver and spleen levels of cAMP and additional elevations in spleen prostaglandin content, compared with irradiated controls at 3-6 hr after radiation treatment. The secondary fluctuations of lysosomal enzyme activities, prostaglandin content, and cyclic nucleotide levels were also altered in irradiated rats pretreated with WR 2721 when compared with irradiated controls. Liver and spleen lysosomal β-glucuronidase activities, spleen cAMP and cGMP levels, and spleen prostaglandin concentrations were closer to physiological levels at 3 days postirradiation in rats given WR 2721 before the radiation treatment