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Sample records for guanylate cyclase sgc

  1. A new small molecule inhibitor of soluble guanylate cyclase

    PubMed Central

    Mota, Filipa; Gane, Paul; Hampden-Smith, Kathryn; Allerston, Charles K.; Garthwaite, John; Selwood, David L.

    2015-01-01

    Soluble guanylate cyclase (sGC) is a haem containing enzyme that regulates cardiovascular homeostasis and multiple mechanisms in the central and peripheral nervous system. Commonly used inhibitors of sGC activity act through oxidation of the haem moiety, however they also bind haemoglobin and this limits their bioavailability for in vivo studies. We have discovered a new class of small molecule inhibitors of sGC and have characterised a compound designated D12 (compound 10) which binds to the catalytic domain of the enzyme with a KD of 11 μM in a SPR assay. PMID:26264842

  2. Nitric oxide-independent vasodilator rescues heme-oxidized soluble guanylate cyclase from proteasomal degradation.

    PubMed

    Meurer, Sabine; Pioch, Sylke; Pabst, Tatjana; Opitz, Nils; Schmidt, Peter M; Beckhaus, Tobias; Wagner, Kristina; Matt, Simone; Gegenbauer, Kristina; Geschka, Sandra; Karas, Michael; Stasch, Johannes-Peter; Schmidt, Harald H H W; Müller-Esterl, Werner

    2009-07-01

    Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the alphabeta heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both alpha and beta subunits. Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC. PMID:19478201

  3. Tetrahydrobiopterin protects soluble guanylate cyclase against oxidative inactivation.

    PubMed

    Schmidt, Kurt; Neubauer, Andrea; Kolesnik, Bernd; Stasch, Johannes-Peter; Werner, Ernst R; Gorren, Antonius C F; Mayer, Bernd

    2012-09-01

    Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress. PMID:22648973

  4. Polymorphism in purified guanylate cyclase from vertebrate rod photoreceptors.

    PubMed Central

    Hayashi, F; Yamazaki, A

    1991-01-01

    Guanylate cyclase from rod photoreceptors of amphibian (toad, Bufo marinus, and frog, Rana catesbeiana) and bovine retinas was solubilized and purified by a single chromatography step on a GTP-agarose column. Silver staining of purified amphibian enzymes in SDS/polyacrylamide gels disclosed a doublet band (110 and 115 kDa), while the bovine enzyme appeared as a singlet band (110 kDa). The identification of these guanylate cyclases was confirmed using three chromatography systems with the purified enzymes. Specific binding to Con A-Sepharose suggested that rod guanylate cyclase is a glycoprotein. Two-dimensional gel electrophoresis of purified toad, frog, and bovine enzymes resolved two, three, and five variants, respectively, that differed in isoelectric point. Two variants of toad guanylate cyclase showed differences in various characterizations. These data suggest multiple mechanisms for regulation of guanylate cyclase activity in vertebrate rod photoreceptors. Images PMID:1675787

  5. Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase.

    PubMed Central

    Roelofs, J; Snippe, H; Kleineidam, R G; Van Haastert, P J

    2001-01-01

    The core of adenylate and guanylate cyclases is formed by an intramolecular or intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a heterodimer and are activated by G-proteins. In contrast, membrane-bound guanylate cyclases have only one transmembrane spanning region and one cyclase domain, and are activated by extracellular ligands to form a homodimer. In the cellular slime mould, Dictyostelium discoideum, membrane-bound guanylate cyclase activity is induced after cAMP stimulation; a G-protein-coupled cAMP receptor and G-proteins are essential for this activation. We have cloned a Dictyostelium gene, DdGCA, encoding a protein with 12 transmembrane spanning regions and two cyclase domains. Sequence alignment demonstrates that the two cyclase domains are transposed, relative to these domains in adenylate cyclases. DdGCA expressed in Dictyostelium exhibits high guanylate cyclase activity and no detectable adenylate cyclase activity. Deletion of the gene indicates that DdGCA is not essential for chemotaxis or osmo-regulation. The knock-out strain still exhibits substantial guanylate cyclase activity, demonstrating that Dictyostelium contains at least one other guanylate cyclase. PMID:11237875

  6. Regulation of soluble guanylate cyclase by matricellular thrombospondins: implications for blood flow.

    PubMed

    Rogers, Natasha M; Seeger, Franziska; Garcin, Elsa D; Roberts, David D; Isenberg, Jeffrey S

    2014-01-01

    Nitric oxide (NO) maintains cardiovascular health by activating soluble guanylate cyclase (sGC) to increase cellular cGMP levels. Cardiovascular disease is characterized by decreased NO-sGC-cGMP signaling. Pharmacological activators and stimulators of sGC are being actively pursued as therapies for acute heart failure and pulmonary hypertension. Here we review molecular mechanisms that modulate sGC activity while emphasizing a novel biochemical pathway in which binding of the matricellular protein thrombospondin-1 (TSP1) to the cell surface receptor CD47 causes inhibition of sGC. We discuss the therapeutic implications of this pathway for blood flow, tissue perfusion, and cell survival under physiologic and disease conditions.

  7. Soluble Guanylate Cyclase: A new therapeutic target for pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension

    PubMed Central

    Das Gupta, Asish; Bowman, Lindsay; D’Arsigny, Christine L.; Archer, Stephen L.

    2014-01-01

    Nitric oxide (NO) activates soluble guanylate cyclase (sGC) by binding its prosthetic heme group, thereby catalyzing cyclic guanosine monophosphate (cGMP) synthesis. cGMP causes vasodilation and may inhibit smooth muscle cell proliferation and platelet aggregation. The NO-sGC-cGMP pathway is disordered in pulmonary arterial hypertension (PAH), a syndrome in which pulmonary vascular obstruction, inflammation, thrombosis, and constriction ultimately lead to death from right heart failure. Expression of sGC is increased in PAH but its function is reduced by decreased NO bioavailability, sGC oxidation and the related loss of sGC’s heme group. Two classes of sGC modulators offer promise in PAH. sGC stimulators (e.g. riociguat) require heme-containing sGC to catalyze cGMP production, whereas sGC activators (e.g. cinaciguat) activate heme-free sGC. Riociguat is approved for PAH and yields functional and hemodynamic benefits similar to other therapies. Its main serious adverse effect is dose-dependent hypotension Riociguat is also approved for inoperable chronic thromboembolic pulmonary hypertension. PMID:25670386

  8. Modulation of soluble guanylate cyclase for the treatment of erectile dysfunction.

    PubMed

    Lasker, George F; Pankey, Edward A; Kadowitz, Philip J

    2013-07-01

    Nitric oxide (NO) is the principal mediator of penile erection, and PDE-5 inhibitors are the first-line agents used to treat erectile dysfunction (ED). When NO formation or bioavailability is decreased by oxidative stress and PDE-5 inhibitors are no longer effective, a new class of agents called soluble guanylate cyclase (sGC) stimulators like BAY 41-8543 will induce erection. sGC stimulators bind to the normally reduced, NO-sensitive form of sGC to increase cGMP formation and promote erection. The sGC stimulators produce normal erectile responses when NO formation is inhibited and the nerves innervating the corpora cavernosa are damaged. However, with severe oxidative stress, the heme iron on sGC can be oxidized, rendering the enzyme unresponsive to NO or sGC stimulators. In this pathophysiological situation, another newly developed class of agents called sGC activators can increase the catalytic activity of the oxidized enzyme, increase cGMP formation, and promote erection. The use of newer agents that stimulate or activate sGC to promote erection and treat ED is discussed in this brief review article. PMID:23817801

  9. Modulation of soluble guanylate cyclase for the treatment of erectile dysfunction.

    PubMed

    Lasker, George F; Pankey, Edward A; Kadowitz, Philip J

    2013-07-01

    Nitric oxide (NO) is the principal mediator of penile erection, and PDE-5 inhibitors are the first-line agents used to treat erectile dysfunction (ED). When NO formation or bioavailability is decreased by oxidative stress and PDE-5 inhibitors are no longer effective, a new class of agents called soluble guanylate cyclase (sGC) stimulators like BAY 41-8543 will induce erection. sGC stimulators bind to the normally reduced, NO-sensitive form of sGC to increase cGMP formation and promote erection. The sGC stimulators produce normal erectile responses when NO formation is inhibited and the nerves innervating the corpora cavernosa are damaged. However, with severe oxidative stress, the heme iron on sGC can be oxidized, rendering the enzyme unresponsive to NO or sGC stimulators. In this pathophysiological situation, another newly developed class of agents called sGC activators can increase the catalytic activity of the oxidized enzyme, increase cGMP formation, and promote erection. The use of newer agents that stimulate or activate sGC to promote erection and treat ED is discussed in this brief review article.

  10. Nitric oxide decreases stability of mRNAs encoding soluble guanylate cyclase subunits in rat pulmonary artery smooth muscle cells.

    PubMed Central

    Filippov, G; Bloch, D B; Bloch, K D

    1997-01-01

    Nitric oxide stimulates soluble guanylate cyclase (sGC) to convert GTP to the intracellular second messenger cGMP. In rat pulmonary artery smooth muscle cells, sGC is an obligate heterodimer composed of alpha1 and beta1 subunits. We investigated the effect of NO donor compounds on sGC subunit gene expression in rat pulmonary artery smooth muscle cells. Sodium nitroprusside and S-nitroso-glutathione decreased sGC subunit mRNA and protein levels, as well as sGC enzyme activity. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an sGC inhibitor, blocked the effect of sodium nitroprusside on sGC subunit gene expression, whereas 8-bromo cGMP decreased subunit mRNA levels, demonstrating that NO-mediated decrease in sGC subunit mRNA levels is cGMP-dependent. sGC subunit mRNA levels decreased more rapidly in rat pulmonary artery smooth muscle cells exposed to NO than in cells exposed to actinomycin D, suggesting that NO decreases sGC subunit mRNA stability. Actinomycin D and cycloheximide blocked the ability of NO to decrease sGC subunit mRNA levels. These results demonstrate that NO decreases sGC subunit mRNA stability via a transcription- and translation-dependent mechanism. PMID:9259594

  11. Crystal Structures of the Catalytic Domain of Human Soluble Guanylate Cyclase

    PubMed Central

    Allerston, Charles K.; von Delft, Frank; Gileadi, Opher

    2013-01-01

    Soluble guanylate cyclase (sGC) catalyses the synthesis of cyclic GMP in response to nitric oxide. The enzyme is a heterodimer of homologous α and β subunits, each of which is composed of multiple domains. We present here crystal structures of a heterodimer of the catalytic domains of the α and β subunits, as well as an inactive homodimer of β subunits. This first structure of a metazoan, heteromeric cyclase provides several observations. First, the structures resemble known structures of adenylate cyclases and other guanylate cyclases in overall fold and in the arrangement of conserved active-site residues, which are contributed by both subunits at the interface. Second, the subunit interaction surface is promiscuous, allowing both homodimeric and heteromeric association; the preference of the full-length enzyme for heterodimer formation must derive from the combined contribution of other interaction interfaces. Third, the heterodimeric structure is in an inactive conformation, but can be superposed onto an active conformation of adenylate cyclase by a structural transition involving a 26° rigid-body rotation of the α subunit. In the modelled active conformation, most active site residues in the subunit interface are precisely aligned with those of adenylate cyclase. Finally, the modelled active conformation also reveals a cavity related to the active site by pseudo-symmetry. The pseudosymmetric site lacks key active site residues, but may bind allosteric regulators in a manner analogous to the binding of forskolin to adenylate cyclase. This indicates the possibility of developing a new class of small-molecule modulators of guanylate cyclase activity targeting the catalytic domain. PMID:23505436

  12. Soluble guanylate cyclase as an alternative target for bronchodilator therapy in asthma

    PubMed Central

    Koziol-White, Cynthia J.; Asosingh, Kewal; Cheng, Georgina; Ruple, Lisa; Groneberg, Dieter; Friebe, Andreas; Comhair, Suzy A. A.; Stasch, Johannes-Peter; Panettieri, Reynold A.; Aronica, Mark A.; Erzurum, Serpil C.; Stuehr, Dennis J.

    2016-01-01

    Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41–2272 and BAY 60–2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41–2272 and BAY 60–2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss. PMID:27071111

  13. Aging does not affect soluble guanylate cyclase redox state in mouse aortas.

    PubMed

    Shimosato, Takashi; Tawa, Masashi; Iwasaki, Hirotaka; Imamura, Takeshi; Okamura, Tomio

    2016-05-01

    Aging is associated with endothelial dysfunction, defined as a reduction in nitric oxide (NO) bioavailability. Although the redox state of the NO acceptor soluble guanylate cyclase (sGC) is another determinant factor for its bioavailability and is disturbed by reactive oxygen species (ROS) known to be increased with age, it is unclear whether aging actually has an impact on vascular sGC redox equilibrium. Therefore, this study investigated this issue using two different types of compounds, the sGC stimulator BAY 41-2272 and the sGC activator BAY 60-2770. Plasma thiobarbituric acid-reactive substances (TBARS) levels were markedly higher in aged (19-20 months old) mice than in young (2-3 months old) mice, whereas superoxide levels in endothelium-denuded aortas were not different between the groups. The relaxant response of endothelium-denuded aortas to either BAY 41-2272 or BAY 60-2770 was identical in aged and young mice. In addition, the vascular cGMP production stimulated with BAY 41-2272 or BAY 60-2770 in aged mice was the same level as that in young mice. These findings suggest that aging accompanied by an increase in systemic oxidative stress does not affect vascular smooth muscle ROS generation and sGC redox equilibrium. Unless ROS are increased in vascular smooth muscle, the sGC redox equilibrium might remain unchanged.

  14. Characterization and expression of soluble guanylate cyclase in skins and melanocytes of sheep.

    PubMed

    Yang, Shanshan; Zhang, Junzhen; Ji, Kaiyuan; Jiao, Dingxing; Fan, Ruiwen; Dong, Changsheng

    2016-04-01

    The study reported the characterization of soluble guanylate cyclase (sGC) with the size of CDS of 1860bp, encoding a protein of 620 amino acids and containing several conserved functional domains including HNOB, HNOBA, and CHD. Quantitative real time PCR analysis of sGC showed that the expression of sGC mRNA is higher (∼5 fold) in white sheep skin relative to black sheep skin with significant difference (P<0.01). Using a rabbit polyclonal anti-sGC antibody, an immune reactive band corresponding to sheep sGC protein was detected in the skin samples by Western blotting analysis, and the expression of sGC protein was significantly higher in white sheep skin compared to black sheep skin (P<0.01). Immunohistochemical analysis revealed that sGC protein was localized in cytoplasm and intercellular substance of upper hair papilla in hair follicles of white sheep skin, but the protein was localized in cytoplasm and intercellular substance of lower hair bulb and outer root sheath cells in hair follicles of black sheep skin. The immunocytochemical analysis revealed that sGC was expressed in melanocytes in vitro of sheep skin. Over expression of sGC in melanocytes resulted in decreased expression of key melanogenic genes including microphthalmia transcription factor (MITF), tyrosinase (TYR), tyrosinase related protein 1(TYRTP1), and tyrosinase related protein 2(TYRP2) both at mRNA and protein level. Moreover, the melanocytes was capable of producing cGMP and cAMP. The observed differential expression and localization of sGC in sheep skins and melanocytes and the capability of producing cGMP and cAMP, which suggested a potential role for this gene in hair color regulation. PMID:26805580

  15. Cardiovascular and pharmacological implications of haem-deficient NO-unresponsive soluble guanylate cyclase knock-in mice.

    PubMed

    Thoonen, Robrecht; Cauwels, Anje; Decaluwe, Kelly; Geschka, Sandra; Tainsh, Robert E; Delanghe, Joris; Hochepied, Tino; De Cauwer, Lode; Rogge, Elke; Voet, Sofie; Sips, Patrick; Karas, Richard H; Bloch, Kenneth D; Vuylsteke, Marnik; Stasch, Johannes-Peter; Van de Voorde, Johan; Buys, Emmanuel S; Brouckaert, Peter

    2015-01-01

    Oxidative stress, a central mediator of cardiovascular disease, results in loss of the prosthetic haem group of soluble guanylate cyclase (sGC), preventing its activation by nitric oxide (NO). Here we introduce Apo-sGC mice expressing haem-free sGC. Apo-sGC mice are viable and develop hypertension. The haemodynamic effects of NO are abolished, but those of the sGC activator cinaciguat are enhanced in apo-sGC mice, suggesting that the effects of NO on smooth muscle relaxation, blood pressure regulation and inhibition of platelet aggregation require sGC activation by NO. Tumour necrosis factor (TNF)-induced hypotension and mortality are preserved in apo-sGC mice, indicating that pathways other than sGC signalling mediate the cardiovascular collapse in shock. Apo-sGC mice allow for differentiation between sGC-dependent and -independent NO effects and between haem-dependent and -independent sGC effects. Apo-sGC mice represent a unique experimental platform to study the in vivo consequences of sGC oxidation and the therapeutic potential of sGC activators. PMID:26442659

  16. Cardiovascular and pharmacological implications of haem-deficient NO-unresponsive soluble guanylate cyclase knock-in mice

    PubMed Central

    Thoonen, Robrecht; Cauwels, Anje; Decaluwe, Kelly; Geschka, Sandra; Tainsh, Robert E.; Delanghe, Joris; Hochepied, Tino; De Cauwer, Lode; Rogge, Elke; Voet, Sofie; Sips, Patrick; Karas, Richard H.; Bloch, Kenneth D.; Vuylsteke, Marnik; Stasch, Johannes-Peter; Van de Voorde, Johan; Buys, Emmanuel S.; Brouckaert, Peter

    2015-01-01

    Oxidative stress, a central mediator of cardiovascular disease, results in loss of the prosthetic haem group of soluble guanylate cyclase (sGC), preventing its activation by nitric oxide (NO). Here we introduce Apo-sGC mice expressing haem-free sGC. Apo-sGC mice are viable and develop hypertension. The haemodynamic effects of NO are abolished, but those of the sGC activator cinaciguat are enhanced in apo-sGC mice, suggesting that the effects of NO on smooth muscle relaxation, blood pressure regulation and inhibition of platelet aggregation require sGC activation by NO. Tumour necrosis factor (TNF)-induced hypotension and mortality are preserved in apo-sGC mice, indicating that pathways other than sGC signalling mediate the cardiovascular collapse in shock. Apo-sGC mice allow for differentiation between sGC-dependent and -independent NO effects and between haem-dependent and -independent sGC effects. Apo-sGC mice represent a unique experimental platform to study the in vivo consequences of sGC oxidation and the therapeutic potential of sGC activators. PMID:26442659

  17. Soluble guanylate cyclase and neuronal nitric oxide synthase colocalize in rat nucleus tractus solitarii.

    PubMed

    Lin, L H; Talman, W T

    2005-03-01

    Nitric oxide has been implicated in transmission of cardiovascular signals in the nucleus tractus solitarii (NTS). Pharmacological studies suggest that activation of neurons by nitric oxide in the NTS may involve soluble guanylate cyclase (sGC). However, anatomical data supporting this suggestion have not been available. In this study, we tested the hypothesis that neurons and fibers containing neuronal nitric oxide synthase (nNOS) lie in close proximity to those containing sGC and the two enzymes colocalize in some neurons and fibers in the NTS. We perfused six rats and obtained brain stem sections for double immunofluorescent staining utilizing antibodies selective for sGC and for nNOS combined with confocal microscopy. The distribution and staining intensity of nNOS-immunoreactivity (IR) was similar to our earlier reports. IR of sGC was present in cell bodies, proximal dendrites and fibers of many brain stem regions. Strong sGC-IR was noted in the hypoglossal, dorsal motor nucleus of vagus and gracilis nuclei. The NTS exhibited moderate sGC-IR. Superimposed images showed that many NTS neurons contained both nNOS-IR and sGC-IR. The percentage of sGC-IR positive cells that were also nNOS-IR positive differed among NTS subnuclei. Similarly, the percentage of nNOS-IR positive cells that were also sGC positive differed among NTS subnuclei. Fibers stained for both nNOS-IR and sGC-IR were also present in NTS subnuclei. In addition, we identified fibers that were stained for nNOS-IR or sGC-IR alone and often found such singly labeled fibers apposed to each other. These data support our hypothesis and provide anatomical support for the suggestion that nitroxidergic activation of the NTS involves sGC.

  18. Dephosphorylation of sperm guanylate cyclase during sea urchin fertilization

    SciTech Connect

    Ward, G.E.

    1985-01-01

    When intact Arbacia punctulata spermatozoa are exposed to solubilized egg jelly, the electrophoretic mobility of an abundant sperm flagellar membrane protein changes from an apparent molecular mass of 160 kDa to 150 kDa. A. punctulata spermatozoa can be labeled in vivo with /sup 32/P-labeled cells it was demonstrated that the mobility shift of the 160-kDa protein is due to dephosphorylation. The peptide resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH/sub 2/) is the component of egg jelly which is responsible for inducing the dephosphorylation. The 160/150-kdal sperm membrane protein has been purified to homogeneity by affinity chromatography on concanavalin A-agarose, and identified as sperm guanylate cyclase. The enzymatic activity of the guanylate cyclase is tightly coupled to its phosphorylation state. Resact has been shown to act as a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration-dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for external calcium. This work represents the first demonstration of animal sperm chemotaxis in response to a precisely-defined molecule of egg origin. The results established a new, biologically meaningful function for resact, and may implicate sperm guanylate cyclase and cGMP in flagellar function and the chemotactic response.

  19. Soluble Guanylate Cyclase Stimulators: a Novel Treatment Option for Heart Failure Associated with Cardiorenal Syndromes?

    PubMed

    Dubin, Ruth F; Shah, Sanjiv J

    2016-06-01

    Heart failure in the setting of chronic kidney disease (CKD) is an increasingly common scenario and carries a poor prognosis. Clinicians lack tools for primary or secondary heart failure prevention in patients with cardiorenal syndromes. In patients without CKD, angiotensin-converting enzyme inhibitors (ACE-I) or angiotensin receptor blockers (ARB) and statins mitigate cardiovascular risk in large part due to salutary effects on the endothelium. In the setting of CKD, use of these therapies is limited by adverse effects of hyperkalemia in pre-dialysis CKD (ACE-I/ARB), or potential increased risk of stroke in end-stage renal disease (statins). The soluble guanylate cyclase (sGC) stimulators are a novel class of medications that promote endothelial and myocardial function with no known risk of hyperkalemia or stroke. In this review, we discuss the evidence emerging from recent clinical trials of sGC stimulators in pulmonary hypertension and heart failure, the diseased pathways involved in cardiorenal syndromes likely to be restored by sGC stimulators, and several strategies for designing future clinical trials of cardiorenal syndromes that might shorten the timeline for discovery and approval of effective cardiovascular therapies in these high-risk patients. PMID:27118234

  20. Efficient expression of human soluble guanylate cyclase in Escherichia coli and its signaling-related interaction with nitric oxide.

    PubMed

    Zhong, Fangfang; Wang, Hongyan; Ying, Tianlei; Huang, Zhong-Xian; Tan, Xiangshi

    2010-07-01

    Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a alpha-subunit (690 AA) and a heme-binding beta-subunit (619 AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5'-triphosphate (GTP) to 3',5'-cyclic guanosine monophosphate (cGMP). cGMP is a second messenger and initiates the nitric oxide signaling, triggering vasodilatation, smooth muscle relaxation, platelet aggregation, and neuronal transmission etc. The breakthrough of the bottle neck problem for sGC-mediated NO singling was made in this study. The recombinant human sGC beta1 subunit (HsGC beta 619) and its truncated N-terminal fragments (HsGC beta 195 and HsGC beta 384) were efficiently expressed in Escherichia coli and purified successfully in quantities. The three proteins in different forms (ferric, ferrous, NO-bound, CO-bound) were characterized by UV-vis and EPR spectroscopy. The homology structure model of the human sGC heme domain was constructed, and the mechanism for NO binding to sGC was proposed. The EPR spectra showed a characteristic of five-coordinated heme-nitrosyl species with triplet hyperfine splitting of NO. The interaction between NO and sGC was investigated and the schematic mechanism was proposed. This study provides new insights into the structure and NO-binding of human sGC. Furthermore, the efficient expression system of E. coli will be beneficial to the further studies on structure and activation mechanism of human sGC. PMID:20063108

  1. Chronic Activation of Heme Free Guanylate Cyclase Leads to Renal Protection in Dahl Salt-Sensitive Rats

    PubMed Central

    Hoffmann, Linda S.; Kretschmer, Axel; Lawrenz, Bettina; Hocher, Berthold; Stasch, Johannes-Peter

    2015-01-01

    The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophasphate (cGMP)-signalling pathway is impaired under oxidative stress conditions due to oxidation and subsequent loss of the prosthetic sGC heme group as observed in particular in chronic renal failure. Thus, the pool of heme free sGC is increased under pathological conditions. sGC activators such as cinaciguat selectively activate the heme free form of sGC and target the disease associated enzyme. In this study, a therapeutic effect of long-term activation of heme free sGC by the sGC activator cinaciguat was investigated in an experimental model of salt-sensitive hypertension, a condition that is associated with increased oxidative stress, heme loss from sGC and development of chronic renal failure. For that purpose Dahl/ss rats, which develop severe hypertension upon high salt intake, were fed a high salt diet (8% NaCl) containing either placebo or cinaciguat for 21 weeks. Cinaciguat markedly improved survival and ameliorated the salt-induced increase in blood pressure upon treatment with cinaciguat compared to placebo. Renal function was significantly improved in the cinaciguat group compared to the placebo group as indicated by a significantly improved glomerular filtration rate and reduced urinary protein excretion. This was due to anti-fibrotic and anti-inflammatory effects of the cinaciguat treatment. Taken together, this is the first study showing that long-term activation of heme free sGC leads to renal protection in an experimental model of hypertension and chronic kidney disease. These results underline the promising potential of cinaciguat to treat renal diseases by targeting the disease associated heme free form of sGC. PMID:26717150

  2. Chronic Activation of Heme Free Guanylate Cyclase Leads to Renal Protection in Dahl Salt-Sensitive Rats.

    PubMed

    Hoffmann, Linda S; Kretschmer, Axel; Lawrenz, Bettina; Hocher, Berthold; Stasch, Johannes-Peter

    2015-01-01

    The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophasphate (cGMP)-signalling pathway is impaired under oxidative stress conditions due to oxidation and subsequent loss of the prosthetic sGC heme group as observed in particular in chronic renal failure. Thus, the pool of heme free sGC is increased under pathological conditions. sGC activators such as cinaciguat selectively activate the heme free form of sGC and target the disease associated enzyme. In this study, a therapeutic effect of long-term activation of heme free sGC by the sGC activator cinaciguat was investigated in an experimental model of salt-sensitive hypertension, a condition that is associated with increased oxidative stress, heme loss from sGC and development of chronic renal failure. For that purpose Dahl/ss rats, which develop severe hypertension upon high salt intake, were fed a high salt diet (8% NaCl) containing either placebo or cinaciguat for 21 weeks. Cinaciguat markedly improved survival and ameliorated the salt-induced increase in blood pressure upon treatment with cinaciguat compared to placebo. Renal function was significantly improved in the cinaciguat group compared to the placebo group as indicated by a significantly improved glomerular filtration rate and reduced urinary protein excretion. This was due to anti-fibrotic and anti-inflammatory effects of the cinaciguat treatment. Taken together, this is the first study showing that long-term activation of heme free sGC leads to renal protection in an experimental model of hypertension and chronic kidney disease. These results underline the promising potential of cinaciguat to treat renal diseases by targeting the disease associated heme free form of sGC. PMID:26717150

  3. Purification and characterization of recombinant human soluble guanylate cyclase produced from baculovirus-infected insect cells.

    PubMed

    Emmons, Thomas L; Mathis, Karl J; Shuck, Mary E; Reitz, Beverly A; Curran, Daniel F; Walker, Mark C; Leone, Joseph W; Day, Jacqueline E; Bienkowski, Michael J; Fischer, H David; Tomasselli, Alfredo G

    2009-06-01

    Soluble guanylate cyclase (sGC) has been purified from 100 L cell culture infected by baculovirus using the newer and highly effective titerless infected-cells preservation and scale-up (TIPS) method. Successive passage of the enzyme through DEAE, Ni(2+)-NTA, and POROS Q columns obtained approximately 100mg of protein. The sGC obtained by this procedure was already about 90% pure and suitable for various studies which include high throughput screening (HTS) and hit follow-up. However, in order to obtain enzyme of greater homogeneity and purity for crystallographic and high precision spectroscopic and kinetic studies of sGC with select stimulators, the sGC solution after the POROS Q step was further purified by GTP-agarose affinity chromatography. This additional step led to the generation of 26 mg of enzyme that was about 99% pure. This highly pure and active enzyme exhibited a M(r)=144,933 by static light scattering supportive of a dimeric structure. It migrated as a two-band protein, each of equal intensity, on SDS-PAGE corresponding to the alpha (M(r) approximately 77,000) and beta (M(r) approximately 70,000) sGC subunits. It showed an A(430)/A(280)=1.01, indicating one heme per heterodimer, and a maximum of the Soret band at 430 nm indicative of a penta-coordinated ferrous heme with a histidine as the axial ligand. The Soret band shifted to 398 nm in the presence of an NO donor as expected for the formation of a penta-coordinated nitrosyl-heme complex. Non-stimulated sGC had k(cat)/K(m)=1.7 x 10(-3)s(-1)microM(-1) that increased to 5.8 x 10(-1)s(-1)microM(-1) upon stimulation with an NO donor which represents a 340-fold increase due to stimulation. The novel combination of using the TIPS method for co-expression of a heterodimeric heme-containing enzyme, along with the application of a reproducible ligand affinity purification method, has enabled us to obtain recombinant human sGC of both the quality and quantity needed to study structure-function relationships

  4. Elevation of Soluble Guanylate Cyclase Suppresses Proliferation and Survival of Human Breast Cancer Cells

    PubMed Central

    Chen, Chen-Yu; Shiah, Shine-Gwo; Kung, Hsing-Jien; King, Kuang-Liang; Su, Liang-Chen; Chang, Shi-Chuan; Chang, Chung-Ho

    2015-01-01

    Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells. PMID:25928539

  5. Loss of α1β1 soluble guanylate cyclase, the major nitric oxide receptor, leads to moyamoya and achalasia.

    PubMed

    Hervé, Dominique; Philippi, Anne; Belbouab, Reda; Zerah, Michel; Chabrier, Stéphane; Collardeau-Frachon, Sophie; Bergametti, Francoise; Essongue, Aurore; Berrou, Eliane; Krivosic, Valérie; Sainte-Rose, Christian; Houdart, Emmanuel; Adam, Frédéric; Billiemaz, Kareen; Lebret, Marilyne; Roman, Sabine; Passemard, Sandrine; Boulday, Gwenola; Delaforge, Audrey; Guey, Stéphanie; Dray, Xavier; Chabriat, Hugues; Brouckaert, Peter; Bryckaert, Maryjke; Tournier-Lasserve, Elisabeth

    2014-03-01

    Moyamoya is a cerebrovascular condition characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and the compensatory development of abnormal "moyamoya" vessels. The pathophysiological mechanisms of this condition, which leads to ischemic and hemorrhagic stroke, remain unknown. It can occur as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes). Here, we describe an autosomal-recessive disease leading to severe moyamoya and early-onset achalasia in three unrelated families. This syndrome is associated in all three families with homozygous mutations in GUCY1A3, which encodes the α1 subunit of soluble guanylate cyclase (sGC), the major receptor for nitric oxide (NO). Platelet analysis showed a complete loss of the soluble α1β1 guanylate cyclase and showed an unexpected stimulatory role of sGC within platelets. The NO-sGC-cGMP pathway is a major pathway controlling vascular smooth-muscle relaxation, vascular tone, and vascular remodeling. Our data suggest that alterations of this pathway might lead to an abnormal vascular-remodeling process in sensitive vascular areas such as ICA bifurcations. These data provide treatment options for affected individuals and strongly suggest that investigation of GUCY1A3 and other members of the NO-sGC-cGMP pathway is warranted in both isolated early-onset achalasia and nonsyndromic moyamoya. PMID:24581742

  6. Loss of α1β1 Soluble Guanylate Cyclase, the Major Nitric Oxide Receptor, Leads to Moyamoya and Achalasia

    PubMed Central

    Hervé, Dominique; Philippi, Anne; Belbouab, Reda; Zerah, Michel; Chabrier, Stéphane; Collardeau-Frachon, Sophie; Bergametti, Francoise; Essongue, Aurore; Berrou, Eliane; Krivosic, Valérie; Sainte-Rose, Christian; Houdart, Emmanuel; Adam, Frédéric; Billiemaz, Kareen; Lebret, Marilyne; Roman, Sabine; Passemard, Sandrine; Boulday, Gwenola; Delaforge, Audrey; Guey, Stéphanie; Dray, Xavier; Chabriat, Hugues; Brouckaert, Peter; Bryckaert, Maryjke; Tournier-Lasserve, Elisabeth

    2014-01-01

    Moyamoya is a cerebrovascular condition characterized by a progressive stenosis of the terminal part of the internal carotid arteries (ICAs) and the compensatory development of abnormal “moyamoya” vessels. The pathophysiological mechanisms of this condition, which leads to ischemic and hemorrhagic stroke, remain unknown. It can occur as an isolated cerebral angiopathy (so-called moyamoya disease) or in association with various conditions (moyamoya syndromes). Here, we describe an autosomal-recessive disease leading to severe moyamoya and early-onset achalasia in three unrelated families. This syndrome is associated in all three families with homozygous mutations in GUCY1A3, which encodes the α1 subunit of soluble guanylate cyclase (sGC), the major receptor for nitric oxide (NO). Platelet analysis showed a complete loss of the soluble α1β1 guanylate cyclase and showed an unexpected stimulatory role of sGC within platelets. The NO-sGC-cGMP pathway is a major pathway controlling vascular smooth-muscle relaxation, vascular tone, and vascular remodeling. Our data suggest that alterations of this pathway might lead to an abnormal vascular-remodeling process in sensitive vascular areas such as ICA bifurcations. These data provide treatment options for affected individuals and strongly suggest that investigation of GUCY1A3 and other members of the NO-sGC-cGMP pathway is warranted in both isolated early-onset achalasia and nonsyndromic moyamoya. PMID:24581742

  7. Soluble guanylate cyclase as a novel treatment target for osteoporosis.

    PubMed

    Joshua, Jisha; Schwaerzer, Gerburg K; Kalyanaraman, Hema; Cory, Esther; Sah, Robert L; Li, Mofei; Vaida, Florin; Boss, Gerry R; Pilz, Renate B

    2014-12-01

    Osteoporosis is a major health problem leading to fractures that cause substantial morbidity and mortality. Current osteoporosis therapies have significant drawbacks, creating a need for novel bone-anabolic agents. We previously showed that the nitric oxide/cyclic GMP (cGMP)/protein kinase G pathway mediates some of the anabolic effects of estrogens and mechanical stimulation in osteoblasts and osteocytes, leading us to hypothesize that cGMP-elevating agents may have bone-protective effects. We tested cinaciguat, a prototype of a novel class of soluble guanylate cyclase activators, in a mouse model of estrogen deficiency-induced osteoporosis. Compared with sham-operated mice, ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with cinaciguat or low-dose 17β-estradiol. Microcomputed tomography of tibiae showed that cinaciguat significantly improved trabecular bone microarchitecture in ovariectomized animals, with effect sizes similar to those obtained with estrogen replacement therapy. Cinaciguat reversed ovariectomy-induced osteocyte apoptosis as efficiently as estradiol and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. Compared with 17β-estradiol, which completely reversed the ovariectomy-induced increase in osteoclast number, cinaciguat had little effect on osteoclasts. Direct guanylate cyclase stimulators have been extremely well tolerated in clinical trials of cardiovascular diseases, and our findings provide proof-of-concept for this new class of drugs as a novel, anabolic treatment strategy for postmenopausal osteoporosis, confirming an important role of nitric oxide/cGMP/protein kinase G signaling in bone. PMID:25188528

  8. Motion of proximal histidine and structural allosteric transition in soluble guanylate cyclase

    PubMed Central

    Yoo, Byung-Kuk; Lamarre, Isabelle; Martin, Jean-Louis; Rappaport, Fabrice; Negrerie, Michel

    2015-01-01

    We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 μs with [NO] = 200 μM) forming a 6c heme with a diffusion-limited rate constant of 2 × 108 M−1⋅s−1. In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 μs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1–50 μs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed. PMID:25831539

  9. [Soluble guanylate cyclase in the molecular mechanism underlying the therapeutic action of drugs].

    PubMed

    Piatakova, N V; Severina, I S

    2012-01-01

    The influence of ambroxol--a mucolytic drug--on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside and Sin-1) were investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin--an antimalarial drug--on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1-100 microM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylate cyclase with an IC50 value 5.6 microM. Artemisinin (10 microM) also inhibited (by 71 +/- 4.0%) the activation of the enzyme by thiol-dependent NO-donor the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 microM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the sygnalling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin.

  10. [Soluble guanylate cyclase in the molecular mechanism underlying the therapeutic action of drugs].

    PubMed

    Piatakova, N V; Severina, I S

    2012-01-01

    The influence of ambroxol--a mucolytic drug--on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside and Sin-1) were investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin--an antimalarial drug--on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1-100 microM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylate cyclase with an IC50 value 5.6 microM. Artemisinin (10 microM) also inhibited (by 71 +/- 4.0%) the activation of the enzyme by thiol-dependent NO-donor the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 microM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the sygnalling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin. PMID:22642150

  11. Soluble Guanylate Cyclase Stimulation Prevents Fibrotic Tissue Remodeling and Improves Survival in Salt-Sensitive Dahl Rats

    PubMed Central

    Geschka, Sandra; Kretschmer, Axel; Sharkovska, Yuliya; Evgenov, Oleg V.; Lawrenz, Bettina; Hucke, Andreas; Hocher, Berthold; Stasch, Johannes-Peter

    2011-01-01

    Background A direct pharmacological stimulation of soluble guanylate cyclase (sGC) is an emerging therapeutic approach to the management of various cardiovascular disorders associated with endothelial dysfunction. Novel sGC stimulators, including riociguat (BAY 63-2521), have a dual mode of action: They sensitize sGC to endogenously produced nitric oxide (NO) and also directly stimulate sGC independently of NO. Little is known about their effects on tissue remodeling and degeneration and survival in experimental malignant hypertension. Methods and Results Mortality, hemodynamics and biomarkers of tissue remodeling and degeneration were assessed in Dahl salt-sensitive rats maintained on a high salt diet and treated with riociguat (3 or 10 mg/kg/d) for 14 weeks. Riociguat markedly attenuated systemic hypertension, improved systolic heart function and increased survival from 33% to 85%. Histological examination of the heart and kidneys revealed that riociguat significantly ameliorated fibrotic tissue remodeling and degeneration. Correspondingly, mRNA expression of the pro-fibrotic biomarkers osteopontin (OPN), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in the myocardium and the renal cortex was attenuated by riociguat. In addition, riociguat reduced plasma and urinary levels of OPN, TIMP-1, and PAI-1. Conclusions Stimulation of sGC by riociguat markedly improves survival and attenuates systemic hypertension and systolic dysfunction, as well as fibrotic tissue remodeling in the myocardium and the renal cortex in a rodent model of pressure and volume overload. These findings suggest a therapeutic potential of sGC stimulators in diseases associated with impaired cardiovascular and renal functions. PMID:21789188

  12. Integrative Signaling Networks of Membrane Guanylate Cyclases: Biochemistry and Physiology

    PubMed Central

    Sharma, Rameshwar K.; Duda, Teresa; Makino, Clint L.

    2016-01-01

    This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs), highlighting contributions made by the authors and their collaborators. Upon resolution of early contentious studies, cyclic GMP emerged alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca2+-sensing subunits that either increase or decrease cyclic GMP synthesis, depending on subunit identity. In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and Transient Receptor Potential (TRP) ion channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory. PMID:27695398

  13. Integrative Signaling Networks of Membrane Guanylate Cyclases: Biochemistry and Physiology

    PubMed Central

    Sharma, Rameshwar K.; Duda, Teresa; Makino, Clint L.

    2016-01-01

    This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs), highlighting contributions made by the authors and their collaborators. Upon resolution of early contentious studies, cyclic GMP emerged alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca2+-sensing subunits that either increase or decrease cyclic GMP synthesis, depending on subunit identity. In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and Transient Receptor Potential (TRP) ion channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory.

  14. Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity.

    PubMed

    Duda, Teresa; Wen, Xiao-Hong; Isayama, Tomoki; Sharma, Rameshwar K; Makino, Clint L

    2015-04-24

    By generating the second messenger cGMP in retinal rods and cones, ROS-GC plays a central role in visual transduction. Guanylate cyclase-activating proteins (GCAPs) link cGMP synthesis to the light-induced fall in [Ca(2+)]i to help set absolute sensitivity and assure prompt recovery of the response to light. The present report discloses a surprising feature of this system: ROS-GC is a sensor of bicarbonate. Recombinant ROS-GCs synthesized cGMP from GTP at faster rates in the presence of bicarbonate with an ED50 of 27 mM for ROS-GC1 and 39 mM for ROS-GC2. The effect required neither Ca(2+) nor use of the GCAPs domains; however, stimulation of ROS-GC1 was more powerful in the presence of GCAP1 or GCAP2 at low [Ca(2+)]. When applied to retinal photoreceptors, bicarbonate enhanced the circulating current, decreased sensitivity to flashes, and accelerated flash response kinetics. Bicarbonate was effective when applied either to the outer or inner segment of red-sensitive cones. In contrast, bicarbonate exerted an effect when applied to the inner segment of rods but had little efficacy when applied to the outer segment. The findings define a new regulatory mechanism of the ROS-GC system that affects visual transduction and is likely to affect the course of retinal diseases caused by cGMP toxicity. PMID:25767116

  15. Murine Guanylate Cyclase C Regulates Colonic Injury and Inflammation1

    PubMed Central

    Steinbrecher, Kris A.; Harmel-Laws, Eleana; Garin-Laflam, Monica P.; Mann, Elizabeth A.; Bezerra, Lucas D.; Hogan, Simon P.; Cohen, Mitchell B.

    2011-01-01

    Guanylate cyclase C (GUCY2C or GC-C) and its ligands, guanylin (GUCA2A or Gn) and uroguanylin (GUCA2B or Ugn), are expressed in intestinal epithelial cells (IECs) and regulate ion secretion, intestinal barrier function, and epithelial monolayer homeostasis via cGMP-dependent signaling pathways. The aim of this study was to determine if GC-C and its ligands direct the course of intestinal inflammation. Here, we show that DSS-induced clinical disease and histological damage to the colonic mucosa were significantly less severe in GC-C−/− mice and moderately reduced in Gn−/− animals. Relative to wildtype controls, GC-C−/− and Gn−/− mice had reduced apoptosis and increased proliferation of IECs during DSS colitis. Basal and DSS-induced production of resistin-like molecule β (RELMβ) was substantially diminished in GC-C−/− mice. RELMβ is thought to stimulate cytokine production in macrophages in this disease model and, consistent with this, TNFα and IFNγ production was minimal in GC-C−/− animals. RELMβ and cytokine levels were similar to wildtype in Gn−/− mice, however. Colonic instillation of recombinant RELMβ by enema into GC-C−/− mice restores sensitivity to DSS-mediated mucosal injury. These findings demonstrate a novel role for GC-C signaling in facilitating mucosal wounding and inflammation and further suggest that this may be mediated, in part, through control of RELMβ production. PMID:21555532

  16. Carbon Monoxide Abrogates Ischemic Insult to Neuronal Cells via the Soluble Guanylate Cyclase-cGMP Pathway

    PubMed Central

    Schallner, Nils; Romão, Carlos C.; Biermann, Julia; Lagrèze, Wolf A.; Otterbein, Leo E.; Buerkle, Hartmut; Loop, Torsten; Goebel, Ulrich

    2013-01-01

    Purpose Carbon monoxide (CO) is an accepted cytoprotective molecule. The extent and mechanisms of protection in neuronal systems have not been well studied. We hypothesized that delivery of CO via a novel releasing molecule (CORM) would impart neuroprotection in vivo against ischemia-reperfusion injury (IRI)-induced apoptosis of retinal ganglion cells (RGC) and in vitro of neuronal SH-SY5Y-cells via activation of soluble guanylate-cyclase (sGC). Methods To mimic ischemic respiratory arrest, SH-SY5Y-cells were incubated with rotenone (100 nmol/L, 4 h) ± CORM ALF186 (10–100 µmol/L) or inactivated ALF186 lacking the potential of releasing CO. Apoptosis and reactive oxygen species (ROS) production were analyzed using flow-cytometry (Annexin V, mitochondrial membrane potential, CM-H2DCFDA) and Western blot (Caspase-3). The impact of ALF186± respiratory arrest on cell signaling was assessed by measuring expression of nitric oxide synthase (NOS) and soluble guanylate-cyclase (sGC) and by analyzing cellular cGMP levels. The effect of ALF186 (10 mg/kg iv) on retinal IRI in Sprague-Dawley rats was assessed by measuring densities of fluorogold-labeled RGC after IRI and by analysis of apoptosis-related genes in retinal tissue. Results ALF186 but not inactivated ALF186 inhibited rotenone-induced apoptosis (Annexin V positive cells: 25±2% rotenone vs. 14±1% ALF186+rotenone, p<0.001; relative mitochondrial membrane potential: 17±4% rotenone vs. 55±3% ALF186+rotenone, p<0.05). ALF186 increased cellular cGMP levels (33±5 nmol/L vs. 23±3 nmol/L; p<0.05) and sGC expression. sGC-inhibition attenuated ALF186-mediated protection (relative mitochondrial membrane potential: 55±3% ALF186+rotenone vs. 20±1% ODQ+ALF186+rotenone, p<0.05). ALF186 protected RGC in vivo (IRI 1255±327 RGC/mm2 vs. ALF186+IRI 2036±83; p<0.05) while sGC inhibition abolished the protective effects of ALF186 (ALF186+IRI 2036±83 RGC/mm2 vs. NS-2028+ALF186+IRI 1263±170, p<0.05). Conclusions The CORM ALF

  17. Soluble guanylate cyclase is required for systemic vasodilation but not positive inotropy induced by nitroxyl in the mouse.

    PubMed

    Zhu, Guangshuo; Groneberg, Dieter; Sikka, Gautam; Hori, Daijiro; Ranek, Mark J; Nakamura, Taishi; Takimoto, Eiki; Paolocci, Nazareno; Berkowitz, Dan E; Friebe, Andreas; Kass, David A

    2015-02-01

    Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO·), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here, we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1α to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-β subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant protein kinase G-1α were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of preconstricted force in aortic rings was ≈400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020, despite its inotropic-vasodilator activity. In protein kinase G-1α(C42S) mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo but are not required for positive inotropic action. Redox modulation of protein kinase G-1α is not a mechanism for HNO-mediated vasodilation.

  18. SOLUBLE GUANYLATE CYCLASE IS REQUIRED FOR SYSTEMIC VASODILATION BUT NOT POSITIVE INOTROPY INDUCED BY NITROXYL (HNO) in the MOUSE

    PubMed Central

    Zhu, Guangshuo; Groneberg, Dieter; Sikka, Gautam; Hori, Daijiro; Ranek, Mark J.; Nakamura, Taishi; Takimoto, Eiki; Paolocci, Nazareno; Berkowitz, Dan E.; Friebe, Andreas; Kass, David A.

    2014-01-01

    Nitroxyl (HNO), the reduced and protonated form of nitric oxide (NO.), confers unique physiological effects including vasorelaxation and enhanced cardiac contractility. These features have spawned current pharmaceutical development of HNO donors as heart failure therapeutics. HNO interacts with selective redox sensitive cysteines to effect signaling, but is also proposed to activate soluble guanylate cyclase (sGC) in vitro to induce vasodilation and potentially enhance contractility. Here we tested whether sGC stimulation is required for these HNO effects in vivo and if HNO also modifies a redox-sensitive cysteine (C42) in protein kinase G-1α (PKG1α) to control vasorelaxation. Intact mice and isolated arteries lacking the sGC-β subunit (sGCKO, results in full sGC deficiency) or expressing solely a redox-dead C42S mutant PKG1α were exposed to the pure HNO donor, CXL-1020. CXL-1020 induced dose-dependent systemic vasodilation while increasing contractility in controls; however, vasodilator effects were absent in sGCKO mice whereas contractility response remained. The CXL-1020 dose reversing 50% of pre-constricted force in aortic rings was ~400-fold greater in sGCKO than controls. Cyclic-GMP and cAMP levels were unaltered in myocardium exposed to CXL-1020 despite its inotropicvasodilator activity. In PKG1αC42S mice, CXL-1020 induced identical vasorelaxation in vivo and in isolated aortic and mesenteric vessels as in littermate controls. In both groups, dilation was near fully blocked by pharmacologically inhibiting sGC. Thus, sGC and cGMP-dependent signaling are necessary and sufficient for HNO-induced vasodilation in vivo, but are not required for positive inotropic action. Redox modulation of PKG1α is not a mechanism for HNO-mediated vasodilation. PMID:25452469

  19. Role of soluble guanylate cyclase in the molecular mechanism underlying the physiological effects of nitric oxide.

    PubMed

    Severina, I S

    1998-07-01

    In this review the molecular mechanisms underlying the antihypertensive and antiaggregatory actions of nitric oxide (NO) are discussed. It has been shown that these effects are directly connected with the activation of soluble guanylate cyclase and the accumulation of cyclic 3;,5;-guanosine monophosphate (cGMP). The mechanism of guanylate cyclase activation by NO is analyzed, especially the role and biological significance of the nitrosyl--heme complex formed as a result of interaction of guanylate cyclase heme with NO and the role of sulfhydryl groups of the enzyme in this process. Using new approaches for studying the antihypertensive and antiaggregatory actions of nitric oxide in combination with the newly obtained data on the regulatory role of guanylate cyclase in the platelet aggregation process, the most important results were obtained regarding the molecular bases providing for a directed search for and creation of new effective antihypertensive and antiaggregatory preparations. In studying the molecular mechanism for directed activation of soluble guanylate cyclase by new NO donors, a series of hitherto unknown enzyme activators generating NO and involved in the regulation of hemostasis and vascular tone were revealed. PMID:9721331

  20. Comparison of soluble guanylate cyclase stimulators and activators in models of cardiovascular disease associated with oxidative stress.

    PubMed

    Costell, Melissa H; Ancellin, Nicolas; Bernard, Roberta E; Zhao, Shufang; Upson, John J; Morgan, Lisa A; Maniscalco, Kristeen; Olzinski, Alan R; Ballard, Victoria L T; Herry, Kenny; Grondin, Pascal; Dodic, Nerina; Mirguet, Olivier; Bouillot, Anne; Gellibert, Francoise; Coatney, Robert W; Lepore, John J; Jucker, Beat M; Jolivette, Larry J; Willette, Robert N; Schnackenberg, Christine G; Behm, David J

    2012-01-01

    Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5-10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible

  1. Comparison of Soluble Guanylate Cyclase Stimulators and Activators in Models of Cardiovascular Disease Associated with Oxidative Stress

    PubMed Central

    Costell, Melissa H.; Ancellin, Nicolas; Bernard, Roberta E.; Zhao, Shufang; Upson, John J.; Morgan, Lisa A.; Maniscalco, Kristeen; Olzinski, Alan R.; Ballard, Victoria L. T.; Herry, Kenny; Grondin, Pascal; Dodic, Nerina; Mirguet, Olivier; Bouillot, Anne; Gellibert, Francoise; Coatney, Robert W.; Lepore, John J.; Jucker, Beat M.; Jolivette, Larry J.; Willette, Robert N.; Schnackenberg, Christine G.; Behm, David J.

    2012-01-01

    Soluble guanylate cyclase (sGC), the primary mediator of nitric oxide (NO) bioactivity, exists as reduced (NO-sensitive) and oxidized (NO-insensitive) forms. We tested the hypothesis that the cardiovascular protective effects of NO-insensitive sGC activation would be potentiated under conditions of oxidative stress compared to those of NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A [a low, non-depressor dose, and a high dose which lowered mean arterial pressure (MAP) by 5–10 mmHg] and those of equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in (1) Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R) and (2) spontaneously hypertensive stroke prone rats (SHR-SP) on a high salt/fat diet (HSFD). In I/R, neither compound reduced infarct size 24 h after reperfusion. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria, and mortality, caused left ventricular hypertrophy with preserved ejection fraction, and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552, but not that of GSK2181236A, decreased urine output, and improved survival. Conversely, the low dose of GSK2181236A, but not that of BAY 60-4552, attenuated the development of cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and improved survival. In addition to these effects, the high dose of BAY 60-4552 reduced urine output and microalbuminuria and attenuated the increase in MAP to a greater extent than did GSK2181236A. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP isolated aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to both GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the oxidative state of sGC is responsible

  2. The Soluble Guanylate Cyclase Stimulator IWP-953 Increases Conventional Outflow Facility in Mouse Eyes

    PubMed Central

    Ge, Pei; Navarro, Iris D.; Kessler, Marco M.; Bernier, Sylvie G.; Perl, Nicholas R.; Sarno, Renee; Masferrer, Jaime; Hannig, Gerhard; Stamer, W. Daniel

    2016-01-01

    Purpose The nitric oxide (NO)–cyclic guanosine-3′,5′-monophosphate (cGMP) pathway regulates aqueous humor outflow and therefore, intraocular pressure. We investigated the pharmacologic effects of the soluble guanylate cyclase (sGC) stimulator IWP-953 on primary human trabecular meshwork (HTM) cells and conventional outflow facility in mouse eyes. Methods Cyclic GMP levels were determined in vitro in HEK-293 cells and four HTM cell strains (HTM120/HTM123: predominantly myofibroblast-like phenotype, HTM130/HTM141: predominantly endothelial-like phenotype), and in HTM cell culture supernatants. Conventional outflow facility was measured following intracameral injection of IWP-953 or DETA-NO using a computerized pressure-controlled perfusion system in enucleated mouse eyes ex vivo. Results IWP-953 markedly stimulated cGMP production in HEK-293 cells in the presence and absence of DETA-NO (half maximal effective concentrations: 17 nM, 9.5 μM). Similarly, IWP-953 stimulated cGMP production in myofibroblast-like HTM120 and HTM123 cells, an effect that was greatly amplified by the presence of DETA-NO. In contrast, IWP-953 stimulation of cGMP production in endothelial-like HTM130 and HTM141 cells was observed, but was markedly less prominent than in HTM120 and HTM123 cells. Notably, cGMP was found in all HTM culture supernatants, following IWP-953/DETA-NO stimulation. In paired enucleated mouse eyes, IWP-953 at 10, 30, 60, and 100 μM concentration-dependently increased outflow facility. This effect (89.5%) was maximal at 100 μM (P = 0.002) and in magnitude comparable to DETA-NO at 100 μM (97.5% increase, P = 0.030). Conclusions These data indicate that IWP-953, via modulation of the sGC–cGMP pathway, increases aqueous outflow facility in mouse eyes, suggesting therapeutic potential for sGC stimulators as novel ocular hypotensive drugs. PMID:26998718

  3. Soluble guanylate cyclase is activated differently by excess NO and by YC-1: Resonance Raman spectroscopic evidence†

    PubMed Central

    Ibrahim, Mohammed; Derbyshire, Emily R.; Soldatova, Alexandra V.; Marletta, Michael A.; Spiro, Thomas G.

    2010-01-01

    Modulation of soluble guanylate cyclase (sGC) activity by nitric oxide (NO) involves two distinct steps. Low level activation of sGC is achieved by the stoichiometric binding of NO (1-NO) to the heme cofactor, while much higher activation is achieved by the binding of additional NO (xsNO) at a non-heme site. Addition of the allosteric activator YC-1 to the 1-NO form leads to activity comparable to xsNO state. In this study the mechanisms of sGC activation were investigated using electronic absorption and resonance Raman (RR) spectroscopic methods. RR spectroscopy confirmed that the 1-NO form contains 5-coordinate NO-heme and showed that the addition of NO to the 1-NO form has no significant effect on the spectrum. In contrast, addition of YC-1 to either the 1-NO or xsNO forms alters the RR spectrum significantly, indicating a protein-induced change in the heme geometry. This change in the heme geometry was also observed when BAY 41-2272 was added to the xsNO form. Bands assigned to bending and stretching motions of the vinyl and propionate substituents change intensity in a pattern suggesting altered tilting of the pyrrole rings to which they are attached. In addition, the N-O stretching frequency increases, with no change in the Fe-NO frequency, an effect modeled via DFT calculations as resulting from a small opening of the Fe-N-O angle. These spectral differences demonstrate different mechanisms of activation by synthetic activators, such as YC-1 and BAY 41-2272, and excess NO. PMID:20459051

  4. Soluble guanylate cyclase is activated differently by excess NO and by YC-1: resonance Raman spectroscopic evidence.

    PubMed

    Ibrahim, Mohammed; Derbyshire, Emily R; Soldatova, Alexandra V; Marletta, Michael A; Spiro, Thomas G

    2010-06-15

    Modulation of soluble guanylate cyclase (sGC) activity by nitric oxide (NO) involves two distinct steps. Low-level activation of sGC is achieved by the stoichiometric binding of NO (1-NO) to the heme cofactor, while much higher activation is achieved by the binding of additional NO (xsNO) at a non-heme site. Addition of the allosteric activator YC-1 to the 1-NO form leads to activity comparable to that of the xsNO state. In this study, the mechanisms of sGC activation were investigated using electronic absorption and resonance Raman (RR) spectroscopic methods. RR spectroscopy confirmed that the 1-NO form contains five-coordinate NO-heme and showed that the addition of NO to the 1-NO form has no significant effect on the spectrum. In contrast, addition of YC-1 to either the 1-NO or xsNO forms alters the RR spectrum significantly, indicating a protein-induced change in the heme geometry. This change in the heme geometry was also observed when BAY 41-2272 was added to the xsNO form. Bands assigned to bending and stretching motions of the vinyl and propionate substituents undergo changes in intensity in a pattern suggesting altered tilting of the pyrrole rings to which they are attached. In addition, the N-O stretching frequency increases, with no change in the Fe-NO stretching frequency, an effect modeled via DFT calculations as resulting from a small opening of the Fe-N-O angle. These spectral differences demonstrate different mechanisms of activation by synthetic activators, such as YC-1 and BAY 41-2272, and excess NO. PMID:20459051

  5. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    PubMed

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-01

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2).

  6. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    PubMed

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-01

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2). PMID:21173231

  7. The calcium-sensor guanylate cyclase activating protein type 2 specific site in rod outer segment membrane guanylate cyclase type 1.

    PubMed

    Duda, Teresa; Fik-Rymarkiewicz, Ewa; Venkataraman, Venkateswar; Krishnan, Ramalingam; Koch, Karl-Wilhelm; Sharma, Rameshwar K

    2005-05-17

    The rod outer segment membrane guanylate cyclase type 1 (ROS-GC1), originally identified in the photoreceptor outer segments, is a member of the subfamily of Ca(2+)-modulated membrane guanylate cyclases. In phototransduction, its activity is tightly regulated by its two Ca(2+)-sensor protein parts, GCAP1 and GCAP2. This study maps the GCAP2-modulatory site in ROS-GC1 through the use of multiple techniques involving surface plasmon resonance binding studies with soluble ROS-GC1 constructs, coimmunoprecipitation, functional reconstitution experiments with deletion mutants, and peptide competition assays. The findings show that the sequence motif of the core GCAP2-modulatory site is Y965-N981 of ROS-GC1. The site is distinct from the GCAP1-modulatory site. It, however, partially overlaps with the S100B-regulatory site. This indicates that the Y965-N981 motif tightly controls the Ca(2+)-dependent specificity of ROS-GC1. Identification of the site demonstrates an intriguing topographical feature of ROS-GC1. This is that the GCAP2 module transmits the Ca(2+) signals to the catalytic domain from its C-terminal side and the GCAP1 module from the distant N-terminal side.

  8. Vasorelaxant effect of isoliquiritigenin, a novel soluble guanylate cyclase activator, in rat aorta.

    PubMed Central

    Yu, S M; Kuo, S C

    1995-01-01

    1. The vasorelaxant activity of isoliquiritigenin, isolated from Dalbergia odorifera T, was investigated in the phenylephrine-precontracted rat aorta by measuring tension, guanylate and adenylate cyclase activities, guanosine 3':5'-cyclic monophosphate (cyclic GMP) and adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 2. Isoliquiritigenin concentration-dependently relaxed rat aorta contracted with phenylephrine, KCl, U-46619, endothelin and 5-hydroxytryptamine, with EC50s of 7.4 +/- 1.6, 10.5 +/- 2.3, 14.3 +/- 3.3, 11.8 +/- 2.0 and 13.6 +/- 3.7 microM, respectively. 3. Isoliquiritigenin caused endothelium-independent relaxation of phenylephrine-precontracted rat aortic rings. Neither NG-monomethyl-L-arginine (L-NMMA) (an inhibitor of the L-arginine-NO pathway) nor oxyhaemoglobin (which binds NO) modified the relaxant effect of isoliquiritigenin. The relaxant action of isoliquiritigenin also persisted in intact aorta in the presence of indomethacin or glibenclamide. However, methylene blue, an inhibitor of soluble guanylate cyclase, abolished relaxation induced by isoliquiritigenin. 4. Incubation of rat aorta with isoliquiritigenin not only increased aortic cyclic GMP content but also caused small increases in aortic cyclic AMP content, and greatly potentiated the increases in cyclic AMP observed in the presence of forskolin. The maximum increase in cyclic GMP by isoliquiritigenin was reached earlier than the increase in cyclic AMP. This result suggests that the increases in cyclic GMP caused by isoliquiritigenin might stimulate the accumulation of cyclic AMP. 5. Concentration-dependent increases in soluble guanylate cyclase activity were observed in isoliquiritigenin (1-100 microM)- or sodium nitroprusside (SNP)-treated rat aortic smooth muscle cells, while adenylate cyclase activity was unchanged in isoliquiritigenin (100 microM)-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7599926

  9. Involvement of H1 and H2 receptors and soluble guanylate cyclase in histamine-induced relaxation of rat mesenteric collecting lymphatics

    PubMed Central

    Kurtz, Kristine H.; Moor, Andrea N.; Souza-Smith, Flavia M.; Breslin, Jerome W.

    2014-01-01

    Objective This study investigated the roles of the H1 and H2 histamine receptors, nitric oxide (NO) synthase, and soluble guanylate (sGC) cyclase in histamine-induced modulation of rat mesenteric collecting lymphatic pumping. Methods Isolated rat mesenteric collecting lymphatics were treated with 1–100 μM histamine. Histamine receptors were blocked with either the H1 antagonist mepyramine or the H2 antagonist cimetidine. The role of NO/sGC signaling was tested using the arginine analog L-NAME, the sGC inhibitor ODQ, and sodium nitroprusside (SNP) as a positive control. Results Histamine applied at 100 μM decreased tone and contraction frequency (CF) of isolated rat mesenteric collecting lymphatics. Pharmacologic blockade of either H1 or H2 histamine receptors significantly inhibited the response to histamine. Pretreatment with ODQ, but not L-NAME, completely inhibited the histamine-induced decrease in tone. ODQ pretreatment also significantly inhibited SNP-induced lymphatic relaxation. Conclusions H1 and H2 histamine receptors are both involved in histamine-induced relaxation of rat mesenteric collecting lymphatics. NO synthesis does not appear to contribute to the histamine-induced response. However, sGC is critical for the histamine-induced decrease in tone and contributes to the drop in CF. PMID:24702851

  10. Oral administration of soluble guanylate cyclase agonists to rats results in osteoclastic bone resorption and remodeling with new bone formation in the appendicular and axial skeleton.

    PubMed

    Homer, Bruce L; Morton, Daniel; Bagi, Cedo M; Warneke, James A; Andresen, Catharine J; Whiteley, Laurence O; Morris, Dale L; Tones, Michael A

    2015-04-01

    Orally administered small molecule agonists of soluble guanylate cyclase (sGC) induced increased numbers of osteoclasts, multifocal bone resorption, increased porosity, and new bone formation in the appendicular and axial skeleton of Sprague-Dawley rats. Similar histopathological bone changes were observed in both young (7- to 9-week-old) and aged (42- to 46-week-old) rats when dosed by oral gavage with 3 different heme-dependent sGC agonist (sGCa) compounds or 1 structurally distinct heme-independent sGCa compound. In a 7-day time course study in 7- to 9-week-old rats, bone changes were observed as early as 2 to 3 days following once daily compound administration. Bone changes were mostly reversed following a 14-day recovery period, with complete reversal after 35 days. The mechanism responsible for the bone changes was investigated in the thyroparathyroidectomized rat model that creates a low state of bone modeling and remodeling due to deprivation of thyroid hormone, calcitonin (CT), and parathyroid hormone (PTH). The sGCa compounds tested increased both bone resorption and formation, thereby increasing bone remodeling independent of calciotropic hormones PTH and CT. Based on these studies, we conclude that the bone changes in rats were likely caused by increased sGC activity.

  11. Regulation of Neuronal Oxygen Responses in C. elegans Is Mediated through Interactions between Globin 5 and the H-NOX Domains of Soluble Guanylate Cyclases

    PubMed Central

    Abergel, Zohar; Chatterjee, Arijit Kumar; Zuckerman, Binyamin

    2016-01-01

    Soluble guanylate cyclases (sGCs) are gas-binding proteins that control diverse physiological processes such as vasodilation, platelet aggregation, and synaptic plasticity. In the nematode Caenorhabditis elegans, a complex of sGCs, GCY-35 and GCY-36, functions in oxygen (O2) sensing. Previous studies suggested that the neuroglobin GLB-5 genetically interacts with GCY-35, and that the inhibitory effect of GLB-5 on GCY-35 function is necessary for fast recovery from prolonged hypoxia. In this study, we identified mutations in gcy-35 and gcy-36 that impact fast recovery and other phenotypes associated with GLB-5, without undermining sGC activity. These mutations, heb1 and heb3, change conserved amino acid residues in the regulatory H-NOX domains of GCY-35 and GCY-36, respectively, and appear to suppress GLB-5 activity by different mechanisms. Moreover, we observed that short exposure to 35% O2 desensitized the neurons responsible for ambient O2 sensing and that this phenomenon does not occur in heb1 animals. These observations may implicate sGCs in neuronal desensitization mechanisms far beyond the specific case of O2 sensing in nematodes. The conservation of functionally important regions of sGCs is supported by examining site-directed mutants of GCY-35, which suggested that similar regions in the H-NOX domains of O2 and NO-sensing sGCs are important for heme/gas interactions. Overall, our studies provide novel insights into sGC activity and regulation, and implicate similar structural determinants in the control of both O2 and NO sensors. SIGNIFICANCE STATEMENT Soluble guanylate cyclases (sGCs) control essential and diverse physiological processes, including memory processing. We used Caenorhabditis elegans to explore how a neuroglobin inhibits a complex of oxygen-sensing sGCs, identifying sGC mutants that resist inhibition. Resistance appears to arise by two different mechanisms: increased basal sGC activity or disruption of an interaction with neuroglobin. Our

  12. Regulation of Neuronal Oxygen Responses in C. elegans Is Mediated through Interactions between Globin 5 and the H-NOX Domains of Soluble Guanylate Cyclases.

    PubMed

    Abergel, Zohar; Chatterjee, Arijit Kumar; Zuckerman, Binyamin; Gross, Einav

    2016-01-20

    Soluble guanylate cyclases (sGCs) are gas-binding proteins that control diverse physiological processes such as vasodilation, platelet aggregation, and synaptic plasticity. In the nematode Caenorhabditis elegans, a complex of sGCs, GCY-35 and GCY-36, functions in oxygen (O2) sensing. Previous studies suggested that the neuroglobin GLB-5 genetically interacts with GCY-35, and that the inhibitory effect of GLB-5 on GCY-35 function is necessary for fast recovery from prolonged hypoxia. In this study, we identified mutations in gcy-35 and gcy-36 that impact fast recovery and other phenotypes associated with GLB-5, without undermining sGC activity. These mutations, heb1 and heb3, change conserved amino acid residues in the regulatory H-NOX domains of GCY-35 and GCY-36, respectively, and appear to suppress GLB-5 activity by different mechanisms. Moreover, we observed that short exposure to 35% O2 desensitized the neurons responsible for ambient O2 sensing and that this phenomenon does not occur in heb1 animals. These observations may implicate sGCs in neuronal desensitization mechanisms far beyond the specific case of O2 sensing in nematodes. The conservation of functionally important regions of sGCs is supported by examining site-directed mutants of GCY-35, which suggested that similar regions in the H-NOX domains of O2 and NO-sensing sGCs are important for heme/gas interactions. Overall, our studies provide novel insights into sGC activity and regulation, and implicate similar structural determinants in the control of both O2 and NO sensors. Significance statement: Soluble guanylate cyclases (sGCs) control essential and diverse physiological processes, including memory processing. We used Caenorhabditis elegans to explore how a neuroglobin inhibits a complex of oxygen-sensing sGCs, identifying sGC mutants that resist inhibition. Resistance appears to arise by two different mechanisms: increased basal sGC activity or disruption of an interaction with neuroglobin. Our

  13. Regulation of Neuronal Oxygen Responses in C. elegans Is Mediated through Interactions between Globin 5 and the H-NOX Domains of Soluble Guanylate Cyclases.

    PubMed

    Abergel, Zohar; Chatterjee, Arijit Kumar; Zuckerman, Binyamin; Gross, Einav

    2016-01-20

    Soluble guanylate cyclases (sGCs) are gas-binding proteins that control diverse physiological processes such as vasodilation, platelet aggregation, and synaptic plasticity. In the nematode Caenorhabditis elegans, a complex of sGCs, GCY-35 and GCY-36, functions in oxygen (O2) sensing. Previous studies suggested that the neuroglobin GLB-5 genetically interacts with GCY-35, and that the inhibitory effect of GLB-5 on GCY-35 function is necessary for fast recovery from prolonged hypoxia. In this study, we identified mutations in gcy-35 and gcy-36 that impact fast recovery and other phenotypes associated with GLB-5, without undermining sGC activity. These mutations, heb1 and heb3, change conserved amino acid residues in the regulatory H-NOX domains of GCY-35 and GCY-36, respectively, and appear to suppress GLB-5 activity by different mechanisms. Moreover, we observed that short exposure to 35% O2 desensitized the neurons responsible for ambient O2 sensing and that this phenomenon does not occur in heb1 animals. These observations may implicate sGCs in neuronal desensitization mechanisms far beyond the specific case of O2 sensing in nematodes. The conservation of functionally important regions of sGCs is supported by examining site-directed mutants of GCY-35, which suggested that similar regions in the H-NOX domains of O2 and NO-sensing sGCs are important for heme/gas interactions. Overall, our studies provide novel insights into sGC activity and regulation, and implicate similar structural determinants in the control of both O2 and NO sensors. Significance statement: Soluble guanylate cyclases (sGCs) control essential and diverse physiological processes, including memory processing. We used Caenorhabditis elegans to explore how a neuroglobin inhibits a complex of oxygen-sensing sGCs, identifying sGC mutants that resist inhibition. Resistance appears to arise by two different mechanisms: increased basal sGC activity or disruption of an interaction with neuroglobin. Our

  14. Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors

    PubMed Central

    Mendez, Ana; Burns, Marie E.; Sokal, Izabela; Dizhoor, Alexander M.; Baehr, Wolfgang; Palczewski, Krzysztof; Baylor, Denis A.; Chen, Jeannie

    2001-01-01

    The retina's photoreceptor cells adjust their sensitivity to allow photons to be transduced over a wide range of light intensities. One mechanism thought to participate in sensitivity adjustments is Ca2+ regulation of guanylate cyclase (GC) by guanylate cyclase-activating proteins (GCAPs). We evaluated the contribution of GCAPs to sensitivity regulation in rods by disrupting their expression in transgenic mice. The GC activity from GCAPs−/− retinas showed no Ca2+ dependence, indicating that Ca2+ regulation of GCs had indeed been abolished. Flash responses from dark-adapted GCAPs−/− rods were larger and slower than responses from wild-type rods. In addition, the incremental flash sensitivity of GCAPs−/− rods failed to be maintained at wild-type levels in bright steady light. GCAP2 expressed in GCAPs−/− rods restored maximal light-induced GC activity but did not restore normal flash response kinetics. We conclude that GCAPs strongly regulate GC activity in mouse rods, decreasing the flash sensitivity in darkness and increasing the incremental flash sensitivity in bright steady light, thereby extending the rod's operating range. PMID:11493703

  15. Regulation of cGMP levels by guanylate cyclase in truncated frog rod outer segments

    PubMed Central

    1989-01-01

    Cyclic GMP is the second messenger in phototransduction and regulates the photoreceptor current. In the present work, we tried to understand the regulation mechanism of cytoplasmic cGMP levels in frog photoreceptors by measuring the photoreceptor current using a truncated rod outer segment (tROS) preparation. Since exogenously applied substance diffuses into tROS from the truncated end, we could examine the biochemical reactions relating to the cGMP metabolism by manipulating the cytoplasmic chemical condition. In tROS, exogenously applied GTP produced a dark current whose amplitude was half-maximal at approximately 0.4 mM GTP. The conductance for this current was suppressed by light in a fashion similar to when it is activated by cGMP. In addition, no current was produced in the absence of Mg2+, which is known to be necessary for the guanylate cyclase activity. These results indicate that guanylate cyclase was present in tROS and synthesized cGMP from exogenously applied GTP. The enzyme activity was distributed throughout the rod outer segment. The amount of synthesized cGMP increased as the cytoplasmic Ca2+ concentration of tROS decreased, which indicated the activation of guanylate cyclase at low Ca2+ concentrations. Half-maximal effect of Ca2+ was observed at approximately 100 nM. tROS contained the proteins involved in the phototransduction mechanism and therefore, we could examine the regulation of the light response waveform by Ca2+. At low Ca2+ concentrations, the time course of the light response was speeded up probably because cGMP recovery was facilitated by activation of the cyclase. Then, if the cytoplasmic Ca2+ concentration of a photoreceptor decreases during light stimulation, the Ca2+ decrease may explain the acceleration of the light response during light adaptation. In tROS, however, we did observe an acceleration during repetitive light flashes when the cytoplasmic Ca2+ concentration increased during the stimulation. This result suggests the

  16. Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase

    PubMed Central

    Sarkar, Anindya; Dai, Yue; Haque, Mohammad Mahfuzul; Seeger, Franziska; Ghosh, Arnab; Garcin, Elsa D.; Montfort, William R.; Hazen, Stanley L.; Misra, Saurav; Stuehr, Dennis J.

    2015-01-01

    Heat shock protein 90 (hsp90) drives heme insertion into the β1 subunit of soluble guanylate cyclase (sGC) β1, which enables it to associate with a partner sGCα1 subunit and mature into a nitric oxide (NO)-responsive active form. We utilized fluorescence polarization measurements and hydrogen-deuterium exchange mass spectrometry to define molecular interactions between the specific human isoforms hsp90β and apo-sGCβ1. hsp90β and its isolated M domain, but not its isolated N and C domains, bind with low micromolar affinity to a heme-free, truncated version of sGCβ1 (sGCβ1(1–359)-H105F). Surprisingly, hsp90β and its M domain bound to the Per-Arnt-Sim (PAS) domain of apo-sGC-β1(1–359), which lies adjacent to its heme-binding (H-NOX) domain. The interaction specifically involved solvent-exposed regions in the hsp90β M domain that are largely distinct from sites utilized by other hsp90 clients. The interaction strongly protected two regions of the sGCβ1 PAS domain and caused local structural relaxation in other regions, including a PAS dimerization interface and a segment in the H-NOX domain. Our results suggest a means by which the hsp90β interaction could prevent apo-sGCβ1 from associating with its partner sGCα1 subunit while enabling structural changes to assist heme insertion into the H-NOX domain. This mechanism would parallel that in other clients like the aryl hydrocarbon receptor and HIF1α, which also interact with hsp90 through their PAS domains to control protein partner and small ligand binding interactions. PMID:26134567

  17. Stimulation of guanylate cyclase by sodium nitroprusside, nitroglycerin and nitric oxide in various tissue preparations and comparison to the effects of sodium azide and hydroxylamine.

    PubMed

    Katsuki, S; Arnold, W; Mittal, C; Murad, F

    1977-02-01

    Sodium nitroprusside, nitroglycerin, sodium azide and hydroxylamine increased guanylate cyclase activity in particulate and/or soluble preparations from various tissues. While sodium nitroprusside increased guanylate cyclase activity in most of the preparations examined, the effects of sodium azide, hydroxylamine and nitroglycerin were tissue specific. Nitroglycerin and hydroxylamine were also less potent. Neither the protein activator factor nor catalase which is required for sodium azide effects altered the stimulatory effect of sodium nitroprusside. In the presence of sodium azide, sodium nitroprusside or hydroxylamine, magnesium ion was as effective as manganese ion as a sole cation cofactor for guanylate cyclase. With soluble guanylate cyclase from rat liver and bovine tracheal smooth muscle the concentrations of sodium nitroprusside that gave half-maximal stimulation with Mn2+ were 0.1 mM and 0.01 mM, respectively. Effective concentrations were slightly less with Mg2+ as a sole cation cofactor. The ability of these agents to increase cyclic GMP levels in intact tissues is probably due to their effects on guanylate cyclase activity. While the precise mechanism of guanylate cyclase activation by these agents is not known, activation may be due to the formation of nitric oxide or another reactive material since nitric oxide also increased guanylate cyclase activity. PMID:14978

  18. Interaction of retinal guanylate cyclase with the alpha subunit of transducin: potential role in transducin localization.

    PubMed

    Rosenzweig, Derek H; Nair, K Saidas; Levay, Konstantin; Peshenko, Igor V; Crabb, John W; Dizhoor, Alexander M; Slepak, Vladlen Z

    2009-02-01

    Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.

  19. Identification of guanylate cyclases and related signaling proteins in sperm tail from sea stars by mass spectrometry.

    PubMed

    Nakachi, Mia; Matsumoto, Midori; Terry, Philip M; Cerny, Ronald L; Moriyama, Hideaki

    2008-01-01

    Marine invertebrates employ external fertilization to take the advantages of sexual reproduction as one of excellent survival strategies. To prevent mismatching, successful fertilization can be made only after going though strictly defined steps in the fertilization. In sea stars, the fertilization process starts with the chemotaxis of sperm followed by hyperactivation of sperm upon arriving onto the egg coat, and then sperm penetrate to the egg coat before achieving the fusion. To investigate whether the initiation of chemotaxis and the following signaling has species specificity, we conducted comparative studies in the protein level among sea stars, Asterias amurensis, A. forbesi, and Asterina pectinifera. Since transcription of messenger ribonucleic acid (mRNA) has been suppressed in gamete, the roles of sperm proteins during the fertilization cannot be investigated by examining the mRNA profile. Therefore, proteomics analysis by mass spectrometry was used in this study. In sea stars, upon receiving asteroidal sperm-activating peptide (asterosap), the receptor membrane-bound guanylate cyclases in the sperm tail trigger sperm chemotaxis. We confirmed the presence of membrane-bound guanylate cyclases in the three sea star species, and they all had the same structural domains including the extracellular domain, kinase-like domain, and guanylate cyclase domain. The majority of peptides recovered were from alpha-helices distributed on the solvent side of the protein. More peptides were recovered from the intracellular domains. The transmembrane domain has not been recovered. The functions of the receptors seemed to be conserved among the species. Furthermore, we identified proteins that may be involved in the guanylate cyclase-triggered signaling pathway.

  20. A role for guanylate cyclase C in acid-stimulated duodenal mucosal bicarbonate secretion.

    PubMed

    Rao, S P; Sellers, Z; Crombie, D L; Hogan, D L; Mann, E A; Childs, D; Keely, S; Sheil-Puopolo, M; Giannella, R A; Barrett, K E; Isenberg, J I; Pratha, V S

    2004-01-01

    Luminal acidification provides the strongest physiological stimulus for duodenal HCO3- secretion. Various neurohumoral mechanisms are believed to play a role in acid-stimulated HCO3- secretion. Previous studies in the rat and human duodenum have shown that guanylin and Escherichia coli heat-stable toxin, both ligands of the transmembrane guanylyl cyclase receptor [guanylate cyclase C (GC-C)], are potent stimulators for duodenal HCO3- secretion. We postulated that the GC-C receptor plays an important role in acid-stimulated HCO3- secretion. In vivo perfusion studies performed in wild-type (WT) and GC-C knockout (KO) mice indicated that acid-stimulated duodenal HCO3- secretion was significantly decreased in the GC-C KO animals compared with the WT counterparts. Pretreatment with PD-98059, an MEK inhibitor, resulted in attenuation of duodenal HCO3- secretion in response to acid stimulation in the WT mice with no further effect in the KO mice. In vitro cGMP generation studies demonstrated a significant and comparable increase in cGMP levels on acid exposure in the duodenum of both WT and KO mice. In addition, a rapid, time-dependent phosphorylation of ERK was observed with acid exposure in the duodenum of WT mice, whereas a marked attenuation in ERK phosphorylation was observed in the KO animals despite equivalent levels of ERK in both groups of animals. On the basis of these studies, we conclude that transmembrane GC-C is a key mediator of acid-stimulated duodenal HCO3- secretion. Furthermore, ERK phosphorylation may be an important intracellular mediator of duodenal HCO3- secretion. PMID:12881226

  1. Genetic modifiers of hypertension in soluble guanylate cyclase α1–deficient mice

    PubMed Central

    Buys, Emmanuel S.; Raher, Michael J.; Kirby, Andrew; Mohd, Shahid; Baron, David M.; Hayton, Sarah R.; Tainsh, Laurel T.; Sips, Patrick Y.; Rauwerdink, Kristen M.; Yan, Qingshang; Tainsh, Robert E.T.; Shakartzi, Hannah R.; Stevens, Christine; Decaluwé, Kelly; Rodrigues-Machado, Maria da Gloria; Malhotra, Rajeev; Van de Voorde, Johan; Wang, Tong; Brouckaert, Peter; Daly, Mark J.; Bloch, Kenneth D.

    2012-01-01

    Nitric oxide (NO) plays an essential role in regulating hypertension and blood flow by inducing relaxation of vascular smooth muscle. Male mice deficient in a NO receptor component, the α1 subunit of soluble guanylate cyclase (sGCα1), are prone to hypertension in some, but not all, mouse strains, suggesting that additional genetic factors contribute to the onset of hypertension. Using linkage analyses, we discovered a quantitative trait locus (QTL) on chromosome 1 that was linked to mean arterial pressure (MAP) in the context of sGCα1 deficiency. This region is syntenic with previously identified blood pressure–related QTLs in the human and rat genome and contains the genes coding for renin. Hypertension was associated with increased activity of the renin-angiotensin-aldosterone system (RAAS). Further, we found that RAAS inhibition normalized MAP and improved endothelium-dependent vasorelaxation in sGCα1-deficient mice. These data identify the RAAS as a blood pressure–modifying mechanism in a setting of impaired NO/cGMP signaling. PMID:22565307

  2. Intestinal Enteroids Model Guanylate Cyclase C-Dependent Secretion Induced by Heat-Stable Enterotoxins.

    PubMed

    Pattison, Amanda M; Blomain, Erik S; Merlino, Dante J; Wang, Fang; Crissey, Mary Ann S; Kraft, Crystal L; Rappaport, Jeff A; Snook, Adam E; Lynch, John P; Waldman, Scott A

    2016-10-01

    Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease. PMID:27481254

  3. Regulation of intraocular pressure by soluble and membrane guanylate cyclases and their role in glaucoma

    PubMed Central

    Buys, Emmanuel S.; Potter, Lincoln R.; Pasquale, Louis R.; Ksander, Bruce R.

    2014-01-01

    Glaucoma is a progressive optic neuropathy characterized by visual field defects that ultimately lead to irreversible blindness (Alward, 2000; Anderson et al., 2006). By the year 2020, an estimated 80 million people will have glaucoma, 11 million of which will be bilaterally blind. Primary open-angle glaucoma (POAG) is the most common type of glaucoma. Elevated intraocular pressure (IOP) is currently the only risk factor amenable to treatment. How IOP is regulated and can be modulated remains a topic of active investigation. Available therapies, mostly geared toward lowering IOP, offer incomplete protection, and POAG often goes undetected until irreparable damage has been done, highlighting the need for novel therapeutic approaches, drug targets, and biomarkers (Heijl et al., 2002; Quigley, 2011). In this review, the role of soluble (nitric oxide (NO)-activated) and membrane-bound, natriuretic peptide (NP)-activated guanylate cyclases that generate the secondary signaling molecule cyclic guanosine monophosphate (cGMP) in the regulation of IOP and in the pathophysiology of POAG will be discussed. PMID:24904270

  4. Membrane guanylate cyclase, a multimodal transduction machine: history, present, and future directions

    PubMed Central

    Sharma, Rameshwar K.; Duda, Teresa

    2014-01-01

    A sequel to these authors' earlier comprehensive reviews which covered the field of mammalian membrane guanylate cyclase (MGC) from its origin to the year 2010, this article contains 13 sections. The first is historical and covers MGC from the year 1963–1987, summarizing its colorful developmental stages from its passionate pursuit to its consolidation. The second deals with the establishment of its biochemical identity. MGC becomes the transducer of a hormonal signal and founder of the peptide hormone receptor family, and creates the notion that hormone signal transduction is its sole physiological function. The third defines its expansion. The discovery of ROS-GC subfamily is made and it links ROS-GC with the physiology of phototransduction. Sections ROS-GC, a Ca2+-Modulated Two Component Transduction System to Migration Patterns and Translations of the GCAP Signals Into Production of Cyclic GMP are Different cover its biochemistry and physiology. The noteworthy events are that augmented by GCAPs, ROS-GC proves to be a transducer of the free Ca2+ signals generated within neurons; ROS-GC becomes a two-component transduction system and establishes itself as a source of cyclic GMP, the second messenger of phototransduction. Section ROS-GC1 Gene Linked Retinal Dystrophies demonstrates how this knowledge begins to be translated into the diagnosis and providing the molecular definition of retinal dystrophies. Section Controlled By Low and High Levels of [Ca2+]i, ROS-GC1 is a Bimodal Transduction Switch discusses a striking property of ROS-GC where it becomes a “[Ca2+]i bimodal switch” and transcends its signaling role in other neural processes. In this course, discovery of the first CD-GCAP (Ca2+-dependent guanylate cyclase activator), the S100B protein, is made. It extends the role of the ROS-GC transduction system beyond the phototransduction to the signaling processes in the synapse region between photoreceptor and cone ON-bipolar cells; in section Ca2

  5. Activation of guanylate cyclase-C attenuates stretch responses and sensitization of mouse colorectal afferents

    PubMed Central

    Feng, Bin; Kiyatkin, Michael E.; La, Jun-Ho; Ge, Pei; Solinga, Robert; Silos-Santiago, Inmaculada; Gebhart, G.F.

    2013-01-01

    Irritable bowel syndrome (IBS) is characterized by altered bowel habits, persistent pain and discomfort, and typically colorectal hypersensitivity. Linaclotide, a peripherally-restricted 14-amino acid peptide approved for the treatment of IBS with constipation, relieves constipation and reduces IBS-associated pain in these patients presumably by activation of guanylate cyclase-C (GC-C), which stimulates production and release of cyclic guanosine monophosphate (cGMP) from intestinal epithelial cells. We investigated whether activation of GC-C by the endogenous agonist uroguanylin or the primary downstream effector of that activation, cGMP, directly modulates responses and sensitization of mechanosensitive colorectal primary afferents. The distal 2 cm of mouse colorectum with attached pelvic nerve was harvested, pinned flat mucosal side up for in vitro single-fiber recordings and the encoding properties of mechanosensitive afferents (serosal, mucosal, muscular and muscular-mucosal) to probing and circumferential stretch studied. Both cGMP (10–300μM) and uroguanylin (1–1000nM) applied directly to colorectal receptive endings significantly reduced responses of muscular and muscular-mucosal afferents to stretch; serosal and mucosal afferents were not affected. Sensitized responses (i.e., increased responses to stretch) of muscular and muscular-mucosal afferents were reversed by cGMP, returning responses to stretch to control. Blocking the transport of cGMP from colorectal epithelia by probenecid, a mechanism validated by studies in cultured intestinal T84 cells, abolished the inhibitory effect of uroguanylin on muscular-mucosal afferents. These results suggest that GC-C agonists like linaclotide alleviate colorectal pain and hypersensitivity by dampening stretch-sensitive afferent mechanosensitivity and normalizing afferent sensitization. PMID:23739979

  6. Effects of hydroxyl radical scavengers KCN and CO on ultraviolet light-induced activation of crude soluble guanylate cyclase

    SciTech Connect

    Karlsson, J.O.; Axelsson, K.L.; Andersson, R.G.

    1985-01-01

    The crude soluble guanylate cyclase (GC) from bovine mesenteric artery was stimulated by ultraviolet (UV) light (366 nm). Addition of free radical scavengers, dimethylsulfoxide or superoxide dismutase and/or catalase to the GC assay did not abolish the stimulatory effect of UV light. On the contrary, the UV light-induced activation was enhanced in the presence of these scavengers. KCN (1 mM) did not affect the UV light-induced activation, while 0.1 mM of CO potentiated the activation. These results may indicate that UV light is operating through a direct interaction with the ferrous form of the GC-heme.

  7. Guanylate cyclase C limits systemic dissemination of a murine enteric pathogen

    PubMed Central

    2013-01-01

    Background Guanylate Cyclase C (GC-C) is an apically-oriented transmembrane receptor that is expressed on epithelial cells of the intestine. Activation of GC-C by the endogenous ligands guanylin or uroguanylin elevates intracellular cGMP and is implicated in intestinal ion secretion, cell proliferation, apoptosis, intestinal barrier function, as well as the susceptibility of the intestine to inflammation. Our aim was to determine if GC-C is required for host defense during infection by the murine enteric pathogen Citrobacter rodentium of the family Enterobacteriacea. Methods GC-C+/+ control mice or those having GC-C genetically ablated (GC-C−/−) were administered C. rodentium by orogastric gavage and analyzed at multiple time points up to post-infection day 20. Commensal bacteria were characterized in uninfected GC-C+/+ and GC-C−/− mice using 16S rRNA PCR analysis. Results GC-C−/− mice had an increase in C. rodentium bacterial load in stool relative to GC-C+/+. C. rodentium infection strongly decreased guanylin expression in GC-C+/+ mice and, to an even greater degree, in GC-C−/− animals. Fluorescent tracer studies indicated that mice lacking GC-C, unlike GC-C+/+ animals, had a substantial loss of intestinal barrier function early in the course of infection. Epithelial cell apoptosis was significantly increased in GC-C−/− mice following 10 days of infection and this was associated with increased frequency and numbers of C. rodentium translocation out of the intestine. Infection led to significant liver histopathology in GC-C−/− mice as well as lymphocyte infiltration and elevated cytokine and chemokine expression. Relative to naïve GC-C+/+ mice, the commensal microflora load in uninfected GC-C−/− mice was decreased and bacterial composition was imbalanced and included outgrowth of the Enterobacteriacea family. Conclusions This work demonstrates the novel finding that GC-C signaling is an essential component of host defense during

  8. Pharmacokinetic interaction profile of riociguat, a new soluble guanylate cyclase stimulator, in vitro.

    PubMed

    Rickert, Verena; Haefeli, Walter Emil; Weiss, Johanna

    2014-08-01

    Riociguat is a new soluble guanylate cyclase stimulator under development for pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension. So far, the interaction potential of riociguat with other drugs is nearly unknown. Therefore, we assessed in vitro the potency of riociguat to inhibit important drug metabolising enzymes (cytochrome P450 (CYP) 3A4, CYP2C19, and CYP2D6) and drug transporters (P-glycoprotein (P-gp/ABCB1), breast cancer resistance protein (BCRP/ABCG2), and organic anion transporting polypeptides (OATP) 1B1 and 1B3). In addition we evaluated its substrate characteristics for P-gp, BCRP, and the multidrug resistance-associated protein 1 (MRP1/ABCC1). We also assessed riociguat's inducing properties on important drug metabolising enzymes and transporters and investigated its ability to activate the pregnane-X-receptor (PXR). Riociguat was identified as a weak to moderate inhibitor of P-gp (f2-value: 11.7 ± 4.8 μM), BCRP (IC50 = 46.2 ± 20.3 μM), OATP1B1 (IC50 = 34.1 ± 3.15 μM), OATP1B3 (IC50 = 50.3 ± 7.5 μM), CYP2D6 (IC50 = 12.4 ± 0.74 μM), and CYP2C19 (IC50 = 46.1 ± 7.14 μM). Furthermore, it induced mRNA expression of BCRP/ABCG2 (3-fold at 20 μM) and to a lesser extent of CYP3A4 (2.3-fold at 20 μM), UGT1A4, and ABCB11. The only weak inducing properties were confirmed by weak activation of PXR. Considering its systemic concentrations its interaction potential as a perpetrator drug seems to be low. In contrast, our data suggest that riociguat is a P-gp substrate and might therefore act as a victim drug when co-administered with strong P-gp inductors or inhibitors. PMID:24657506

  9. A constitutively activated mutant of human soluble guanylyl cyclase (sGC): implication for the mechanism of sGC activation

    NASA Technical Reports Server (NTRS)

    Martin, Emil; Sharina, Iraida; Kots, Alexander; Murad, Ferid

    2003-01-01

    Heterodimeric alphabeta soluble guanylyl cyclase (sGC) is a recognized receptor for nitric oxide (NO) and mediates many of its physiological functions. Although it has been clear that the heme moiety coordinated by His-105 of the beta subunit is crucial for mediating the activation of the enzyme by NO, it is not understood whether the heme moiety plays any role in the function of the enzyme in the absence of NO. Here we analyze the effects of biochemical and genetic removal of heme and its reconstitution on the activity of the enzyme. Detergent-induced loss of heme from the wild-type alphabeta enzyme resulted in several-fold activation of the enzyme. This activation was inhibited after hemin reconstitution. A heme-deficient mutant alphabetaCys-105 with Cys substituted for His-105 was constitutively active with specific activity approaching the activity of the wild-type enzyme activated by NO. However, reconstitution of mutant enzyme with heme and/or DTT treatment significantly inhibited the enzyme. Mutant enzyme reconstituted with ferrous heme was activated by NO and CO alone and showed additive effects between gaseous effectors and the allosteric activator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrim idin-4-ylamine. We propose that the heme moiety through its coordination with His-105 of the beta subunit acts as an endogenous inhibitor of sGC. Disruption of the heme-coordinating bond induced by binding of NO releases the restrictions imposed by this bond and allows the formation of an optimally organized catalytic center in the heterodimer.

  10. Mutations in a guanylate cyclase GCY-35/GCY-36 modify Bardet-Biedl syndrome-associated phenotypes in Caenorhabditis elegans.

    PubMed

    Mok, Calvin A; Healey, Michael P; Shekhar, Tanvi; Leroux, Michel R; Héon, Elise; Zhen, Mei

    2011-10-01

    Ciliopathies are pleiotropic and genetically heterogeneous disorders caused by defective development and function of the primary cilium. Bardet-Biedl syndrome (BBS) proteins localize to the base of cilia and undergo intraflagellar transport, and the loss of their functions leads to a multisystemic ciliopathy. Here we report the identification of mutations in guanylate cyclases (GCYs) as modifiers of Caenorhabditis elegans bbs endophenotypes. The loss of GCY-35 or GCY-36 results in suppression of the small body size, developmental delay, and exploration defects exhibited by multiple bbs mutants. Moreover, an effector of cGMP signalling, a cGMP-dependent protein kinase, EGL-4, also modifies bbs mutant defects. We propose that a misregulation of cGMP signalling, which underlies developmental and some behavioural defects of C. elegans bbs mutants, may also contribute to some BBS features in other organisms. PMID:22022287

  11. Digital Holographic Tomography and Fluorescence Used to Localize sGC in HEK293 Cells

    NASA Astrophysics Data System (ADS)

    Sheldrake, Eric; Mann, Christopher; Gage, Matthew

    2014-03-01

    Digital Holographic Tomography (DHT) is used to analyze and localize the intracellular protein soluble guanylate cyclase (sGC) in human embryonic kidney 293 (HEK293) cells. DHT is a non-invasive phase microscopy technique that provides three dimensional quantitative information of HEK293 cells including variance of index of refraction or physical thickness. A fluorescence component will be added to the microscope to further studies of sGC localization. The signaling pathway including nitric oxide (NO) and sGC is studied and has been linked to various cardiovascular diseases, platelet aggregation, and variations in blood pressure via vasodilation. sGC will be labeled using a fluorescent antibody and analyzed using the DHT microscope. DHT will be used to analyze changes in sGC localization in its natural environment and when stimulated by NO. An understanding of how sGC interacts with its surroundings is vital to further research in cardiovascular disease.

  12. Kinetics of nitric oxide dissociation from five- and six-coordinate nitrosyl hemes and heme proteins, including soluble guanylate cyclase.

    PubMed

    Kharitonov, V G; Sharma, V S; Magde, D; Koesling, D

    1997-06-01

    Kinetics of NO dissociation were characterized for three five-coordinate systems, heme-NO, HSA-heme-NO (human serum albumin), GC-NO (soluble guanylate cyclase), and for the six-coordinate system, Im-heme-NO. Nitrosyl myoglobin was redetermined for comparison. Previously known, six-coordinate R and T state nitrosyl hemoglobins are also included in the comparison. The data indicate that NO dissociates more than 1000 times faster from five-coordinate model heme than it does from the six-coordinate analog. Such a negative trans-effect between NO and a proximal base is in sharp contrast to carboxy heme derivatives, in which ligand dissociation rates are greatly slowed in when a trans base is present. As a result of opposite trans-effects, six-coordinate carboxy and nitrosyl derivatives have comparable dissociation rates, even though the five-coordinate species are very different. In proteins, five- and six-coordinate forms do not show a large difference in dissociation rates. Part of the reason may be due to different probabilities for geminate recombination in the different proteins, but this cannot explain all the facts. There must also be influences of the protein structure on bond-breaking rate constants themselves. With the exception of hemoglobin in the T state, nitrosyl guanylate cyclase shows the highest NO dissociation rate constant, k(obs) = 6 x 10(-4) s(-1). This would yield a half-life of about 2 min at 37 degrees C for dissociation of NO from GC-NO, a number that has implications for the mechanism of regulation of the activity of this key heme enzyme.

  13. Overexpression of Guanylate Cyclase Activating Protein 2 in Rod Photoreceptors In Vivo Leads to Morphological Changes at the Synaptic Ribbon

    PubMed Central

    López-Begines, Santiago; Fernández-Sánchez, Laura; Cuenca, Nicolás; Llorens, Jordi; de la Villa, Pedro; Méndez, Ana

    2012-01-01

    Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic

  14. Tumor necrosis factor alpha activates soluble guanylate cyclase in bovine glomerular mesangial cells via an L-arginine-dependent mechanism

    PubMed Central

    1990-01-01

    Endothelium-derived nitric oxide (NO) causes vasodilatation by activating soluble guanylate cyclase, and glomerular mesangial cells respond to NO with elevations of intracellular guanosine 3',5'-cyclic monophosphate (cGMP). We explored whether mesangial cells can be stimulated to produce NO and whether NO modulates mesangial cell function in an autocrine or paracrine fashion. Tumor necrosis factor alpha (TNF-alpha) raised mesangial cell cGMP levels in a time- and concentration-dependent manner (threshold dose 1 ng/ml, IC50 13.8 ng/ml, maximal response 100 ng/ml). TNF-alpha-induced increases in mesangial cGMP content were evident at 8 h and maximal at 18-24 h. The TNF-alpha-induced stimulation of mesangial cell cGMP production was abrogated by actinomycin D or cycloheximide suggesting dependence on new RNA or protein synthesis. Hemoglobin and methylene blue, both known to inhibit NO action, dramatically reduced TNF-alpha-induced mesangial cell cGMP production. Superoxide dismutase, known to potentiate NO action, augmented the TNF-alpha-induced effect. Ng-monomethyl-L- arginine (L-NMMA) decreased cGMP levels in TNF-alpha-treated, but not vehicle-treated mesangial cells in a concentration-dependent manner (IC50 53 microM). L-arginine had no effect on cGMP levels in control or TNF-alpha-treated mesangial cells but reversed L-NMMA-induced inhibition. Interleukin 1 beta and lipopolysaccharide (LPS), but not interferon gamma, also increased mesangial cell cGMP content. Transforming growth factor beta 1 blunted the mesangial cell response to TNF-alpha. TNF-alpha-induced L-arginine-dependent increases in cGMP were also evident in bovine renal artery vascular smooth muscle cells, COS-1 cells, and 1502 human fibroblasts. These findings suggest that TNF-alpha induces expression in mesangial cell of an enzyme(s) involved in the formation of L-arginine-derived NO. Moreover, the data indicate that NO acts in an autocrine and paracrine fashion to activate mesangial cell soluble

  15. Effects of a novel guanylate cyclase inhibitor on nitric oxide-dependent inhibitory neurotransmission in canine proximal colon

    PubMed Central

    Franck, H; Sweeney, K M; Sanders, K M; Shuttleworth, C W R

    1997-01-01

    Previous studies suggested that nitric oxide (NO) may cause hyperpolarization and relaxation of canine colonic smooth muscle by both cGMP-dependent and cGMP-independent mechanisms. This hypothesis was tested using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a novel inhibitor of NO-stimulated guanylate cyclase. In the presence of histamine (30 μM), atropine and indomethacin (both at 1 μM), electrical field stimulation of intrinsic neurons (EFS; 5 Hz) produced inhibition of phasic contractile activity that is due to NO synthesis. ODQ caused a concentration-dependent block of this response (10 nM to 10 μM). Inhibitory junction potentials (IJPs) due to NO synthesis were recorded from muscle cells located near the myenteric border of the circular muscle layer, using intracellular microelectrodes. IJPs were abolished by ODQ (1–10 μM). EFS (10–20 Hz) produced frequency-dependent inhibition of electrical slow waves recorded from cells located near the submucosal surface of the circular muscle layer. This inhibition is due to NO synthesis, and it was abolished by ODQ (1–10 μM). Hyperpolarization and relaxation produced by an NO donor, sodium nitroprusside, were abolished by ODQ pretreatment (1–10 μM). In contrast, inhibitory responses to 8-Br-cGMP (1 mM) were unaffected by ODQ. ODQ alone (1–10 μM) had no significant effect on spontaneous electrical or phasic contractile activity. In tissues pre-treated with L-NAME (300 μM), ODQ decreased the amplitude of spontaneous or histamine-stimulated phasic contractile activity. These results suggest that electrical and mechanical effects of endogenously released and exogenously applied NO in canine colon are largely due to cGMP synthesis by ODQ-sensitive soluble guanylate cyclase. No evidence to support a direct (cGMP-independent) mechanism of NO action was found. ODQ also appears to cause a non-specific inhibition of muscle contractile activity; however, this effect does not

  16. A gain-of-function screen in zebrafish identifies a guanylate cyclase with a role in neuronal degeneration

    PubMed Central

    Maddison, Lisette A.; Lu, Jianjun; Victoroff, Tristan; Scott, Ethan; Baier, Herwig; Chen, Wenbiao

    2013-01-01

    Manipulation of gene expression is one of the most informative ways to study gene function. Genetic screens have been an informative method to identify genes involved in developmental processes. In the zebrafish, loss-of-function screens have been the primary approach for these studies. We sought to complement loss-of-function screens using an unbiased approach to overexpress genes with a Gal4-UAS based system, similar to the gain-of-function screens in Drosophila. Using MMLV as a mutagenic vector, a cassette containing a UAS promoter was readily inserted in the genome, often at the 5′ end of genes, allowing Gal4-dependent overexpression. We confirmed that genes downstream of the viral insertions were overexpressed in a Gal4-VP16 dependent manner. We further demonstrate that misexpression of one such downstream gene gucy2F, a membrane-bound guanylate cyclase, throughout the nervous system results in multiple defects including a loss of forebrain neurons. This suggests proper control of cGMP production is important in neuronal survival. From this study we propose that this gain-of-function approach can be applied to large-scale genetic screens in a vertebrate model organism and may reveal previously unknown gene function. PMID:19221799

  17. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    SciTech Connect

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S.

    2011-06-01

    Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures of the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.

  18. Androgen-sensitive hypertension associated with soluble guanylate cyclase-α1 deficiency is mediated by 20-HETE.

    PubMed

    Dordea, Ana C; Vandenwijngaert, Sara; Garcia, Victor; Tainsh, Robert E T; Nathan, Daniel I; Allen, Kaitlin; Raher, Michael J; Tainsh, Laurel T; Zhang, Fan; Lieb, Wolfgang S; Mikelman, Sarah; Kirby, Andrew; Stevens, Christine; Thoonen, Robrecht; Hindle, Allyson G; Sips, Patrick Y; Falck, John R; Daly, Mark J; Brouckaert, Peter; Bloch, Kenneth D; Bloch, Donald B; Malhotra, Rajeev; Schwartzman, Michal L; Buys, Emmanuel S

    2016-06-01

    Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1 (-/-) mice) display sex- and strain-specific hypertension: male but not female sGCα1 (-/-) mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex- and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGCα1 (-/-)S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGCα1 (-/-)S6 than of male sGCα1 (-/-)B6 mice. Furthermore, treating male sGCα1 (-/-)S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background- and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling.

  19. The process of S-nitrosation in sGC β1(1-194) revealed by infrared spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, Li; Wang, Dandan; Xu, Haoran; Mi, Mengdan; Li, Zhengqiang

    2015-06-01

    Soluble guanylate cyclase (sGC) is the most important receptor for the signaling molecule NO. NO activates sGC by binding to its heme cofactor and reacts with free thiols in the protein itself. The S-nitrosation of cysteine thiols affects the activity of soluble guanylate cyclase (sGC). In this study, infrared (IR) spectroscopy and site-directed mutagenesis were used to investigate S-H vibration and the process of S-nitrosation in the β1 subunit (amino acids 1-194) of sGC. Fourier transform IR spectroscopy revealed that wild-type and mutants (C78S and C122S) of sGC β1(1-194) exhibited S-H peaks around 2560 cm-1. The signals were attenuated in the IR spectra of S-nitrosoglutathione-treated mutants, demonstrating that S-nitrosation in sGC β1(1-194) occured at residues C78 and C122, and the process of the reaction was GSNO concentration-dependent.

  20. Expression of natriuretic peptides, nitric oxide synthase, and guanylate cyclase activity in frog mesonephros during the annual cycle.

    PubMed

    Fenoglio, Carla; Visai, Livia; Addario, Concetta; Gerzeli, Giuseppe; Milanesi, Gloria; Vaccarone, Rita; Barni, Sergio

    2004-06-01

    Natriuretic peptides (NPs), a family of structurally related hormones and nitric oxide (NO), generated by nitric oxide synthase (NOS), are believed to be involved in the regulation of fluid balance and sodium homeostasis. Differential expression and regulation of these factors depend on both physiological and pathological conditions. Both NPs and NO act in target organs through the activation of guanylate cyclase (GC) and the generation of guanosine 3',5'-cyclic monophosphate (cGMP), which is considered a common messenger for the action of these factors. The present study was designed to investigate--by histochemical methods--the expression of some NPs (proANP and ANP) and isoforms of NOS (neuronal NOS, nNOS, and inducible NOS, iNOS) in the mesonephros of Rana esculenta in different periods of the year including hibernation, to evaluate possible seasonal changes in their expression. We also studied the enzyme activity of NOS-related nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) and of GC. The experiments were performed on pieces of kidney of R. esculenta collected in their natural environment during active and hibernating life. The study was carried out using immunohistochemical techniques to demonstrate proANP, ANP, and some NOS isoforms. Antigen capture by enzyme linked immunosorbent assay (ELISA) was also performed to determine the presence of NPs in the frog kidney extract. Enzyme histochemistry was used to demonstrate the NOS-related NADPHd activity at light microscopy; GC activity was visualized at the electron microscope, using cerium as capture agent. The application of the immunohistochemical techniques demonstrated that frog mesonephros tubules express different patterns of distribution and/or expression of ANP and NOS during the annual cycle. Comparing the results obtained on active and hibernating frogs has provided interesting data; the NOS/NADPHd and GC activities showed some variations as well. Furthermore, the presence of NPs in

  1. Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

    PubMed Central

    Purohit, Rahul; Weichsel, Andrzej; Montfort, William R

    2013-01-01

    Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ∼150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme-Nitric Oxide Oxygen (H-NOX), Per-ARNT-Sim (PAS), coiled-coil and cyclase domains. Here, we present the crystal structure of the α1 PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β1 H-NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction. PMID:23934793

  2. GLOBIN-5-Dependent O2 Responses Are Regulated by PDL-1/PrBP That Targets Prenylated Soluble Guanylate Cyclases to Dendritic Endings

    PubMed Central

    Soltesz, Zoltan; Oda, Shigekazu; Zelmanovich, Veronica; Abergel, Zohar

    2014-01-01

    Aerobic animals constantly monitor and adapt to changes in O2 levels. The molecular mechanisms involved in sensing O2 are, however, incompletely understood. Previous studies showed that a hexacoordinated globin called GLB-5 tunes the dynamic range of O2-sensing neurons in natural C. elegans isolates, but is defective in the N2 lab reference strain (McGrath et al., 2009; Persson et al., 2009). GLB-5 enables a sharp behavioral switch when O2 changes between 21 and 17%. Here, we show that GLB-5 also confers rapid behavioral and cellular recovery from exposure to hypoxia. Hypoxia reconfigures O2-evoked Ca2+ responses in the URX O2 sensors, and GLB-5 enables rapid recovery of these responses upon re-oxygenation. Forward genetic screens indicate that GLB-5's effects on O2 sensing require PDL-1, the C. elegans ortholog of mammalian PrBP/PDE6δ protein. In mammals, PDE6δ regulates the traffic and activity of prenylated proteins (Zhang et al., 2004; Norton et al., 2005). PDL-1 promotes localization of GCY-33 and GCY-35, atypical soluble guanylate cyclases that act as O2 sensors, to the dendritic endings of URX and BAG neurons, where they colocalize with GLB-5. Both GCY-33 and GCY-35 are predicted to be prenylated. Dendritic localization is not essential for GCY-35 to function as an O2 sensor, but disrupting pdl-1 alters the URX neuron's O2 response properties. Functional GLB-5 can restore dendritic localization of GCY-33 in pdl-1 mutants, suggesting GCY-33 and GLB-5 are in a complex. Our data suggest GLB-5 and the soluble guanylate cyclases operate in close proximity to sculpt O2 responses. PMID:25505325

  3. 7-Nitroindazole and methylene blue, inhibitors of neuronal nitric oxide synthase and NO-stimulated guanylate cyclase, block MK-801-elicited behaviors in mice.

    PubMed

    Deutsch, S I; Rosse, R B; Paul, S M; Tomasino, V; Koetzner, L; Morn, C B; Mastropaolo, J

    1996-07-01

    We examined the abilities of 7-nitroindazole and methylene blue, inhibitors of the neuronal isoform of nitric oxide synthase (NOS) and nitric oxide-stimulated guanylate cyclase activity respectively, to attenuate explosive episodic jumping behavior(s) ("popping") elicited by MK-801 in mice. MK-801, like phencyclidine (PCP), is a high-affinity, noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. We have postulated that MK-801-elicited popping behavior in mice represents an animal model of schizophrenia, because popping behavior is markedly inhibited/antagonized by both typical and atypical antipsychotic drugs. In the present study, popping behavior induced by MK-801 was measured using an automated detection system that quantifies vertical displacements on the testing platform. 7-Nitroindazole (100 mg/kg) and methylene blue (32 and 100 mg/kg) significantly reduced the number and force of MK-801-elicited popping behavior. Mouse rotorod performance did not differ between animals receiving 7-nitroindazole, methylene blue, or their respective vehicles, suggesting that attenuation of MK-801-elicited popping behavior was not due to either sedation or ataxia caused by 7-nitroindazole or methylene blue. Our findings suggest that nitric oxide may, in part, mediate behaviors induced by NMDA receptor antagonists, like MK-801, and that inhibitors of NOS may have antipsychotic actions.

  4. Lentiviral Expression of Retinal Guanylate Cyclase-1 (RetGC1) Restores Vision in an Avian Model of Childhood Blindness

    PubMed Central

    Haire, Shannon E; Aleman, Tomas S; Cideciyan, Artur V; Sokal, Izabel; Palczewski, Krzysztof; Jacobson, Samuel G; Semple-Rowland, Susan L

    2006-01-01

    Background Leber congenital amaurosis (LCA) is a genetically heterogeneous group of retinal diseases that cause congenital blindness in infants and children. Mutations in the GUCY2D gene that encodes retinal guanylate cyclase–1 (retGC1) were the first to be linked to this disease group (LCA type 1 [LCA1]) and account for 10%–20% of LCA cases. These mutations disrupt synthesis of cGMP in photoreceptor cells, a key second messenger required for function of these cells. The GUCY1*B chicken, which carries a null mutation in the retGC1 gene, is blind at hatching and serves as an animal model for the study of LCA1 pathology and potential treatments in humans. Methods and Findings A lentivirus-based gene transfer vector carrying the GUCY2D gene was developed and injected into early-stage GUCY1*B embryos to determine if photoreceptor function and sight could be restored to these animals. Like human LCA1, the avian disease shows early-onset blindness, but there is a window of opportunity for intervention. In both diseases there is a period of photoreceptor cell dysfunction that precedes retinal degeneration. Of seven treated animals, six exhibited sight as evidenced by robust optokinetic and volitional visual behaviors. Electroretinographic responses, absent in untreated animals, were partially restored in treated animals. Morphological analyses indicated there was slowing of the retinal degeneration. Conclusions Blindness associated with loss of function of retGC1 in the GUCY1*B avian model of LCA1 can be reversed using viral vector-mediated gene transfer. Furthermore, this reversal can be achieved by restoring function to a relatively low percentage of retinal photoreceptors. These results represent a first step toward development of gene therapies for one of the more common forms of childhood blindness. PMID:16700630

  5. Primary response of the sGC heme binding domain to the cleavage of the Fe-His bond

    PubMed Central

    Zhang, Huali; Lu, Ming; Zhang, Yuebin; Li, Zhengqiang

    2008-01-01

    Soluble guanylate cyclase (sGC) is an important heme sensor protein. Regulation of the status of heme in the heme binding domain (or HNOX domain) by various gaseous activators can increase the catalytic efficiency of the cyclase domain. Several studies have demonstrated that the full activation of sGC is directly related to the cleavage of the Fe-His bond of the HNOX domain. To expand the primary response of the sGC HNOX domain to the cleavage event, a structural model of the sGC HNOX domain was constructed using homology modeling and the Fe-His bond was released at 6 ns of a 10-ns molecular dynamics simulation. An instant increment of Cα-RMSD over L2 (Loop2, residues 124-130) was found after the cleavage of the Fe-His bond, which was consistent with the principle component analysis (PCA). The energy analysis results suggest that the motions of L2 are energetic. Based on the results, energetic conformational transformation of L2 is identified as the primary response of the sGC HNOX domain to the cleavage of the Fe-His bond. PMID:18478082

  6. Meconium Ileus Caused by Mutations in GUCY2C, Encoding the CFTR-Activating Guanylate Cyclase 2C

    PubMed Central

    Romi, Hila; Cohen, Idan; Landau, Daniella; Alkrinawi, Suliman; Yerushalmi, Baruch; Hershkovitz, Reli; Newman-Heiman, Nitza; Cutting, Garry R.; Ofir, Rivka; Sivan, Sara; Birk, Ohad S.

    2012-01-01

    Meconium ileus, intestinal obstruction in the newborn, is caused in most cases by CFTR mutations modulated by yet-unidentified modifier genes. We now show that in two unrelated consanguineous Bedouin kindreds, an autosomal-recessive phenotype of meconium ileus that is not associated with cystic fibrosis (CF) is caused by different homozygous mutations in GUCY2C, leading to a dramatic reduction or fully abrogating the enzymatic activity of the encoded guanlyl cyclase 2C. GUCY2C is a transmembrane receptor whose extracellular domain is activated by either the endogenous ligands, guanylin and related peptide uroguanylin, or by an external ligand, Escherichia coli (E. coli) heat-stable enterotoxin STa. GUCY2C is expressed in the human intestine, and the encoded protein activates the CFTR protein through local generation of cGMP. Thus, GUCY2C is a likely candidate modifier of the meconium ileus phenotype in CF. Because GUCY2C heterozygous and homozygous mutant mice are resistant to E. coli STa enterotoxin-induced diarrhea, it is plausible that GUCY2C mutations in the desert-dwelling Bedouin kindred are of selective advantage. PMID:22521417

  7. Trpc2-expressing sensory neurons in the mouse main olfactory epithelium of type B express the soluble guanylate cyclase Gucy1b2

    PubMed Central

    Omura, Masayo; Mombaerts, Peter

    2015-01-01

    Chemoreception in the mouse olfactory system occurs primarily at two chemosensory epithelia in the nasal cavity: the main olfactory epithelium (MOE) and the vomeronasal epithelium. The canonical chemosensory neurons in the MOE, the olfactory sensory neurons (OSNs), express the odorant receptor (OR) gene repertoire, and depend on Adcy3 and Cnga2 for chemosensory signal transduction. The canonical chemosensory neurons in the vomeronasal epithelium, the vomeronasal sensory neurons (VSNs), express two unrelated vomeronasal receptor (VR) gene repertoires, and involve Trpc2 for chemosensory signal transduction. Recently we reported the discovery of two types of neurons in the mouse MOE that express Trcp2 in addition to Cnga2. These cell types can be distinguished at the single-cell level by expression of Adcy3: positive, type A and negative, type B. Some type A cells express OR genes. Thus far there is no specific gene or marker for type B cells, hampering further analyses such as physiological recordings. Here, we show that among MOE cells, type B cells are unique in their expression of the soluble guanylate cyclase Gucy1b2. We came across Gucy1b2 in an explorative approach based on Long Serial Analysis of Gene Expression (LongSAGE) that we applied to single red-fluorescent cells isolated from whole olfactory mucosa and vomeronasal organ of mice of a novel Trcp2-IRES-taumCherry gene-targeted strain. The generation of a novel Gucy1b2-IRES-tauGFP gene-targeted strain enabled us to visualize coalescence of axons of type B cells into glomeruli in the main olfactory bulb. Our molecular and anatomical analyses define Gucy1b2 as a marker for type B cells within the MOE. The Gucy1b2-IRES-tauGFP strain will be useful for physiological, molecular, cellular, and anatomical studies of this newly described chemosensory subsystem. PMID:25701815

  8. Assessment of the effects of hepatic impairment and smoking on the pharmacokinetics of a single oral dose of the soluble guanylate cyclase stimulator riociguat (BAY 63-2521)

    PubMed Central

    Becker, Corina; Unger, Sigrun; Schmidt, Anja; Wensing, Georg; Mück, Wolfgang

    2016-01-01

    Abstract Riociguat, a soluble guanylate cyclase stimulator developed for the treatment of pulmonary hypertension, is metabolized in part by the liver. Expression of one of the metabolizing enzymes, CYP1A1, is induced by aromatic hydrocarbons in tobacco smoke. Two nonrandomized, nonblinded studies were conducted to investigate the pharmacokinetics of riociguat in individuals with mild (Child-Pugh A) or moderate (Child-Pugh B) hepatic impairment associated with liver cirrhosis compared with that in age-, weight-, and sex-matched healthy controls: study 1 included smokers and nonsmokers, and study 2 included nonsmokers only. Data from these studies were integrated for analysis. All participants (N = 64) received a single oral dose of riociguat 1.0 mg. Riociguat exposure was significantly higher in individuals with Child-Pugh B hepatic impairment than in healthy controls (ratio: 153% [90% confidence interval: 103%–228%]) but was similar in those with Child-Pugh A hepatic impairment and controls. The half-life of the riociguat metabolite M1 was prolonged in patients with Child-Pugh B or A hepatic impairment compared with that in controls by approximately 43% and 24%, respectively. Impaired hepatic function was associated with higher riociguat exposure in nonsmokers compared with the population of smokers and nonsmokers combined. Riociguat’s safety profile was similar in individuals with impaired or normal liver function. In conclusion, moderate hepatic impairment was associated with increased riociguat exposure compared with that in controls, probably as a result of reduced clearance of the metabolite M1. This suggests that dose titration of riociguat should be administered with particular care in patients with moderate hepatic impairment. PMID:27162628

  9. The sGC activator BAY 60-2770 has potent erectile activity in the rat.

    PubMed

    Lasker, George F; Pankey, Edward A; Frink, Terrence J; Zeitzer, Jonathan R; Walter, Korey A; Kadowitz, Philip J

    2013-06-15

    Nitric oxide (NO) is the principal mediator of penile erection, and soluble guanylate cyclase (sGC) is the receptor for NO. In pathophysiological conditions when sGC is inactivated and not responsive to NO or sGC stimulators a new class of agents called sGC activators increase the activity of NO-insensitive sGC and produce erection. The aim of this study was to investigate erectile responses to BAY 60-2770, a sGC activator, under physiological and pathophysiological conditions. In the present study increases in intracavernosal pressure (ICP) in response to intracavernosal (ic) injections of BAY 60-2770 were investigated under baseline conditions, when sGC was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), when nitric oxide synthase (NOS) was inhibited by N-nitro-L-arginine methyl ester (L-NAME), and after cavernosal nerve crush injury. Under baseline conditions ic injections of BAY 60-2770 increase ICP, ICP/mean arterial pressure (MAP), and area under the ICP curve (AUC) and produce small decreases in MAP at the highest doses studied. BAY 60-2770 was very potent in its ability to induce erection and responses to BAY 60-2770 were enhanced by ODQ which attenuates erectile responses to sodium nitroprusside (SNP), diethylamine NONOate (DEA/NO), and cavernosal nerve stimulation. Responses to BAY 60-2770 were not altered by L-NAME or cavernosal nerve crush injury. These data indicate that BAY 60-2770 has potent erectile activity that is enhanced by ODQ and show that responses to BAY 60-2770 are not attenuated by NOS inhibition or cavernosal nerve injury. These results suggest that BAY 60-2770 would be effective in the treatment of erectile dysfunction when NO bioavailability is reduced, after pelvic nerve injury, and when sGC is oxidized.

  10. YC-1 binding to the β subunit of soluble guanylyl cyclase overcomes allosteric inhibition by the α subunit.

    PubMed

    Purohit, Rahul; Fritz, Bradley G; The, Juliana; Issaian, Aaron; Weichsel, Andrzej; David, Cynthia L; Campbell, Eric; Hausrath, Andrew C; Rassouli-Taylor, Leida; Garcin, Elsa D; Gage, Matthew J; Montfort, William R

    2014-01-14

    Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remain unknown. Using linked equilibrium binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the β subunit. In the absence of CO, YC-1 binds with a Kd of 9-21 μM, depending on the construct. In the presence of CO, these values decrease to 0.6-1.1 μM. Pfizer compound 25 bound ∼10-fold weaker than YC-1 in the absence of CO, whereas compound BAY 41-2272 bound particularly tightly in the presence of CO (Kd = 30-90 nM). Additionally, we found that CO binds much more weakly to heterodimeric sGC proteins (Kd = 50-100 μM) than to the isolated heme domain (Kd = 0.2 μM for Manduca β H-NOX/PAS). YC-1 greatly enhanced binding of CO to heterodimeric sGC, as expected (Kd ∼ 1 μM). These data indicate the α subunit induces a heme pocket conformation with a lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the α subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.

  11. Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

    NASA Technical Reports Server (NTRS)

    Lee, Y. C.; Martin, E.; Murad, F.

    2000-01-01

    The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

  12. Endogenous occurrence of protein S-guanylation in Escherichia coli: Target identification and genetic regulation.

    PubMed

    Tsutsuki, Hiroyasu; Jung, Minkyung; Zhang, Tianli; Ono, Katsuhiko; Ida, Tomoaki; Kunieda, Kohei; Ihara, Hideshi; Akaike, Takaaki; Sawa, Tomohiro

    2016-09-01

    8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated cGMP derivative formed in response to nitric oxide (NO) and reactive oxygen species (ROS). It can cause a post-translational modification (PTM) of protein thiols through cGMP adduction (protein S-guanylation). Accumulating evidence has suggested that, in mammals, S-guanylation of redox-sensor proteins may implicate in regulation of adaptive responses against ROS-associated oxidative stress. Occurrence as well as protein targets of S-guanylation in bacteria remained unknown, however. Here we demonstrated, for the first time, the endogenous occurrence of protein S-guanylation in Escherichia coli (E. coli). Western blotting using anti-S-guanylation antibody clearly showed that multiple proteins were S-guanylated in E. coli. Interestingly, some of those proteins were more intensely S-guanylated when bacteria were cultured under static culture condition than shaking culture condition. It has been known that E. coli is deficient of guanylate cyclase, an enzyme indispensable for 8-nitro-cGMP formation in mammals. We found that adenylate cyclase from E. coli potentially catalyzed 8-nitro-cGMP formation from its precursor 8-nitroguanosine 5'-triphosphate. More importantly, E. coli lacking adenylate cyclase showed significantly reduced formation of S-guanylated proteins. Our S-guanylation proteomics successfully identified S-guanylation protein targets in E. coli, including chaperons, ribosomal proteins, and enzymes which associate with protein synthesis, redox regulation and metabolism. Understanding of functional impacts for protein S-guanylation in bacterial signal transduction is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of pathogenic bacterial infections. PMID:27473654

  13. Endogenous occurrence of protein S-guanylation in Escherichia coli: Target identification and genetic regulation.

    PubMed

    Tsutsuki, Hiroyasu; Jung, Minkyung; Zhang, Tianli; Ono, Katsuhiko; Ida, Tomoaki; Kunieda, Kohei; Ihara, Hideshi; Akaike, Takaaki; Sawa, Tomohiro

    2016-09-01

    8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is a nitrated cGMP derivative formed in response to nitric oxide (NO) and reactive oxygen species (ROS). It can cause a post-translational modification (PTM) of protein thiols through cGMP adduction (protein S-guanylation). Accumulating evidence has suggested that, in mammals, S-guanylation of redox-sensor proteins may implicate in regulation of adaptive responses against ROS-associated oxidative stress. Occurrence as well as protein targets of S-guanylation in bacteria remained unknown, however. Here we demonstrated, for the first time, the endogenous occurrence of protein S-guanylation in Escherichia coli (E. coli). Western blotting using anti-S-guanylation antibody clearly showed that multiple proteins were S-guanylated in E. coli. Interestingly, some of those proteins were more intensely S-guanylated when bacteria were cultured under static culture condition than shaking culture condition. It has been known that E. coli is deficient of guanylate cyclase, an enzyme indispensable for 8-nitro-cGMP formation in mammals. We found that adenylate cyclase from E. coli potentially catalyzed 8-nitro-cGMP formation from its precursor 8-nitroguanosine 5'-triphosphate. More importantly, E. coli lacking adenylate cyclase showed significantly reduced formation of S-guanylated proteins. Our S-guanylation proteomics successfully identified S-guanylation protein targets in E. coli, including chaperons, ribosomal proteins, and enzymes which associate with protein synthesis, redox regulation and metabolism. Understanding of functional impacts for protein S-guanylation in bacterial signal transduction is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of pathogenic bacterial infections.

  14. Proatherosclerotic Effect of the α1-Subunit of Soluble Guanylyl Cyclase by Promoting Smooth Muscle Phenotypic Switching.

    PubMed

    Segura-Puimedon, Maria; Mergia, Evanthia; Al-Hasani, Jaafar; Aherrahrou, Redouane; Stoelting, Stephanie; Kremer, Felix; Freyer, Jennifer; Koesling, Doris; Erdmann, Jeanette; Schunkert, Heribert; de Wit, Cor; Aherrahrou, Zouhair

    2016-08-01

    Soluble guanylate cyclase (sGC), a key enzyme of the nitric oxide signaling pathway, is formed as a heterodimer by various isoforms of its α and β subunit. GUCY1A3, encoding the α1 subunit, was identified as a risk gene for coronary artery disease and myocardial infarction, but its specific contribution to atherosclerosis remains unclear. This study sought to decipher the role of Gucy1a3 in atherosclerosis in mice. At age 32 weeks and after 20 weeks of standard or high-fat diet, Gucy1a3(-/-)/Ldlr(-/-) mice exhibited a significant reduction of the atherosclerotic plaque size at the aortic root and the aorta for high-fat diet animals as compared with Ldlr(-/-) control mice. Collagen content in plaques in the aortic root was reduced, suggesting an alteration of smooth muscle cell function. Proliferation and migration were reduced in Gucy1a3(-/-) primary aortic smooth muscle cells (AoSMCs), and proliferation was also reduced in human AoSMCs after inhibition of sGC by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one. Gucy1a3 deficiency in AoSMCs prevents their phenotypic switching, as indicated by the differential expression of marker proteins. The inherited Gucy1a3(-/-) loss exerts an atheroprotective effect. We suggest that sGC activity promotes the phenotypic switching of smooth muscle cells from a contractile to a synthetic state, fostering the formation of atherosclerosis. Preventing this switch by sGC inhibition may provide a novel target in atherosclerotic disease. PMID:27315776

  15. Alternative splicing impairs soluble guanylyl cyclase function in aortic aneurysm.

    PubMed

    Martin, Emil; Golunski, Eva; Laing, Susan T; Estrera, Anthony L; Sharina, Iraida G

    2014-12-01

    Nitric oxide (NO) receptor soluble guanylyl cyclase (sGC) is a key regulator of several important vascular functions and is important for maintaining cardiovascular homeostasis and vascular plasticity. Diminished sGC expression and function contributes to pathogenesis of several cardiovascular diseases. However, the processes that control sGC expression in vascular tissue remain poorly understood. Previous work in animal and cell models revealed the complexity of alternative splicing of sGC genes and demonstrated its importance in modulation of sGC function. The aim of this study was to examine the role of alternative splicing of α1 and β1 sGC in healthy and diseased human vascular tissue. Our study found a variety of α1 and β1 sGC splice forms expressed in human aorta. Their composition and abundance were different between samples of aortic tissue removed during surgical repair of aortic aneurysm and samples of aortas without aneurysm. Aortas with aneurysm demonstrated decreased sGC activity, which correlated with increased expression of dysfunctional sGC splice variants. In addition, the expression of 55-kDa oxidation-resistant α1 isoform B sGC (α1-IsoB) was significantly lower in aortic samples with aneurysm. The α1-IsoB splice variant was demonstrated to support sGC activity in aortic lysates. Together, our results suggest that alternative splicing contributes to diminished sGC function in vascular dysfunction. Precise understanding of sGC splicing regulation could help to design new therapeutic interventions and to personalize sGC-targeting therapies in treatments of vascular disease.

  16. Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F, Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

    PubMed Central

    Beste, Kerstin Y.; Spangler, Corinna M.; Burhenne, Heike; Koch, Karl-Wilhelm; Shen, Yuequan; Tang, Wei-Jen; Kaever, Volkhard; Seifert, Roland

    2013-01-01

    Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides. PMID:23922959

  17. Nitric Oxide Receptor Soluble Guanylyl Cyclase Undergoes Splicing Regulation in Differentiating Human Embryonic Cells

    PubMed Central

    Sharin, Vladislav G.; Mujoo, Kalpana; Kots, Alexander Y.; Martin, Emil; Murad, Ferid

    2011-01-01

    Nitric oxide (NO), an important mediator molecule in mammalian physiology, initiates a number of signaling mechanisms by activating the enzyme soluble guanylyl cyclase (sGC). Recently, a new role for NO/cyclic guanosine monophosphate signaling in embryonic development and cell differentiation has emerged. The changes in expression of NO synthase isoforms and various sGC subunits has been demonstrated during human and mouse embryonic stem (ES) cells differentiation. Previously, our laboratory demonstrated that nascent α1 sGC transcript undergoes alternative splicing and that expression of α1 sGC splice forms directly affects sGC activity. Expression of sGC splice variants in the process of human ES (hES) cells differentiation has not been investigated. In this report, we demonstrate that α1 sGC undergoes alternative splicing during random hES differentiation for the first time. Our results indicate that C-α1 sGC splice form is expressed at high levels in differentiating cells and its intracellular distribution varies from canonical α1 sGC subunit. Together, our data suggest that alternative splicing of sGC subunits is associated with differentiation of hES cells. PMID:20964618

  18. Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

    NASA Technical Reports Server (NTRS)

    Krumenacker, J. S.; Hyder, S. M.; Murad, F.

    2001-01-01

    Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

  19. NO and CO Differentially Activate Soluble Guanylyl Cyclase via a Heme Pivot-bend Mechanism

    SciTech Connect

    Ma,X.; Sayed, N.; Beuve, A.; van den Akker, F.

    2007-01-01

    Diatomic ligand discrimination by soluble guanylyl cyclase (sGC) is paramount to cardiovascular homeostasis and neuronal signaling. Nitric oxide (NO) stimulates sGC activity 200-fold compared with only four-fold by carbon monoxide (CO). The molecular details of ligand discrimination and differential response to NO and CO are not well understood. These ligands are sensed by the heme domain of sGC, which belongs to the heme nitric oxide oxygen (H-NOX) domain family, also evolutionarily conserved in prokaryotes. Here we report crystal structures of the free, NO-bound, and CO-bound H-NOX domains of a cyanobacterial homolog. These structures and complementary mutational analysis in sGC reveal a molecular ruler mechanism that allows sGC to favor NO over CO while excluding oxygen, concomitant to signaling that exploits differential heme pivoting and heme bending. The heme thereby serves as a flexing wedge, allowing the N-terminal subdomain of H-NOX to shift concurrent with the transition of the six- to five-coordinated NO-bound state upon sGC activation. This transition can be modulated by mutations at sGC residues 74 and 145 and corresponding residues in the cyanobacterial H-NOX homolog.

  20. CCAAT-binding factor regulates expression of the beta1 subunit of soluble guanylyl cyclase gene in the BE2 human neuroblastoma cell line

    NASA Technical Reports Server (NTRS)

    Sharina, Iraida G.; Martin, Emil; Thomas, Anthony; Uray, Karen L.; Murad, Ferid

    2003-01-01

    Soluble guanylyl cyclase (sGC) is a cytosolic enzyme producing the intracellular messenger cyclic guanosine monophosphate (cGMP) on activation with nitric oxide (NO). sGC is an obligatory heterodimer composed of alpha and beta subunits. We investigated human beta1 sGC transcriptional regulation in BE2 human neuroblastoma cells. The 5' upstream region of the beta1 sGC gene was isolated and analyzed for promoter activity by using luciferase reporter constructs. The transcriptional start site of the beta1 sGC gene in BE2 cells was identified. The functional significance of consensus transcriptional factor binding sites proximal to the transcriptional start site was investigated by site deletions in the 800-bp promoter fragment. The elimination of CCAAT-binding factor (CBF) and growth factor independence 1 (GFI1) binding cores significantly diminished whereas deletion of the NF1 core elevated the transcription. Electrophoretic mobility-shift assay (EMSA) and Western analysis of proteins bound to biotinated EMSA probes confirmed the interaction of GFI1, CBF, and NF1 factors with the beta1 sGC promoter. Treatment of BE2 cells with genistein, known to inhibit the CBF binding to DNA, significantly reduced protein levels of beta1 sGC by inhibiting transcription. In summary, our study represents an analysis of the human beta1 sGC promoter regulation in human neuroblastoma BE2 cells and identifies CBF as a critically important factor in beta1 sGC expression.

  1. 21 CFR 172.530 - Disodium guanylate.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Disodium guanylate. 172.530 Section 172.530 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION (CONTINUED) FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR HUMAN...

  2. YC-1 activation of human soluble guanylyl cyclase has both heme-dependent and heme-independent components

    NASA Technical Reports Server (NTRS)

    Martin, E.; Lee, Y. C.; Murad, F.

    2001-01-01

    YC-1 [3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole] is an allosteric activator of soluble guanylyl cyclase (sGC). YC-1 increases the catalytic rate of the enzyme and sensitizes the enzyme toward its gaseous activators nitric oxide or carbon monoxide. In other studies the administration of YC-1 to experimental animals resulted in the inhibition of the platelet-rich thrombosis and a decrease of the mean arterial pressure, which correlated with increased cGMP levels. However, details of YC-1 interaction with sGC and enzyme activation are incomplete. Although evidence in the literature indicates that YC-1 activation of sGC is strictly heme-dependent, this report presents evidence for both heme-dependent and heme-independent activation of sGC by YC-1. The oxidation of the sGC heme by 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one completely inhibited the response to NO, but only partially attenuated activation by YC-1. We also observed activation by YC-1 of a mutant sGC, which lacks heme. These findings indicate that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme.

  3. Restoring Soluble Guanylyl Cyclase Expression and Function Blocks the Aggressive Course of GliomaS⃞

    PubMed Central

    Zhu, Haifeng; Li, Jessica Tao; Zheng, Fang; Martin, Emil; Kots, Alexander Y.; Krumenacker, Joshua S.; Choi, Byung-Kwon; McCutcheon, Ian E.; Weisbrodt, Norman; Bögler, Oliver; Murad, Ferid

    2011-01-01

    The NO and cGMP signaling pathways are of broad physiological and pathological significance. We compared the NO/soluble guanylyl cyclase (sGC)/cGMP pathway in human glioma tissues and cell lines with that of healthy control samples and demonstrated that sGC expression is significantly lower in glioma preparations. Our analysis of GEO databases (National Cancer Institute) further revealed a statistically significant reduction of sGC transcript levels in human glioma specimens. On the other hand, the expression levels of particulate (membrane) guanylyl cyclases (pGC) and cGMP-specific phosphodiesterase (PDE) were intact in the glioma cells that we have tested. Pharmacologically manipulating endogenous cGMP generation in glioma cells through either stimulating pGC by ANP/BNP, or blocking PDE by 3-isobutyl-1-methylxanthine/zaprinast caused significant inhibition of proliferation and colony formation of glioma cells. Genetically restoring sGC expression also correlated inversely with glioma cells growth. Orthotopic implantation of glioma cells transfected with an active mutant form of sGC (sGCα1β1Cys105) in athymic mice increased the survival time by 4-fold over the control. Histological analysis of xenografts overexpressing α1β1Cys105 sGC revealed changes in cellular architecture that resemble the morphology of normal cells. In addition, a decrease in angiogenesis contributed to glioma inhibition by sGC/cGMP therapy. Our study proposes the new concept that suppressed expression of sGC, a key enzyme in the NO/cGMP pathway, may be associated with an aggressive course of glioma. The sGC/cGMP signaling-targeted therapy may be a favorable alternative to chemotherapy and radiotherapy for glioma and perhaps other tumors. PMID:21908708

  4. The fibrate gemfibrozil is a NO- and haem-independent activator of soluble guanylyl cyclase: in vitro studies

    PubMed Central

    Sharina, I G; Sobolevsky, M; Papakyriakou, A; Rukoyatkina, N; Spyroulias, G A; Gambaryan, S; Martin, E

    2015-01-01

    Background and Purpose Fibrates are a class of drugs widely used to treat dyslipidaemias. They regulate lipid metabolism and act as PPARα agonists. Clinical trials demonstrate that besides changes in lipid profiles, fibrates decrease the incidence of cardiovascular events, with gemfibrozil exhibiting the most pronounced benefit. This study aims to characterize the effect of gemfibrozil on the activity and function of soluble guanylyl cyclase (sGC), the key mediator of NO signalling. Experimental Approach High-throughput screening of a drug library identified gemfibrozil as a direct sGC activator. Activation of sGC is unique to gemfibrozil and is not shared by other fibrates. Key Results Gemfibrozil activated purified sGC, induced endothelium-independent relaxation of aortic rings and inhibited platelet aggregation. Gemfibrozil-dependent activation was absent when the sGC haem domain was deleted, but was significantly enhanced when sGC haem was lacking or oxidized. Oxidation of sGC haem enhanced the vasoactive and anti-platelet effects of gemfibrozil. Gemfibrozil competed with the haem-independent sGC activators ataciguat and cinaciguat. Computational modelling predicted that gemfibrozil occupies the space of the haem group and interacts with residues crucial for haem stabilization. This is consistent with structure-activity data which revealed an absolute requirement for gemfibrozil's carboxyl group. Conclusions and Implications These data suggest that in addition to altered lipid and lipoprotein state, the cardiovascular preventive benefits of gemfibrozil may derive from direct activation and protection of sGC function. A sGC-directed action may explain the more pronounced cardiovascular benefit of gemfibrozil observed over other fibrates and some of the described side effects of gemfibrozil. PMID:25536881

  5. Evidence for a dissociable protein subunit required for calmodulin stimulation of brain adenylate cyclase.

    PubMed Central

    Toscano, W A; Westcott, K R; LaPorte, D C; Storm, D R

    1979-01-01

    An adenylate cyclase [ATP pyrophosphatelyase (cyclizing), EC 4.6.1.1] preparation that is not stimulated by NaF,5'-guanylyl imidodiphosphate, or Ca2+.calmodulin has been isolated from bovine cerebral cortex by Affi-Gel Blue chromatography and calmodulin-Sepharose chromatography. Sensitivity to these effectors was restored by incubation of the adenylate cyclase preparation with detergent-solubilized protein from bovine cerebral cortex. Reconstitution of of Ca2+.calmodulin activation required the presence of 5'-guanylyl imidodiphosphate. The factor required for restoration of Ca2+.calmodulin stimulation was sensitive to heat, trypsin digestion, and N-ethylmaleimide. These observations suggest that this adenylate cyclase activity requires the presence of one or more guanyl nucleotide binding subunits for calmodulin sensitivity. PMID:293663

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

    PubMed Central

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

    2010-01-01

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

  7. Reduced expression of NO-sensitive guanylyl cyclase in reactive astrocytes of Alzheimer disease, Creutzfeldt-Jakob disease, and multiple sclerosis brains.

    PubMed

    Baltrons, María Antonia; Pifarré, Paula; Ferrer, Isidre; Carot, José Miguel; García, Agustina

    2004-12-01

    In Alzheimer's disease (AD) brains increased NO synthase (NOS) expression is found in reactive astrocytes surrounding amyloid plaques. We have recently shown that treatment with beta-amyloid peptides or IL-1beta down-regulates NO-sensitive soluble guanylyl cyclase (sGC) in cultured astrocytes and in adult rat brain. In this work, we have examined sGC activity and expression in postmortem brain tissue of AD patients and matched controls. No significant alteration was observed in basal or NO-stimulated sGC activity, nor in sGC beta1 and alpha1 subunit levels in cortical extracts of AD brains. Immunohistochemistry showed intense and widespread labeling of sGC beta1 in cortical and hippocampal neurons and white matter fibrillar astrocytes, while grey matter astrocytes were faintly stained. In AD, expression of sGC in neurons and fibrillar astrocytes is not altered but is markedly reduced in reactive astrocytes surrounding amyloid plaques. Immunostaining for sGC beta1 was also lacking in reactive astrocytes in cortex and subcortical white matter in Creutzfeldt-Jakob disease brains and in subacute and chronic plaques in multiple sclerosis (MS) brains. Thus, induction of astrocyte reactivity is associated with decreased capacity to generate cGMP in response to NO both in vitro and in vivo. This effect may be related to the development of the astroglial inflammatory response. PMID:15571982

  8. Bacterial terpene cyclases.

    PubMed

    Dickschat, Jeroen S

    2016-01-01

    Covering: up to 2015. This review summarises the accumulated knowledge about characterised bacterial terpene cyclases. The structures of identified products and of crystallised enzymes are included, and the obtained insights into enzyme mechanisms are discussed. After a summary of mono-, sesqui- and diterpene cyclases the special cases of the geosmin and 2-methylisoborneol synthases that are both particularly widespread in bacteria will be presented. A total number of 63 enzymes that have been characterised so far is presented, with 132 cited references. PMID:26563452

  9. Stimulators of the soluble guanylyl cyclase: promising functional insights from rare coding atherosclerosis-related GUCY1A3 variants.

    PubMed

    Wobst, Jana; von Ameln, Simon; Wolf, Bernhard; Wierer, Michael; Dang, Tan An; Sager, Hendrik B; Tennstedt, Stephanie; Hengstenberg, Christian; Koesling, Doris; Friebe, Andreas; Braun, Siegmund L; Erdmann, Jeanette; Schunkert, Heribert; Kessler, Thorsten

    2016-07-01

    Stimulators of the soluble guanylyl cyclase (sGC) are emerging therapeutic agents in cardiovascular diseases. Genetic alterations of the GUCY1A3 gene, which encodes the α1 subunit of the sGC, are associated with coronary artery disease. Studies investigating sGC stimulators in subjects with CAD and carrying risk-related variants in sGC are, however, lacking. Here, we functionally investigate the impact of coding GUCY1A3 variants on sGC activity and the therapeutic potential of sGC stimulators in vitro. In addition to a known loss-of-function variant, eight coding variants in GUCY1A3 were cloned and expressed in HEK 293 cells. Protein levels and dimerization capability with the β1 subunit were analysed by immunoblotting and co-immunoprecipitation, respectively. All α1 variants found in MI patients dimerized with the β1 subunit. Protein levels were reduced by 72 % in one variant (p < 0.01). Enzymatic activity was analysed using cGMP radioimmunoassay after stimulation with a nitric oxide (NO) donor. Five variants displayed decreased cGMP production upon NO stimulation (p < 0.001). The addition of the sGC stimulator BAY 41-2272 increased cGMP formation in all of these variants (p < 0.01). Except for the variant leading to decreased protein level, cGMP amounts reached the wildtype NO-induced level after addition of BAY 41-2272. In conclusion, rare coding variants in GUCY1A3 lead to reduced cGMP formation which can be rescued by a sGC stimulator in vitro. These results might therefore represent the starting point for discovery of novel treatment strategies for patients at risk with coding GUCY1A3 variants. PMID:27342234

  10. Reactive oxygen species induce tyrosine phosphorylation of and Src kinase recruitment to NO-sensitive guanylyl cyclase.

    PubMed

    Meurer, Sabine; Pioch, Sylke; Gross, Steffen; Müller-Esterl, Werner

    2005-09-30

    Soluble guanylyl cyclase (sGC) is the major cytosolic receptor for nitric oxide (NO) that converts GTP into the second messenger cGMP in a NO-dependent manner. Other factors controlling this key enzyme are intracellular proteins such as Hsp90 and PSD95, which bind to sGC and modulate its activity, stability, and localization. To date little is known about the effects of posttranslational modifications of sGC, although circumstantial evidence suggests that reversible phosphorylation may contribute to sGC regulation. Here we demonstrate that inhibitors of protein-tyrosine phosphatases such as pervanadate and bisperoxo(1,10-phenanthroline)oxovanadate(V) as well as reactive oxygen species such as H2O2 induce specific tyrosine phosphorylation of the beta1 but not of the alpha1 subunit of sGC. Tyrosine phosphorylation of sGCbeta1 is also inducible by pervanadate and H2O2 in intact PC12 cells, rat aortic smooth muscle cells, and in rat aortic tissues, indicating that tyrosine phosphorylation of sGC may also occur in vivo. We have mapped the major tyrosine phosphorylation site to position 192 of beta1, where it forms part of a highly acidic phospho-acceptor site for Src-like kinases. In the phosphorylated state Tyr(P)-192 exposes a docking site for SH2 domains and efficiently recruits Src and Fyn to sGCbeta1, thereby promoting multiple phosphorylation of the enzyme. Our results demonstrate that sGC is subject to tyrosine phosphorylation and interaction with Src-like kinases, revealing an unexpected cross-talk between the NO/cGMP and tyrosine kinase signaling pathways at the level of sGC.

  11. Insights into BAY 60-2770 activation and S-nitrosylation-dependent desensitization of soluble guanylyl cyclase via crystal structures of homologous Nostoc H-NOX domain complexes

    PubMed Central

    Kumar, Vijay; Martin, Faye; Hahn, Michael G.; Schaefer, Martina; Stamler, Jonathan S.; Stasch, Johannes-Peter; van den Akker, Focco

    2013-01-01

    The soluble guanylyl cyclase (sGC) is an important receptor for nitric oxide (NO). Nitric oxide activates sGC several hundred fold to generate cGMP from GTP. Because of sGC’s salutary roles in cardiovascular physiology, it has received substantial attention as a drug target. The heme domain of sGC is key to its regulation as it not only contains the NO activation site but also harbors sites for NO-independent sGC activators as well an S-nitrosylation site (β1 C122) involved in desensitization. Here we report the crystal structure of the activator BAY 60-2770 bound to the Nostoc H-NOX domain that is homologous to sGC. The structure reveals that BAY 60-2770 has displaced the heme and acts as a heme mimetic via carboxylate-mediated interactions with the conserved YxSxR motif as well as hydrophobic interactions. Comparisons with the previously determined BAY 58-2667 bound structure reveals that BAY 60-2770 is more ordered in its hydrophobic tail region. sGC activity assays demonstrate that BAY 60-2770 has about 10% higher fold maximal stimulation compared to BAY 58-2667. S-nitrosylation of the BAY 60-2770 substituted Nostoc H-NOX domain causes subtle changes in the vicinity of the S-nitrosylated C122 residue. These shifts could impact the adjacent YxSxR motif and αF helix and as such potentially inhibit either heme incorporation or NO-activation of sGC and thus provide a structural basis for desensitization. PMID:23614626

  12. Crystal Structure of the Signaling Helix Coiled-coil Domain of the b1 Subunit of the Soluble guanylyl Cyclase

    SciTech Connect

    Ma, X.; Beuve, A; van den Akker, F

    2010-01-01

    The soluble guanylyl cyclase (sGC) is a heterodimeric enzyme that, upon activation by nitric oxide, stimulates the production of the second messenger cGMP. Each sGC subunit harbor four domains three of which are used for heterodimerization: H-NOXA/H-NOBA domain, coiled-coil domain (CC), and catalytic guanylyl cyclase domain. The CC domain has previously been postulated to be part of a larger CC family termed the signaling helix (S-helix) family. Homodimers of sGC have also been observed but are not functionally active yet are likely transient awaiting their intended heterodimeric partner. To investigate the structure of the CC S-helix region, we crystallized and determined the structure of the CC domain of the sGC{beta}1 subunit comprising residues 348-409. The crystal structure was refined to 2.15 {angstrom} resolution. The CC structure of sGC{beta}1 revealed a tetrameric arrangement comprised of a dimer of CC dimers. Each monomer is comprised of a long a-helix, a turn near residue P399, and a short second a-helix. The CC structure also offers insights as to how sGC homodimers are not as stable as (functionally) active heterodimers via a possible role for inter-helix salt-bridge formation. The structure also yielded insights into the residues involved in dimerization. In addition, the CC region is also known to harbor a number of congenital and man-made mutations in both membrane and soluble guanylyl cyclases and those function-affecting mutations have been mapped onto the CC structure. This mutant analysis indicated an importance for not only certain dimerization residue positions, but also an important role for other faces of the CC dimer which might perhaps interact with adjacent domains. Our results also extend beyond guanylyl cyclases as the CC structure is, to our knowledge, the first S-helix structure and serves as a model for all S-helix containing family members.

  13. Activation of soluble guanylyl cyclase by BAY 58-2667 improves bladder function in cyclophosphamide-induced cystitis in mice.

    PubMed

    de Oliveira, Mariana G; Calmasini, Fabiano B; Alexandre, Eduardo C; De Nucci, Gilberto; Mónica, Fabíola Z; Antunes, Edson

    2016-07-01

    Activators of soluble guanylyl cyclase (sGC) interact directly with its prosthetic heme group, enhancing the enzyme responsiveness in pathological conditions. This study aimed to evaluate the effects of the sGC activator BAY 58-2667 on voiding dysfunction, protein expressions of α1 and β1 sGC subunits and cGMP levels in the bladder tissues after cyclophosphamide (CYP) exposure. Female C57BL/6 mice (20-25 g) were injected with CYP (300 mg/kg ip) to induce cystitis. Mice were pretreated or not with BAY 58-2667 (1 mg/kg, gavage), given 1 h before CYP injection. The micturition patterns and in vitro bladder contractions were evaluated at 24 h. In freely moving mice, the CYP injection produced reduced the micturition volume and increased the number of urine spots. Cystometric recordings in CYP-injected mice revealed significant increases in basal pressure, voiding frequency, and nonvoiding contractions (NVCs), along with decreases in bladder capacity, intercontraction interval, and compliance. BAY 58-2667 significantly prevented the micturition alterations observed in both freely moving mice and cystometry and normalized the reduced in vitro carbachol-induced contractions in the CYP group. Reduced protein expressions of α1 and β1 sGC subunits and of cGMP levels were observed in the CYP group, all of which were prevented by BAY 58-2667. CYP exposure significantly increased reactive-oxygen species (ROS) generation in both detrusor and urothelium, and this was normalized by BAY 58-2667. The increased myeloperoxidase and cyclooxygenase-2 activities in the bladders of the CYP group remained unchanged by BAY 58-2667. Activators of sGC may constitute a novel and promising therapeutic approach for management of interstitial cystitis. PMID:27122537

  14. Ontogeny of fetal adenylate cyclase; mechanisms for regulation of beta-adrenergic receptors.

    PubMed

    Maier, J A; Roberts, J M; Jacobs, M M

    1989-11-01

    Transmembrane second messenger signalling systems regulate differentiation, growth and homeostatic responses during fetal development. The beta-adrenergic adenylate cyclase system is the best studied of these and has been used as a model to investigate the control of developmental processes. In tissues such as lung, heart and parotid, beta-adrenergic responsiveness of adenylate cyclase increases during development. In the developing fetal lung beta-receptor concentration increases during gestation or after glucocorticoid treatment, but cannot fully explain enhanced adrenergic responsiveness. To probe developmental and hormonal effects on beta-receptor function, we asked if advancing gestation or glucocorticoid treatment alters beta-receptor-Gs interactions in fetal rabbit lung membrane particulates. Before 25 days gestation, 1-isoproterenol competes for 3H-dihydroalprenolol (DHA), a radiolabelled beta-antagonist, with a single low affinity, later in gestation, high and low affinities of isoproterenol for the beta-receptor are present which can be shifted to the lower affinity by addition of guanyl nucleotide. High affinity binding is precociously induced in 25 days--fetal lung particulates as early as 3 h after maternal betamethasone treatment, but beta-adrenoreceptor concentration in treated fetuses was increased over controls only after 24 h of treatment. Cholera toxin catalyzed ADP ribosylation of membrane particulates showed cholera toxin substrate (Gs) was not altered by glucocorticoid treatment. Stimulation of adenylate cyclase activity with isoproterenol (100mM) and GTP (100mM) resulted in no incremental increase over that produced by GTP (100mM) alone in glucocorticoid treated or control particulates, either early or late in gestation. These data demonstrate that beta-receptor-Gs interactions are not sufficient to produce full agonist responses. Although both beta-adrenergic receptors and Gs are present in fetal rabbit lung early in gestation, interaction

  15. Heterologous desensitization of adenylate cyclase from pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

    SciTech Connect

    Popov, K.M.; Bulargina, T.V.; Severin, E.S.

    1985-09-20

    Preincubation of the plasma membranes from pigeon erythrocytes with the catalytic subunit of cAMP-dependent protein kinase leads to desensitization of adenylate cyclase of the erythrocytes. The adenylate cyclase activity, measured in the presence of 10 ..mu..M isoproterenol and 50 ..mu..M GTP-..gamma..-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 ..mu..M GTP-..gamma..-S is decreased by 35% in 20 min. The decrease in the adenylate cyclase activity is due to an increase in the lag phase of activation of the enzyme in the presence of a GTP analog stable to hydrolysis and a decrease in the activity in the steady-state phase of activation. Heterologous desensitization of adenylate cyclase under the action of cAMP-dependent protein kinase is coupled with a decrease in the number of ..beta..-adrenoreceptors capable of passing into a state of high affinity for antagonists in the absence of guanylic nucleotides. The influence of the catalytic subunit on adenylate cyclase entirely models the process of desensitization of the enzyme absorbed in the influence of isoproterenol or cAMP on erythrocytes.

  16. Nitric oxide from vascular smooth muscle cells: regulation of platelet reactivity and smooth muscle cell guanylate cyclase.

    PubMed Central

    Mollace, V.; Salvemini, D.; Anggard, E.; Vane, J.

    1991-01-01

    1. Incubation of smooth muscle cells (SMC) from bovine aorta for 3 min with human washed platelets treated with indomethacin (10 microM) promoted a cell number-related inhibition of platelet aggregation induced by thrombin (40 mu ml-1). This inhibition was not attributable to products of the cyclo-oxygenase pathway for the SMC were also treated with indomethacin (10 microM). 2. The inhibitory activity of the SMC on platelet aggregation was enhanced by incubating the SMC with E. coli lipopolysaccharide (LPS, 0.5 micrograms ml-1) for a period of 9 to 24 h. This effect was attenuated when cycloheximide (10 micrograms ml-1) was incubated together with LPS. Cycloheximide did not prevent the inhibitory activity of the non-treated cells. 3. The inhibition of platelet aggregation obtained with non-treated or LPS-treated SMC was potentiated by superoxide dismutase (SOD, 60 u ml-1) and ablated by oxyhaemoglobin (OxyHb, 10 microM). Preincubation of the SMC with NG-monomethyl-L-arginine (L-NMMA, 30-300 microM) for 60 min prevented their antiaggregatory activity. This effect was reversed by concurrent incubation with L-arginine (L-Arg, 100 microM) but not with D-arginine (D-Arg, 100 microM). 4. Exposure of the non-treated SMC (5 x 10(5) cells) to stirring (1000 r.p.m., 37 degrees C) for 10 min led to a significant increase in their levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) but not adenosine 3':5'-cyclic monophosphate (cyclic AMP). L-NMMA (300 microM) attenuated the increase in cyclic GMP induced by stirring but did not affect the basal levels of cyclic GMP in the cells.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1724627

  17. Digitonin effects on photoreceptor adenylate cyclase.

    PubMed

    Bitensky, M W; Gorman, R E; Miller, W H

    1972-03-24

    Adenylate cyclase is described in a number of photoreceptor membranes. Vertebrate rod outer segments contain light-regulated cyclase, and light regulation is abolished by digitonin. Disruption of microvilli in cone and rhabdomphotoreceptors is also associated with loss of light regulation and retention of full enzymic activity. The data suggest that inhibitory constraint provides regulation in cyclase systems and that disruption of membrane structure uncouples catalytic and regulatory elements.

  18. Apoptosis of human gastric cancer SGC-7901 cells induced by podophyllotoxin

    PubMed Central

    JI, CHEN-FENG; JI, YU-BIN

    2014-01-01

    Numerous studies have demonstrated that podophyllotoxin and its derivatives exhibit antitumor effects. The aim of the present study was to investigate SGC-7901 cell apoptosis and the underlying mechanism induced by podophyllotoxin. SGC-7901 cells were treated with varying concentrations of podophyllotoxin. MTT assays and flow cytometry were used to evaluate the effects of podophyllotoxin on the proliferation and apoptosis of SGC-7901 cells, while fluorescence inverted microscopy was used to observe the morphology of SGC-7901 cells that had been dyed with Hoechst 33258. In addition, laser scanning confocal microscopy was used to analyze the mitochondrial membrane potential (MMP) of SGC-7901 cells dyed with Rhodamine 123. Western blotting was performed to analyze the expression levels of cytochrome c (cyt-c), caspase-9 and caspase-3 in the SGC-7901 cells. The results indicated that podophyllotoxin was capable of inhibiting growth and inducing the apoptosis of SGC-7901 cells in a dose-dependent manner, causing cell cycle arrest at the G2/M phase. After 48 h of treatment, the apoptotic morphology of SGC-7901 cells was clear, exhibiting cell protuberance, concentrated cytoplasms and apoptotic bodies. Following 24 h of treatment, the MMP of the SGC-7901 cells decreased. In addition, after 48 h, the expression of cyt-c was shown to be upregulated, while the expression levels of pro-caspase-9 and pro-caspase-3 in the SGC-7901 cells were shown to be downregulated. In conclusion, apoptosis can be induced in SGC-7901 cells by podophyllotoxin, potentially via a mitochondrial pathway, indicating that podophyllotoxin may be a potent agent for cancer treatment. PMID:24940431

  19. Lessons from ten years of crystallization experiments at the SGC

    PubMed Central

    Ng, Jia Tsing; Dekker, Carien; Reardon, Paul; von Delft, Frank

    2016-01-01

    Although protein crystallization is generally considered more art than science and remains significantly trial-and-error, large-scale data sets hold the promise of providing general learning. Observations are presented here from retrospective analyses of the strategies actively deployed for the extensive crystallization experiments at the Oxford site of the Structural Genomics Consortium (SGC), where comprehensive annotations by SGC scientists were recorded on a customized database infrastructure. The results point to the importance of using redundancy in crystallizing conditions, specifically by varying the mixing ratios of protein sample and precipitant, as well as incubation temperatures. No meaningful difference in performance could be identified between the four most widely used sparse-matrix screens, judged by the yield of crystals leading to deposited structures; this suggests that in general any comparison of screens will be meaningless without extensive cross-testing. Where protein sample is limiting, exploring more conditions has a higher likelihood of being informative by yielding hits than does redundancy of either mixing ratio or temperature. Finally, on the logistical question of how long experiments should be stored, 98% of all crystals that led to deposited structures appeared within 30 days. Overall, these analyses serve as practical guidelines for the design of initial screening experiments for new crystallization targets. PMID:26894670

  20. Identification of a gamma subunit associated with the adenylyl cyclase regulatory proteins Ns and Ni.

    PubMed

    Hildebrandt, J D; Codina, J; Risinger, R; Birnbaumer, L

    1984-02-25

    The subunit composition of the Ns and Ni, the human erythrocyte stimulatory and inhibitory regulatory proteins of adenylyl cyclase, respectively, were analyzed by a sodium dodecyl sulfate-containing discontinuous urea and polyacrylamide gradient gel electrophoresis system designed for the study of low molecular weight polypeptides. This system disclosed that these proteins, in addition to their known alpha and beta subunits, contain an additional small peptide of apparent molecular weight of 5,000 (5K). This "5K peptide" is also present in preparations of another protein which we termed "40K protein" on the basis of its hydrodynamic behavior and whose primary protein constituent is the Mr 35,000 beta subunit of the above regulatory proteins. Analyzing Ni, the 5K peptide was functionally related to the protein by showing that its apparent Stokes radius changes from 5.9 to 5.1 nm after treatment with guanyl-5'-yl imidodiphosphate and magnesium in parallel with the alpha and beta subunits. These data are interpreted as evidence for the existence of a third subunit associated with the regulatory proteins of adenylyl cyclase. We call this subunit gamma and propose a minimum subunit structure for these proteins of the alpha beta gamma type. PMID:6321456

  1. Mechano-chemistry of the enzyme Guanylate Kinase

    NASA Astrophysics Data System (ADS)

    Tseng, C.-Y.; Wang, A.; Zocchi, G.

    2010-07-01

    We address the coupling of mechanics and chemistry in an enzyme through equilibrium experiments where we mechanically deform the enzyme and measure the effect on the chemical reaction it catalyzes. We use the DNA molecular spring method to exert stresses at three different specific locations on the enzyme Guanylate Kinase, and for each case determine the changes in substrates binding affinities and catalytic rate. We find that the enzyme kinetics parameters can be affected separately, depending on where the mechanical stress is applied. For one configuration the applied stress mainly affects the catalytic rate kcat, for another it mainly affects the binding affinity of the substrate GMP. These experiments show that a stress applied by pulling two residues on the surface of the protein generally results in a strain propagating into the structure.

  2. Lethal Accumulation of Guanylic Nucleotides in Saccharomyces cerevisiae HPT1-Deregulated Mutants

    PubMed Central

    Breton, Annick; Pinson, Benoît; Coulpier, Fanny; Giraud, Marie-France; Dautant, Alain; Daignan-Fornier, Bertrand

    2008-01-01

    Guanylic nucleotide biosynthesis is a conserved and highly regulated process. Drugs reducing GMP synthesis affect the immunological response and mutations enabling guanylic-derivative recycling lead to severe mental retardation. While the effects of decreased GMP synthesis have been well documented, the consequences of GMP overproduction in eukaryotes are poorly understood. In this work, we selected and characterized several mutations making yeast hypoxanthine–guanine phosphoribosyltransferase insensitive to feedback inhibition by GMP. In these mutants, accumulation of guanylic nucleotides can be triggered by addition of extracellular guanine. We show that such an accumulation is highly toxic for yeast cells and results in arrest of proliferation and massive cell death. This growth defect could be partially suppressed by overexpression of Rfx1p, a transcriptional repressor of the DNA damage response pathway. Importantly, neither guanylic nucleotide toxicity nor its suppression by Rfx1p was associated with an alteration of forward mutation frequency. PMID:18245832

  3. Pathophysiological role of guanylate-binding proteins in gastrointestinal diseases.

    PubMed

    Britzen-Laurent, Nathalie; Herrmann, Christian; Naschberger, Elisabeth; Croner, Roland S; Stürzl, Michael

    2016-07-28

    Guanylate-binding proteins (GBPs) are interferon-stimulated factors involved in the defense against cellular pathogens and inflammation. These proteins, particularly GBP-1, the most prominent member of the family, have been established as reliable markers of interferon-γ-activated cells in various diseases, including colorectal carcinoma (CRC) and inflammatory bowel diseases (IBDs). In CRC, GBP-1 expression is associated with a Th1-dominated angiostatic micromilieu and is correlated with a better outcome. Inhibition of tumor growth by GBP-1 is the result of its strong anti-angiogenic activity as well as its direct anti-tumorigenic effect on tumor cells. In IBD, GBP-1 mediates the anti-proliferative effects of interferon-γ on intestinal epithelial cells. In addition, it plays a protective role on the mucosa by preventing cell apoptosis, by inhibiting angiogenesis and by regulating the T-cell receptor signaling. These functions rely to a large extent on the ability of GBP-1 to interact with and remodel the actin cytoskeleton. PMID:27605879

  4. Pathophysiological role of guanylate-binding proteins in gastrointestinal diseases

    PubMed Central

    Britzen-Laurent, Nathalie; Herrmann, Christian; Naschberger, Elisabeth; Croner, Roland S; Stürzl, Michael

    2016-01-01

    Guanylate-binding proteins (GBPs) are interferon-stimulated factors involved in the defense against cellular pathogens and inflammation. These proteins, particularly GBP-1, the most prominent member of the family, have been established as reliable markers of interferon-γ-activated cells in various diseases, including colorectal carcinoma (CRC) and inflammatory bowel diseases (IBDs). In CRC, GBP-1 expression is associated with a Th1-dominated angiostatic micromilieu and is correlated with a better outcome. Inhibition of tumor growth by GBP-1 is the result of its strong anti-angiogenic activity as well as its direct anti-tumorigenic effect on tumor cells. In IBD, GBP-1 mediates the anti-proliferative effects of interferon-γ on intestinal epithelial cells. In addition, it plays a protective role on the mucosa by preventing cell apoptosis, by inhibiting angiogenesis and by regulating the T-cell receptor signaling. These functions rely to a large extent on the ability of GBP-1 to interact with and remodel the actin cytoskeleton.

  5. Pathophysiological role of guanylate-binding proteins in gastrointestinal diseases

    PubMed Central

    Britzen-Laurent, Nathalie; Herrmann, Christian; Naschberger, Elisabeth; Croner, Roland S; Stürzl, Michael

    2016-01-01

    Guanylate-binding proteins (GBPs) are interferon-stimulated factors involved in the defense against cellular pathogens and inflammation. These proteins, particularly GBP-1, the most prominent member of the family, have been established as reliable markers of interferon-γ-activated cells in various diseases, including colorectal carcinoma (CRC) and inflammatory bowel diseases (IBDs). In CRC, GBP-1 expression is associated with a Th1-dominated angiostatic micromilieu and is correlated with a better outcome. Inhibition of tumor growth by GBP-1 is the result of its strong anti-angiogenic activity as well as its direct anti-tumorigenic effect on tumor cells. In IBD, GBP-1 mediates the anti-proliferative effects of interferon-γ on intestinal epithelial cells. In addition, it plays a protective role on the mucosa by preventing cell apoptosis, by inhibiting angiogenesis and by regulating the T-cell receptor signaling. These functions rely to a large extent on the ability of GBP-1 to interact with and remodel the actin cytoskeleton. PMID:27605879

  6. Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier Protein-Tethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus.

    PubMed

    Chang, Chin-Yuan; Lohman, Jeremy R; Cao, Hongnan; Tan, Kemin; Rudolf, Jeffrey D; Ma, Ming; Xu, Weijun; Bingman, Craig A; Yennamalli, Ragothaman M; Bigelow, Lance; Babnigg, Gyorgy; Yan, Xiaohui; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

    2016-09-13

    C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-β-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 Å resolution, respectively. SgcE6 shares a similar β-barrel fold with the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few other known FADH2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. The crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate. PMID:27560143

  7. Bifunctional Homodimeric Triokinase/FMN Cyclase

    PubMed Central

    Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

    2014-01-01

    Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity. PMID:24569995

  8. Nitric oxide sets off an antioxidant response in adrenal cells: involvement of sGC and Nrf2 in HO-1 induction.

    PubMed

    Astort, F; Mercau, M; Giordanino, E; Degese, M S; Caldareri, L; Coso, O; Cymeryng, C B

    2014-02-15

    Induction of microsomal heme oxygenase 1 (HO-1) activity is considered a cytoprotective mechanism in different cell types. In adrenal cells, HO-1 induction by ACTH exerts a modulatory effect on steroid production as well. As nitric oxide (NO) has been also regarded as an autocrine/paracrine modulator of adrenal steroidogenesis we sought to study the effects of NO on the induction of HO-1 and the mechanism involved. We hereby analyzed the time and dose-dependent effect of a NO-donor (DETA/NO) on HO-1 induction in a murine adrenocortical cell line. We showed that this effect is mainly exerted at a transcriptional level as it is inhibited by actinomycin D and HO-1 mRNA degradation rates were not affected by DETA/NO treatment. HO-1 induction by NO does not appear to involve the generation of oxidative stress as it was not affected by antioxidant treatment. We also demonstrated that NO-treatment results in the nuclear translocation of the nuclear factor-erythroid 2-related factor (Nrf2), an effect that is attenuated by transfecting the cells with a dominant negative isoform of Nrf2. We finally show that the effects of the NO-donor are reproduced by a permeable analog of cGMP and that a soluble guanylate cyclase specific inhibitor blocked both the induction of HO-1 by NO and the nuclear translocation of Nrf2. PMID:24361900

  9. Nitric oxide sets off an antioxidant response in adrenal cells: involvement of sGC and Nrf2 in HO-1 induction.

    PubMed

    Astort, F; Mercau, M; Giordanino, E; Degese, M S; Caldareri, L; Coso, O; Cymeryng, C B

    2014-02-15

    Induction of microsomal heme oxygenase 1 (HO-1) activity is considered a cytoprotective mechanism in different cell types. In adrenal cells, HO-1 induction by ACTH exerts a modulatory effect on steroid production as well. As nitric oxide (NO) has been also regarded as an autocrine/paracrine modulator of adrenal steroidogenesis we sought to study the effects of NO on the induction of HO-1 and the mechanism involved. We hereby analyzed the time and dose-dependent effect of a NO-donor (DETA/NO) on HO-1 induction in a murine adrenocortical cell line. We showed that this effect is mainly exerted at a transcriptional level as it is inhibited by actinomycin D and HO-1 mRNA degradation rates were not affected by DETA/NO treatment. HO-1 induction by NO does not appear to involve the generation of oxidative stress as it was not affected by antioxidant treatment. We also demonstrated that NO-treatment results in the nuclear translocation of the nuclear factor-erythroid 2-related factor (Nrf2), an effect that is attenuated by transfecting the cells with a dominant negative isoform of Nrf2. We finally show that the effects of the NO-donor are reproduced by a permeable analog of cGMP and that a soluble guanylate cyclase specific inhibitor blocked both the induction of HO-1 by NO and the nuclear translocation of Nrf2.

  10. SGC tests for influence of material composition on compaction characteristic of asphalt mixtures.

    PubMed

    Chen, Qun; Li, Yuzhi

    2013-01-01

    Compaction characteristic of the surface layer asphalt mixture (13-type gradation mixture) was studied using Superpave gyratory compactor (SGC) simulative compaction tests. Based on analysis of densification curve of gyratory compaction, influence rules of the contents of mineral aggregates of all sizes and asphalt on compaction characteristic of asphalt mixtures were obtained. SGC Tests show that, for the mixture with a bigger content of asphalt, its density increases faster, that there is an optimal amount of fine aggregates for optimal compaction and that an appropriate amount of mineral powder will improve workability of mixtures, but overmuch mineral powder will make mixtures dry and hard. Conclusions based on SGC tests can provide basis for how to adjust material composition for improving compaction performance of asphalt mixtures, and for the designed asphalt mixture, its compaction performance can be predicted through these conclusions, which also contributes to the choice of compaction schemes. PMID:23818830

  11. SGC Tests for Influence of Material Composition on Compaction Characteristic of Asphalt Mixtures

    PubMed Central

    Chen, Qun

    2013-01-01

    Compaction characteristic of the surface layer asphalt mixture (13-type gradation mixture) was studied using Superpave gyratory compactor (SGC) simulative compaction tests. Based on analysis of densification curve of gyratory compaction, influence rules of the contents of mineral aggregates of all sizes and asphalt on compaction characteristic of asphalt mixtures were obtained. SGC Tests show that, for the mixture with a bigger content of asphalt, its density increases faster, that there is an optimal amount of fine aggregates for optimal compaction and that an appropriate amount of mineral powder will improve workability of mixtures, but overmuch mineral powder will make mixtures dry and hard. Conclusions based on SGC tests can provide basis for how to adjust material composition for improving compaction performance of asphalt mixtures, and for the designed asphalt mixture, its compaction performance can be predicted through these conclusions, which also contributes to the choice of compaction schemes. PMID:23818830

  12. Oridonin induces apoptosis through the mitochondrial pathway in human gastric cancer SGC-7901 cells.

    PubMed

    Gao, Shiyong; Tan, Huixin; Zhu, Nan; Gao, Haiyu; Lv, Chunyu; Gang, Jian; Ji, Yubin

    2016-06-01

    Oridonin is one of the most important antitumor active ingredients of Rabdosia rubescens. Recently published studies from our laboratory have demonstrated that oridonin was able to arrest human gastric cancer SGC-7901 cells at G2/M phase. However, little is known about inducing apoptosis in gastric cancer. The aim of this study was to investigate the effect of oridonin on antineoplastic capability of SGC-7901 cells and the detailed molecular mechanism of oridonin-mediated intrinsic pathway of apoptosis. Cell proliferation was assessed by MTT assay while apoptosis induced by oridonin was determined by Hoechst 33342 staining assay and Annexin V/PI double staining assay. Early apoptotic rate was stained by Annexin V/PI and detected by flow cytometry. Apoptosis pathway was analyzed by western blot analysis of Bcl-2, Bax, cytochrome c and caspase-3 expression. The results showed that oridonin was able to inhibit the SGC-7901 cell proliferation, the 50% growth inhibition (IC50) was 22.74 µM. Oridonin could induce cell apoptosis of SGC-7901 cells and the early apoptotic rates induced by 0, 20, 40, 80 µmol/l oridonin were 1.53±0.67, 3.33±0.29, 84.80±0.82 and 96.43±0.51%, respectively. Western blot analysis revealed that oridonin downregulated Bcl-2 protein (the anti-apoptotic factor) and upregulated Bax protein (pro-apoptotic factor), eventually leading to a reduction in the ratio of Bcl-2/Bax proteins. Furthermore, oridonin induced the release of cytochrome c from the mitochondria to the cytosol and the activation of caspase-3. Taken together, the current study suggested that oridonin induced apoptosis in SGC-7901 cells via the mitochondrial signal pathway, which may represent one of the major mechanisms of oridonin-mediated apoptosis in SGC-7901 cells. PMID:27082253

  13. Tributyrin inhibits human gastric cancer SGC-7901 cell growth by inducing apoptosis and DNA synthesis arrest

    PubMed Central

    Yan, Jun; Xu, Yong-Hua

    2003-01-01

    AIM: To evaluate the effects of tributyrin, a pro-drug of natural butyrate and a neutral short-chain fatty acid triglyceride, on the growth inhibition of human gastric cancer SGC-7901 cell. METHODS: Human gastric cancer SGC-7901 cells were exposed to tributyrin at 0.5, 1, 2, 5, 10 and 50 mmol·L-1 for 24-72 h. MTT assay was applied to detect the cell proliferation. [3H]-TdR uptake was measured to determine DNA synthesis. Apoptotic morphology was observed by electron microscopy and Hoechst-33258 staining. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to detect tributyrin-triggered apoptosis. The expressions of PARP, Bcl-2 and Bax were examined by Western blot assay. RESULTS: Tributyrin could initiate growth inhibition of SGC-7901 cell in a dose- and time-dependent manner. [3H]-TdR uptake by SGC-7901 cells was reduced to 33.6% after 48 h treatment with 2 mmol·L-1 tributyrin, compared with the control (P < 0.05). Apoptotic morphology was detected by TUNEL assay. Flow cytometry revealed that tributyrin could induce apoptosis of SGC-7901 cells in dose-dependent manner. After 48 hours incubation with tributyrin at 2 mmol·L-1, the level of Bcl-2 protein was lowered, and the level of Bax protein was increased in SGC-7901, accompanied by PARP cleavage. CONCLUSION: Tributyrin could inhibit the growth of gastric cancer cells effectively in vitro by inhibiting DNA synthesis and inducing apoptosis, which was associated with the down-regulated Bcl-2 expression and the up-regulated Bax expression. Therefore, tributyrin might be a promising chemopreventive and chemotherapeutic agent against human gastric carcinogenesis. PMID:12679905

  14. Stimulatory and inhibitory effects of guanine nucleotides on arginine-vasotocin-sensitive adenylate cyclase in the epithelial cell membranes of the bullfrog bladder.

    PubMed

    Mishina, T; Shimada, H; Marumo, F

    1983-11-01

    The effects of arginine-vasotocin and nucleotides on the steady-state kinetics of the adenylate cyclase activity in the epithelial cell membranes of the bullfrog (Rana catesbiana) bladder were studied. Arginine-vasotocin stimulated adenylate cyclase more effectively than oxytocin or arginine-vasopressin, with respect to both the maximal hormonal activation ratio relative to basal, and the hormone concentration yielding a half-maximal response (apparent Km). Arginine-vasotocin, GTP and its analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p) increased the Vmax of the basal adenylate cyclase activity, but showed no effect of the apparent Km of the system for ATP. In addition, Gpp(NH)p enhanced the arginine-vasotocin-stimulated adenylate cyclase activity, further increasing the Vmax, while GTP showed no statistically significant effect. Dual effects of GDP were apparent: it was stimulatory at 1 x 10(-5) mol/l and inhibitory at 1 x 10(-3) mol/l, on both the basal and the arginine-vasotocin-stimulated adenylate cyclase activity. Guanosine 5'-monophosphate, CTP, UTP and ITP showed no apparent effect on the enzyme activity. Sodium fluoride acted in the same manner as GTP on the adenylate cyclase system, increasing only basal activity. Adenylate cyclase activities exhibited pH optima that were less distinct in the presence than in the absence of Gpp(NH)p. The Arrhenius plot of the temperature experiment showed that a high-energy step was involved for activation by Gpp(NH)p or arginine-vasotocin. When the relative activation ratios by arginine-vasotocin at different ATP concentrations were studied, a distinct activation optimum was shown at 2.5 x 10(-4) mol ATP/l, either in the absence or presence of Gpp(NH)p. The possibility that GTP, GDP nd ATP play a regulatory role in the epithelial cells of the bullfrog bladder by adjusting the responsiveness of the system to a natural hormone, arginine-vasotocin, is discussed. PMID:6606697

  15. Diterpene Cyclases and the Nature of the Isoprene Fold

    PubMed Central

    Cao, Rong; Zhang, Yonghui; Mann, Francis M.; Huang, Cancan; Mukkamala, Dushyant; Hudock, Michael P.; Mead, Matthew; Prisic, Sladjana; Wang, Ke; Lin, Fu-Yang; Chang, Ting-Kai; Peters, Reuben; Oldfield, Eric

    2013-01-01

    The structures and mechanism of action of many terpene cyclases are known, but there are no structures of diterpene cyclases. Here, we propose structural models based on bioinformatics, site-directed mutagenesis, domain swapping, enzyme inhibition and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain ∼20 α-helices and the same conserved “QW” and DxDD motifs as in triterpene cyclases, indicating the presence of a βγ barrel structure. Plant diterpene cyclases have a similar catalytic motif and βγ-domain structure together with a third, α-domain, forming an αβγ structure, and in H+-initiated cyclases, there is an EDxxD-like Mg2+/diphosphate binding motif located in the γ-domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (βγ) bacterial triterpene cyclases to (βγ) bacterial and thence to (αβγ) plant diterpene cyclases. PMID:20602361

  16. R-Phycoerythrin Induces SGC-7901 Apoptosis by Arresting Cell Cycle at S Phase

    PubMed Central

    Tan, Huixin; Gao, Shiyong; Zhuang, Yan; Dong, Yanhong; Guan, Wenhui; Zhang, Kun; Xu, Jian; Cui, Jingru

    2016-01-01

    R-Phycoerythrin (R-PE), one of the chemical constituents of red algae, could produce singlet oxygen upon excitation with the appropriate radiation and possibly be used in photodynamic therapy (PDT) for cancer. Documents reported that R-PE could inhibit cell proliferation in HepG2 and A549 cells, which was significative for cancer therapy. This is due to the fact that R-PE could kill cancer cells directly as well as by PDT. However, little is known about the cytotoxicity of R-PE to the SGC-7901 cell. In this study, it has been found that R-PE could inhibit SGC-7901 proliferation and induce cell apoptosis, which was achieved by arresting the SGC-7901 cell at S phase. CyclinA, CDK2 and CDC25A are proteins associated with the S phase, and it was found that R-PE could increase the expression of cyclin A protein and decrease the expression of CDK2 and CDC25A proteins. Thus, it was concluded that R-PE reduced the CDK2 protein activated through decreasing the CDC25A factor, which reduced the formation of Cyclin-CDK complex. The reduction of Cyclin-CDK complex made the SGC-7901 cells arrest at the S phase. Therefore, R-PE induced apoptosis by arresting the SGC-7901 cell at S phase was successful, which was achieved by the expression of the CDC25A protein, which reduced the CDK2 protein actived and the formation of Cyclin-CDK complex. PMID:27626431

  17. R-Phycoerythrin Induces SGC-7901 Apoptosis by Arresting Cell Cycle at S Phase.

    PubMed

    Tan, Huixin; Gao, Shiyong; Zhuang, Yan; Dong, Yanhong; Guan, Wenhui; Zhang, Kun; Xu, Jian; Cui, Jingru

    2016-01-01

    R-Phycoerythrin (R-PE), one of the chemical constituents of red algae, could produce singlet oxygen upon excitation with the appropriate radiation and possibly be used in photodynamic therapy (PDT) for cancer. Documents reported that R-PE could inhibit cell proliferation in HepG2 and A549 cells, which was significative for cancer therapy. This is due to the fact that R-PE could kill cancer cells directly as well as by PDT. However, little is known about the cytotoxicity of R-PE to the SGC-7901 cell. In this study, it has been found that R-PE could inhibit SGC-7901 proliferation and induce cell apoptosis, which was achieved by arresting the SGC-7901 cell at S phase. CyclinA, CDK2 and CDC25A are proteins associated with the S phase, and it was found that R-PE could increase the expression of cyclin A protein and decrease the expression of CDK2 and CDC25A proteins. Thus, it was concluded that R-PE reduced the CDK2 protein activated through decreasing the CDC25A factor, which reduced the formation of Cyclin-CDK complex. The reduction of Cyclin-CDK complex made the SGC-7901 cells arrest at the S phase. Therefore, R-PE induced apoptosis by arresting the SGC-7901 cell at S phase was successful, which was achieved by the expression of the CDC25A protein, which reduced the CDK2 protein actived and the formation of Cyclin-CDK complex. PMID:27626431

  18. Soluble Adenylyl Cyclase in Health and Disease

    PubMed Central

    Schmid, Andreas; Meili, Dimirela; Salathe, Matthias

    2014-01-01

    The second messenger cAMP is integral for many physiological processes. Soluble adenylyl cyclase (sAC) was recently identified as a widely expressed intracellular source of cAMP in mammalian cells. sAC is evolutionary, structurally, and biochemically distinct from the G-protein-responsive transmembranous adenylyl cyclases (tmAC). The structure of the catalytic unit of sAC is similar to tmAC, but sAC does not contain transmembranous domains, allowing localizations independent of the membranous compartment. sAC activity is stimulated by HCO3-, Ca2+ and is sensitive to physiologically relevant ATP fluctuations. sAC functions as a physiological sensor for carbon dioxide and bicarbonate, and therefore indirectly for pH. Here we review the physiological role of sAC in different human tissues with a major focus on the lung. PMID:25064591

  19. Membrane guanylyl cyclase receptors: an update

    PubMed Central

    Garbers, David L.; Chrisman, Ted D.; Wiegn, Phi; Katafuchi, Takeshi; Albanesi, Joseph P.; Bielinski, Vincent; Barylko, Barbara; Redfield, Margaret M.; Burnett, John C.

    2007-01-01

    Recent studies have demonstrated key roles for several membrane guanylyl cyclase receptors in the regulation of cell hyperplasia, hypertrophy, migration and extracellular matrix production, all of which having an impact on clinically relevant diseases, including tissue remodeling after injury. Additionally, cell differentiation, and even tumor progression, can be profoundly influenced by one or more of these receptors. Some of these receptors also mediate important communication between the heart and intestine, and the kidney to regulate blood volume and Na+ balance. PMID:16815030

  20. Adenylyl cyclases in the digestive system.

    PubMed

    Sabbatini, Maria Eugenia; Gorelick, Fred; Glaser, Shannon

    2014-06-01

    Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca(2+) and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed.

  1. Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum.

    PubMed

    Silva, Fábio H; Lanaro, Carolina; Leiria, Luiz Osório; Rodrigues, Renata Lopes; Davel, Ana Paula; Claudino, Mário A; Toque, Haroldo A; Antunes, Edson

    2014-11-15

    Impairment of nitric oxide (NO)-mediated cavernosal relaxations in middle age contributes to erectile dysfunction. However, little information is available about the alterations of sympathetic neurotransmission and contraction in erectile tissue at middle age. This study aimed to evaluate the alterations of the contractile machinery associated with tyrosine hydroxylase (TH) in rat corpus cavernosum (RCC) at middle age, focusing on the role of superoxide anion. Male Wistar young (3.5-mo) and middle-aged (10-mo) rats were used. Electrical-field stimulation (EFS)- and phenylephrine-induced contractions were obtained in RCC strips. Levels of reactive-oxygen species (ROS) and TH mRNA expression, as well as protein expressions for α₁/β₁-subunits of soluble guanylyl cyclase (sGC), in RCC were evaluated. The neurogenic contractile responses elicited by EFS (4-32 Hz) were greater in RCC from the middle-aged group that was accompanied by elevated TH mRNA expression (P < 0.01). Phenylephrine-induced contractions were also greater in the middle-aged group. A 62% increase in ROS generation in RCC from middle-aged rats was observed. The mRNA expression for the α₁A-adrenoceptor remained unchanged among groups. Protein levels of α₁/β₁-sGC subunits were decreased in RCC from the middle-aged compared with young group. The NADPH oxidase inhibitor apocynin (85 mg·rat(-1)·day(-1), 4 wk) fully restored the enhanced ROS production, TH mRNA expressions, and α₁/β₁-subunit sGC expression, indicating that excess of superoxide anion plays a major role in the sympathetic hyperactivity and hypercontractility in erectile tissue at middle age. Reduction of oxidative stress by dietary antioxidants may be an interesting approach to treat erectile dysfunction in aging population.

  2. GMP reductase and genetic uncoupling of adenylate and guanylate metabolism in Leishmania donovani parasites.

    PubMed

    Boitz, Jan M; Jardim, Armando; Ullman, Buddy

    2016-08-01

    Purine acquisition is an essential nutritional process for Leishmania. Although purine salvage into adenylate nucleotides has been investigated in detail, little attention has been focused on the guanylate branch of the purine pathway. To characterize guanylate nucleotide metabolism in Leishmania and create a cell culture model in which the pathways for adenylate and guanylate nucleotide synthesis can be genetically uncoupled for functional studies in intact cells, we created and characterized null mutants of L. donovani that were deficient in either GMP reductase alone (Δgmpr) or in both GMP reductase and its paralog IMP dehydrogenase (Δgmpr/Δimpdh). Whereas wild type parasites were capable of utilizing virtually any purine nucleobase/nucleoside, the Δgmpr and Δgmpr/Δimpdh null lines exhibited highly restricted growth phenotypes. The Δgmpr single mutant could not grow in xanthine, guanine, or their corresponding nucleosides, while no purine on its own could support the growth of Δgmpr/Δimpdh cells. Permissive growth conditions for the Δgmpr/Δimpdh necessitated both xanthine, guanine, or the corresponding nucleosides, and additionally, a second purine that could serve as a source for adenylate nucleotide synthesis. Interestingly, GMPR, like its paralog IMPDH, is compartmentalized to the leishmanial glycosome, a process mediated by its COOH-terminal peroxisomal targeting signal. The restricted growth phenotypes displayed by the L. donovani Δgmpr and Δgmpr/Δimpdh null mutants confirms the importance of GMPR in the purine interconversion processes of this parasite.

  3. Estrogen reduces beta-adrenoceptor-mediated cAMP production and the concentration of the guanyl nucleotide-regulatory protein, Gs, in rabbit myometrium.

    PubMed

    Riemer, R K; Wu, Y Y; Bottari, S P; Jacobs, M M; Goldfien, A; Roberts, J M

    1988-04-01

    The uterine contractile response to adrenergic agonists or sympathetic stimulation is influenced dramatically by the hormonal milieu. Rabbit uterine contraction is mediated by alpha 1-adrenoceptors, whereas relaxation in response to the same stimulus is mediated by beta 2-adrenoceptors. Whether uterine contractility is increased or decreased by adrenergic stimulation is determined by the gonadal steroids estrogen and progesterone: uterine contraction prevails in the estrogen-dominant or the ovariectomized animal, but in the progesterone-dominant rabbit, uterine relaxation is observed. In previous studies, we have demonstrated that changes in the concentration or agonist affinity of these adrenoceptors cannot account for the changes in contractile response. In the present studies, we tested whether sex steroids might alter beta-adrenergic response by acting on events distal to receptor occupancy, and whether this could explain the conversion of contractile response. We found that myometrial cAMP generation is potently stimulated by beta-agonists in progesterone-treated and also in ovariectomized animals, but this stimulation is absent after estrogen treatment. Similar, but smaller, changes were observed for cAMP generation in response to prostaglandin E2 and forskolin. Stimulation of adenylate cyclase in uterine particulates by agents which act on the guanyl nucleotide-sensitive stimulatory transducer, Gs, is unchanged after estrogen treatment. However, specific labeling of Gs catalyzed by cholera toxin is reduced in membrane particulates from estrogen-treated animals. Recombination of extracts of uterine membranes from the differently treated animals also suggested qualitative differences in Gs. We conclude that at least one component of the adenylate cyclase cascade beyond the beta-adrenoceptor, i.e., Gs, is a target for ovarian steroids; estrogen reduces Gs labeling and beta-adrenoceptor-mediated cAMP production. However, uterine Gs labeling and cAMP production

  4. Silencing of FRAT1 by siRNA inhibits the proliferation of SGC7901 human gastric adenocarcinoma cells

    PubMed Central

    YU, QINGGONG; SHANG, LU; YU, HONGBO; YANG, ZIRONG; XU, DEKUI

    2016-01-01

    Frequently rearranged in advanced T cell lymphomas-1 (FRAT1) positively regulates the Wnt/β-catenin signaling pathway by inhibiting glycogen synthase kinase-3 mediated phosphorylation of β-catenin. FRAT1 is a proto-oncogene, implicated in tumorigenesis. The present study aimed to investigate the effects of FRAT1 silencing on the proliferation and apoptosis of SGC7901 cells. FRAT1 in SGC7901 cells was silenced by RNA interference. Reverse transcription-quantitative polymerase chain reaction was used for the analysis of FRAT1 mRNA and western blotting was used to evaluate FRAT1 and β-catenin protein levels. Cell proliferation was analyzed by the MTT assay. Cell cycle distribution and apoptosis were analyzed by flow cytometry. The expression of FRAT1 mRNA, FRAT1 and β-catenin protein in FRAT1-silenced SGC7901 cells were reduced significantly compared to untreated cells. The proliferation of FRAT1 silenced SGC7901 cells decreased significantly The FRAT1 silenced SGC7901 cells were arrested at G0/G1 stage to a greater degree, and apoptosis was increased. In summary, silencing of FRAT1 inhibits SGC7901 cell proliferation and induces apoptosis, possible through a reduction in β-catenin expression. FRAT1 may serve as a prognostic biomarker and therapeutic target for gastric cancer. PMID:26893843

  5. Comparative analysis of plant lycopene cyclases.

    PubMed

    Koc, Ibrahim; Filiz, Ertugrul; Tombuloglu, Huseyin

    2015-10-01

    Carotenoids are essential isoprenoid pigments produced by plants, algae, fungi and bacteria. Lycopene cyclase (LYC) commonly cyclize carotenoids, which is an important branching step in the carotenogenesis, at one or both end of the backbone. Plants have two types of LYC (β-LCY and ϵ-LCY). In this study, plant LYCs were analyzed. Based on domain analysis, all LYCs accommodate lycopene cyclase domain (Pf05834). Furthermore, motif analysis indicated that motifs were conserved among the plants. On the basis of phylogenetic analysis, β-LCYs and ϵ-LCYs were classified in β and ϵ groups. Monocot and dicot plants separated from each other in the phylogenetic tree. Subsequently, Oryza sativa Japonica Group and Zea mays of LYCs as monocot plants and Vitis vinifera and Solanum lycopersicum of LYCs as dicot plants were analyzed. According to nucleotide diversity analysis of β-LCY and ϵ-LCY genes, nucleotide diversities were found to be π: 0.30 and π: 0.25, respectively. The result highlighted β-LCY genes showed higher nucleotide diversity than ϵ-LCY genes. LYCs interacting genes and their co-expression partners were also predicted using String server. The obtained data suggested the importance of LYCs in carotenoid metabolism. 3D modeling revealed that depicted structures were similar in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs. Likewise, the predicted binding sites were highly similar between O. sativa, Z mays, S. lycopersicum, and V. vinifera LCYs. Most importantly, analysis elucidated the V/IXGXGXXGXXXA motif for both type of LYC (β-LCY and ϵ-LCY). This motif related to Rossmann fold domain and probably provides a flat platform for binding of FAD in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs with conserved structure. In addition to lycopene cyclase domain, the V/IXGXGXXGXXXA motif can be used for exploring LYCs proteins and to annotate the function of unknown proteins containing lycopene cyclase

  6. Comparative analysis of plant lycopene cyclases.

    PubMed

    Koc, Ibrahim; Filiz, Ertugrul; Tombuloglu, Huseyin

    2015-10-01

    Carotenoids are essential isoprenoid pigments produced by plants, algae, fungi and bacteria. Lycopene cyclase (LYC) commonly cyclize carotenoids, which is an important branching step in the carotenogenesis, at one or both end of the backbone. Plants have two types of LYC (β-LCY and ϵ-LCY). In this study, plant LYCs were analyzed. Based on domain analysis, all LYCs accommodate lycopene cyclase domain (Pf05834). Furthermore, motif analysis indicated that motifs were conserved among the plants. On the basis of phylogenetic analysis, β-LCYs and ϵ-LCYs were classified in β and ϵ groups. Monocot and dicot plants separated from each other in the phylogenetic tree. Subsequently, Oryza sativa Japonica Group and Zea mays of LYCs as monocot plants and Vitis vinifera and Solanum lycopersicum of LYCs as dicot plants were analyzed. According to nucleotide diversity analysis of β-LCY and ϵ-LCY genes, nucleotide diversities were found to be π: 0.30 and π: 0.25, respectively. The result highlighted β-LCY genes showed higher nucleotide diversity than ϵ-LCY genes. LYCs interacting genes and their co-expression partners were also predicted using String server. The obtained data suggested the importance of LYCs in carotenoid metabolism. 3D modeling revealed that depicted structures were similar in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs. Likewise, the predicted binding sites were highly similar between O. sativa, Z mays, S. lycopersicum, and V. vinifera LCYs. Most importantly, analysis elucidated the V/IXGXGXXGXXXA motif for both type of LYC (β-LCY and ϵ-LCY). This motif related to Rossmann fold domain and probably provides a flat platform for binding of FAD in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs with conserved structure. In addition to lycopene cyclase domain, the V/IXGXGXXGXXXA motif can be used for exploring LYCs proteins and to annotate the function of unknown proteins containing lycopene cyclase

  7. Effects of culture media on metabolic profiling of the human gastric cancer cell line SGC7901.

    PubMed

    Huang, Zicheng; Shao, Wei; Gu, Jinping; Hu, Xiaomin; Shi, Yuanzhi; Xu, Wenqi; Huang, Caihua; Lin, Donghai

    2015-07-01

    Cell culture metabolomics has demonstrated significant advantages in cancer research. However, its applications have been impeded by some influencing factors such as culture media, which could significantly affect cellular metabolic profiles and lead to inaccuracy and unreliability of comparative metabolomic analysis of cells. To evaluate the effects of different culture media on cellular metabolic profiling, we performed NMR-based metabolomic analysis of the human gastric cancer cell line SGC7901 cultured in both RPMI1640 and DMEM. We found that SGC7901 cultured in the two media exhibited distinct metabolic profiles with obviously different levels of discrepant metabolites, even though they showed almost the same cellular morphology and proliferation rates. When SGC7901 originally cultured in RPMI1640 was gradually acclimated in DMEM, both the metabolic profiles and most of the discrepant metabolite levels gradually converged toward those of the cells originally cultured in DMEM without significantly altered cell proliferation rates. However, several metabolite levels did not show the converging trends. Our results indicate that the effects of culture media on metabolic profiling must be carefully taken into account for comparative metabolomic analysis of cell lines. This work may be of benefit to the development of cell culture metabolomics.

  8. Effects of ophiopogonin B on the proliferation and apoptosis of SGC-7901 human gastric cancer cells

    PubMed Central

    ZHANG, WEIYUE; ZHANG, QIAOYAN; JIANG, YIPING; LI, FENG; XIN, HAILIANG

    2016-01-01

    Ophiopogonin B (OP-B) is a bioactive component of Radix Ophiopogon japonicus, which is often used in traditional Chinese medicine to treat cancer. The present study aimed to investigate the antitumor activity of OP-B in gastric cancer. Cell Counting kit-8, flow cytometry with Annexin V-fluorescein isothiocyanate, Hoechst staining, mitochondrial membrane potential (MMP) detection, and reactive oxygen species (ROS) assay were used to detect the biological function of SGC-7901 gastric cancer cells. The results demonstrated that high concentrations of OP-B (5, 10 and 20 μmol/l) exerted potent antiproliferative effects on SGC-7901 cells in a dose-dependent manner. Furthermore, apoptotic rates were increased and cell morphology was altered following treatment with OP-B. In addition, OP-B-induced apoptosis of SGC-7901 cells was associated with loss of MMP and increased ROS generation. Western blotting indicated that treatment with OP-B increased the protein expression levels of caspase-3 and B-cell lymphoma 2 (Bcl-2)-associated X protein, whereas the expression levels of Bcl-2 and the phosphorylation levels of extracellular signal-regulated kinases 1/2 and c-Jun N-terminal kinases 1/2 were decreased. These results suggest that OP-B may be considered a potential inhibitor of gastric cancer progression, and may be used as an alternative compound for its treatment. PMID:27121658

  9. The triterpene cyclase protein family: a systematic analysis.

    PubMed

    Racolta, Silvia; Juhl, P Benjamin; Sirim, Demet; Pleiss, Jürgen

    2012-08-01

    Triterpene cyclases catalyze a broad range of cyclization reactions to form polycyclic triterpenes. Triterpene cyclases that convert squalene to hopene are named squalene-hopene cyclases (SHC) and triterpene cyclases that convert oxidosqualene are named oxidosqualene cyclases (OSC). Many sequences have been published, but there is only one structure available for each of SHCs and OSCs. Although they catalyze a similar reaction, the sequence similarity between SHCs and OSCs is low. A family classification based on phylogenetic analysis revealed 20 homologous families which are grouped into two superfamilies, SHCs and OSCs. Based on this family assignment, the Triterpene Cyclase Engineering Database (TTCED) was established. It integrates available information on sequence and structure of 639 triterpene cyclases as well as on structurally and functionally relevant amino acids. Family specific multiple sequence alignments were generated to identify the functionally relevant residues. Based on sequence alignments, conserved residues in SHCs and OSCs were analyzed and compared to experimentally confirmed mutational data. Functional schematic models of the central cavities of OSCs and SHCs were derived from structure comparison and sequence conservation analysis. These models demonstrate the high similarity of the substrate binding cavity of SHCs and OSCs and the equivalences of the respective residues. The TTCED is a novel source for comprehensive information on the triterpene cyclase family, including a compilation of previously described mutational data. The schematic models present the conservation analysis in a readily available fashion and facilitate the correlation of residues to a specific function or substrate interaction.

  10. Soluble variants of human recombinant glutaminyl cyclase.

    PubMed

    Castaldo, Cristiana; Ciambellotti, Silvia; de Pablo-Latorre, Raquel; Lalli, Daniela; Porcari, Valentina; Turano, Paola

    2013-01-01

    Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of (1)H-(15)N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer's disease. PMID:23977104

  11. Adenylate cyclases involvement in pathogenicity, a minireview.

    PubMed

    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

    Cyclic AMP (cAMP), one of the most important secondary messengers, is produced by adenylate cyclase (AC) from adenosine triphosphate (ATP). AC is a widespread enzyme, being present both in prokaryotes and eukaryotes. Although they have the same enzymatic activity (ATP cyclization), the structure of these proteins varies, depending on their function and the producing organism. Some pathogenic bacteria utilize these enzymes as toxins which interact with calmodulin (or another eukaryote activator), causing intense cAMP synthesis and disruption of infected cell functions. In contrast, other pathogenic bacteria benefit of augmentation of AC activity for their own function. Based on sequence analysis ofAC catalytic domain from two pathogenic bacteria (Bacillus anthracis and Bordetellapertussis) with known three-dimensional structures, a possible secondary structure for 1-255 amino acid fragment from Pseudomonas aeruginosa AC (with 80TKGFSVKGKSS90 as the ATP binding site) is proposed.

  12. Soluble Variants of Human Recombinant Glutaminyl Cyclase

    PubMed Central

    Castaldo, Cristiana; Ciambellotti, Silvia; de Pablo-Latorre, Raquel; Lalli, Daniela; Porcari, Valentina; Turano, Paola

    2013-01-01

    Recombinant human Glutaminyl Cyclase expressed in E. coli is produced as inclusion bodies. Lack of glycosylation is the main origin of its accumulation in insoluble aggregates. Mutation of single isolated hydrophobic amino acids into negative amino acids was not able to circumvent inclusion bodies formation. On the contrary, substitution with carboxyl-terminal residues of two or three aromatic residues belonging to extended hydrophobic patches on the protein surface provided soluble but still active forms of the protein. These mutants could be expressed in isotopically enriched forms for NMR studies and the maximal attainable concentration was sufficient for the acquisition of 1H-15N HSQC spectra that represent the starting point for future drug development projects targeting Alzheimer’s disease. PMID:23977104

  13. Diuretics and the renal adenylate cyclase system

    PubMed Central

    Dawborn, J.K.; Macneil, S.; Martin, T.J.

    1977-01-01

    1 The relationship between the diuretic effectiveness and the effect on the renal adenylate cyclase of three diuretics, acetazolamide, frusemide and ethacrynic acid, was examined. The hypothesis that acetazolamide and parathyroid hormone (PTH), inhibit renal carbonic anhydrase by a cyclic adenosine 3′,5′-monophosphate (cyclic AMP)-dependent mechanism was also tested. 2 In vitro, acetazolamide, frusemide and ethacrynic acid at high concentrations (10-3M) all produced some inhibition of basal and stimulated rat kidney plasma membrane adenylate cyclase. The effect of acetazolamide was much less than that of frusemide and ethacrynic acid. These plasma membrane effects were reproduced in studies of cyclic AMP formation in isolated kidney tubules of rats. 3 Intravenous injections of acetazolamide did not change the total cyclic AMP content of the kidneys of rats killed by microwave irradiation. 4 Acetazolamide produced a diuresis in the rat and a slight inhibition of the antidiuretic effect of Pitressin. Frusemide produced a diuresis and greatly reduced the antidiuretic response to Pitressin. Ethacrynic acid was ineffective as a diuretic in the rat and actually enhanced the antidiuretic response to Pitressin. 5 In investigating the possible influence of diuretics and PTH on the activity and state of phosphorylation of carbonic anhydrase it was found that: there was no correlation between the ability of diuretics to inhibit carbonic anhydrase activity and to inhibit carbonic anhydrase phosphorylation; neither PTH nor cyclic AMP (in the presence of adenosine triphosphate, Mg2+, K+ and incubation at 37°C) inhibited rat cortex homogenate carbonic anhydrase activity. 6 It seems unlikely that any of the tested diuretics exerts its pharmacological effect by means of changes in kidney cyclic AMP metabolism. PMID:202362

  14. Carnosine inhibits the proliferation of human gastric cancer SGC-7901 cells through both of the mitochondrial respiration and glycolysis pathways.

    PubMed

    Shen, Yao; Yang, Jianbo; Li, Juan; Shi, Xiaojie; Ouyang, Li; Tian, Yueyang; Lu, Jianxin

    2014-01-01

    Carnosine, a naturally occurring dipeptide, has been recently demonstrated to possess anti-tumor activity. However, its underlying mechanism is unclear. In this study, we investigated the effect and mechanism of carnosine on the cell viability and proliferation of the cultured human gastric cancer SGC-7901 cells. Carnosine treatment did not induce cell apoptosis or necrosis, but reduced the proliferative capacity of SGC-7901 cells. Seahorse analysis showed SGC-7901 cells cultured with pyruvate have active mitochondria, and depend on mitochondrial oxidative phosphorylation more than glycolysis pathway for generation of ATP. Carnosine markedly decreased the absolute value of mitochondrial ATP-linked respiration, and reduced the maximal oxygen consumption and spare respiratory capacity, which may reduce mitochondrial function correlated with proliferative potential. Simultaneously, carnosine also reduced the extracellular acidification rate and glycolysis of SGC-7901 cells. Our results suggested that carnosine is a potential regulator of energy metabolism of SGC-7901 cells both in the anaerobic and aerobic pathways, and provided a clue for preclinical and clinical evaluation of carnosine for gastric cancer therapy.

  15. Matrine Suppresses Proliferation and Invasion of SGC7901 Cells through Inactivation of PI3K/Akt/uPA Pathway.

    PubMed

    Peng, Xiaochun; Zhou, Dawei; Wang, Xianwang; Hu, Zhifan; Yan, Yan; Huang, Jiangrong

    2016-09-01

    This study was to examine the inhibitory effect of matrine on the proliferation and metastasis of gastric cancer cells, and to explore the possible mechanisms involved in these processes. MTT was used to evaluate the proliferation ability of SGC7901 cells. A two and three-dimensional cell migration assay were performed to determine the effect of matrine on the migration of SGC7901 cells. Then, the changes of the uPA protein and other possible signal molecules were detected by western blot. We found that the proliferation ability of SGC 7901 cells was suppressed by matrine (p<0.05), and the average cell migration velocity was also significantly inhibited by matrine when compared to the control in a two-dimensional cell migration assay. In addition, SGC7901cells treated with matrine (50μg/ml) migrated less than the control cells in a three-dimensional cell migration assay. At the meantime, the decreased uPA protein expression in SGC7901 cells treated with matrine was observed, and the PI3K/Akt pathway was inhibited. These results suggested that matrine can inhibit the proliferation and metastasis of gastric cancer cells through the PI3K/Akt/uPA pathway, indicating that matrine might be a potential molecular target for treatment of gastric carcinoma.

  16. TFF3 mediated induction of VEGF via hypoxia in human gastric cancer SGC-7901 cells.

    PubMed

    Guleng, Bayasi; Han, Jia; Yang, Jin-Qiu; Huang, Qing-Wen; Huang, Jian-Kun; Yang, Xiao-Ning; Liu, Jing-Jing; Ren, Jian-Lin

    2012-04-01

    Increasing evidence indicates that in gastric epithelial cells, induction of TFF3 by hypoxia is mediated by HIF-1. Since VEGF is one of the most important angiogenic factors on cancer progression, we have started to investigate the possible link among HIF-1α, VEGF, and TFF3 in gastric cancer cells. We induced the hypoxic condition in SGC-7901cells using hypoxia-mimetic agent of CoCI2. SGC7901 cells were transfected with pcPUR + U6 plasmid carrying RNAi targeted to human TFF3 and selected puromycin-resistant pools to establish the stable knockdown of TFF3 cells. Our results showed the induction of HIF-1a via hypoxia and consequences of increased expressions of the TFF3 and VEGF in gastric cancer SGC-7901 cells. Overexpression of TFF3 upregulated the mRNA expressions of VEGF and HIF-1a induced by hypoxia, and stable knockdown of TFF3 impaired the mRNA upregulations of VEGF and HIF-1a induced by hypoxia. Furthermore, knockdown of TFF3 reduced the VEGF protein secretion: as VEGF secretion was increased time dependent manner in response to the hypoxia induction in TFF3-WT cells; however, VEGF production was significantly decreased in TFF3-KD cells (621 ± 89 vs. 264 ± 73 at 6 h and 969 ± 97 vs. 508 ± 69 at 12 h, P < 0.05). Our data demonstrated the TFF3 mediated regulation of VEGF expression induced by hypoxia, and implicated that TFF3 might be applied as a potential anti-angiogenic target for treatment of gastric cancer.

  17. Cellular and subcellular localization of Ras guanyl nucleotide-releasing protein in the rat hippocampus.

    PubMed

    Pierret, P; Vallée, A; Mechawar, N; Dower, N A; Stone, J C; Richardson, P M; Dunn, R J

    2001-01-01

    Ras guanyl nucleotide-releasing protein (RasGRP) is a recently discovered Ras guanyl nucleotide exchange factor that is expressed in selected regions of the rodent CNS, with high levels of expression in the hippocampus. Biochemical studies suggest that RasGRP can activate the Ras signal pathway in response to changes in diacylglycerol and possibly calcium. To investigate potential sites for RasGRP signaling, we have determined the cellular and subcellular localization of RasGRP protein in adult rat hippocampus, and have also examined the appearance of RasGRP mRNA and protein during hippocampal development. RasGRP immunoreactivity is predominately localized to those neurons participating in the direct cortico-hippocampo-cortical loop. In both hippocampal and entorhinal neurons, RasGRP protein appeared to be localized to both dendrites and somata, but not to axons. Electron microscopy of hippocampal pyramidal cells confirmed RasGRP immunoreactivity in neuronal cell bodies and dendrites, where it appeared to be associated with microtubules. The localization of RasGRP to dendrites suggests a role for this pathway in the regulation of dendritic function. Examination of developing hippocampal structures indicated that RasGRP mRNA and protein appear synchronously during the first 2 weeks of postnatal development as these neurons become fully mature. This result indicates that the RasGRP signal transduction pathway is not required during early hippocampal development, but is a feature of mature neurons during the later stages of development.

  18. Farnesyloxycoumarins, a new class of squalene-hopene cyclase inhibitors.

    PubMed

    Cravotto, Giancarlo; Balliano, Gianni; Robaldo, Bruna; Oliaro-Bosso, Simonetta; Chimichi, Stefano; Boccalini, Marco

    2004-04-19

    A few naturally occurring prenyl- and prenyloxycoumarins and several new related synthetic derivatives were evaluated as inhibitors of squalene-hopene cyclase (SHC), a useful model enzyme, to predict their interactions with oxidosqualene cyclase (OSC). Umbelliprenin-10',11'-monoepoxide (IC(50) 2.5 microM) and the corresponding 6',7'-10',11' diepoxide (IC(50) 1.5 microM) were the most active enzyme inhibitors.

  19. Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

    PubMed Central

    Carricarte, V C; Bianchini, G M; Muschietti, J P; Téllez-Iñón, M T; Perticari, A; Torres, N; Flawiá, M M

    1988-01-01

    An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase. PMID:3128270

  20. Matrine alters microRNA expression profiles in SGC-7901 human gastric cancer cells.

    PubMed

    Li, Hailong; Xie, Shoupin; Liu, Xiaojun; Wu, Hongyan; Lin, Xingyao; Gu, Jing; Wang, Huping; Duan, Yongqiang

    2014-11-01

    Matrine, a major alkaloid extracted from Sophora flavescens, has been reported to possess antitumor properties in several types of cancers, including gastric cancer. However, its mechanisms of action on gastric cancer remain poorly understood. Dysregulation of microRNAs, a class of small, non-coding, regulatory RNA molecules involved in gene expression, is strongly correlated with cancer. The aim of the present study was to demonstrate that matrine treatment altered miRNA expression in SGC7901 cells. Using miRCURY™ microarray analysis, we identified 128 miRNAs substantially exhibiting >2-fold expression changes in matrine-treated cells relative to their expression levels in untreated cells. RT-qPCR was used to show that the levels of 8 miRNAs whose target genes were clustered in the cell cycle pathway increased, while levels of 14 miRNAs whose target genes were clustered in the MAPK signaling pathway decreased. These results were consistent with those from the miRNA microarray experiment. Bioinformatical analysis revealed that the majority of 57 identified enrichment pathways were highly involved in tumorigenesis. In conclusion, the results demonstrated that matrine induces considerable changes in the miRNA expression profiles of SGC7901 cells, suggesting miRNA microarray combined with RT-qPCR validation and bioinformatical analysis provide a novel and promising approach to identify anticancer targets and the mechanisms of matrine involved.

  1. Novel hopanoid cyclases from the environment.

    PubMed

    Pearson, Ann; Flood Page, Sarah R; Jorgenson, Tyler L; Fischer, Woodward W; Higgins, Meytal B

    2007-09-01

    Hopanoids are ubiquitous isoprenoid lipids found in modern biota, in recent sediments and in low-maturity sedimentary rocks. Because these lipids primarily are derived from bacteria, they are used as proxies to help decipher geobiological communities. To date, much of the information about sources of hopanoids has come from surveys of culture collections, an approach that does not address the vast fraction of prokaryotic communities that remains uncharacterized. Here we investigated the phylogeny of hopanoid producers using culture-independent methods. We obtained 79 new sequences of squalene-hopene cyclase genes (sqhC) from marine and lacustrine bacterioplankton and analysed them along with all 31 sqhC fragments available from existing metagenomics libraries. The environmental sqhCs average only 60% translated amino acid identity to their closest relatives in public databases. The data imply that the sources of these important geologic biomarkers remain largely unknown. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analysed here, yet the geologic record contains abundant hopanoids apparently of cyanobacterial origin. The data also suggest that hopanoid biosynthesis is uncommon: < 10% of bacterial species may be capable of producing hopanoids. A better understanding of the contemporary distribution of hopanoid biosynthesis may reveal fundamental insight about the function of these compounds, the organisms in which they are found, and the environmental signals preserved in the sedimentary record.

  2. Novel hopanoid cyclases from the environment.

    PubMed

    Pearson, Ann; Flood Page, Sarah R; Jorgenson, Tyler L; Fischer, Woodward W; Higgins, Meytal B

    2007-09-01

    Hopanoids are ubiquitous isoprenoid lipids found in modern biota, in recent sediments and in low-maturity sedimentary rocks. Because these lipids primarily are derived from bacteria, they are used as proxies to help decipher geobiological communities. To date, much of the information about sources of hopanoids has come from surveys of culture collections, an approach that does not address the vast fraction of prokaryotic communities that remains uncharacterized. Here we investigated the phylogeny of hopanoid producers using culture-independent methods. We obtained 79 new sequences of squalene-hopene cyclase genes (sqhC) from marine and lacustrine bacterioplankton and analysed them along with all 31 sqhC fragments available from existing metagenomics libraries. The environmental sqhCs average only 60% translated amino acid identity to their closest relatives in public databases. The data imply that the sources of these important geologic biomarkers remain largely unknown. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analysed here, yet the geologic record contains abundant hopanoids apparently of cyanobacterial origin. The data also suggest that hopanoid biosynthesis is uncommon: < 10% of bacterial species may be capable of producing hopanoids. A better understanding of the contemporary distribution of hopanoid biosynthesis may reveal fundamental insight about the function of these compounds, the organisms in which they are found, and the environmental signals preserved in the sedimentary record. PMID:17686016

  3. Interferon-induced guanylate-binding proteins in inflammasome activation and host defense.

    PubMed

    Kim, Bae-Hoon; Chee, Jonathan D; Bradfield, Clinton J; Park, Eui-Soon; Kumar, Pradeep; MacMicking, John D

    2016-05-01

    Traditional views of the inflammasome highlight the assembly of pre-existing core components shortly after infection or tissue damage. Emerging work, however, suggests that the inflammasome machinery is also subject to 'tunable' or inducible signals that might accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these signals operate downstream of interferon receptors to elicit inflammasome regulators, including a new family of interferon-induced GTPases called 'guanylate-binding proteins' (GBPs). Here we investigate the critical roles of interferon-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity to a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential effect of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases. PMID:27092805

  4. Interferon-induced guanylate-binding proteins in inflammasome activation and host defense.

    PubMed

    Kim, Bae-Hoon; Chee, Jonathan D; Bradfield, Clinton J; Park, Eui-Soon; Kumar, Pradeep; MacMicking, John D

    2016-05-01

    Traditional views of the inflammasome highlight the assembly of pre-existing core components shortly after infection or tissue damage. Emerging work, however, suggests that the inflammasome machinery is also subject to 'tunable' or inducible signals that might accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these signals operate downstream of interferon receptors to elicit inflammasome regulators, including a new family of interferon-induced GTPases called 'guanylate-binding proteins' (GBPs). Here we investigate the critical roles of interferon-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity to a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential effect of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases.

  5. IFN-induced Guanylate Binding Proteins in Inflammasome Activation and Host Defense

    PubMed Central

    Kim, Bae-Hoon; Chee, Jonathan D.; Bradfield, Clinton J.; Park, Eui-Soon; Kumar, Pradeep; MacMicking, John D.

    2016-01-01

    Traditional views of the inflammasome highlight pre-existing core components being assembled under basal conditions shortly after infection or tissue damage. Recent work, however, suggests the inflammasome machinery is also subject to tunable or inducible signals that may accelerate its autocatalytic properties and dictate where inflammasome assembly takes place in the cell. Many of these immune signals operate downstream of interferon (IFN) receptors to elicit inflammasome regulators, including a new family of IFN-induced GTPases termed guanylate binding proteins (GBPs). Here, we examine the critical roles for IFN-induced GBPs in directing inflammasome subtype-specific responses and their consequences for cell-autonomous immunity against a wide variety of microbial pathogens. We discuss emerging mechanisms of action and the potential impact of these GBPs on predisposition to sepsis and other infectious or inflammatory diseases. PMID:27092805

  6. Anisotropic mechanical response of the enzyme Guanylate Kinase perturbed by the DNA molecular spring.

    NASA Astrophysics Data System (ADS)

    Tseng, Chiao-Yu; Wang, Andrew; Zocchi, Giovanni

    2010-03-01

    Protein molecules are semi-rigid objects with organized but fluctuating conformation. For Guanylate Kinase, which catalyzes phosphoryl transfer between ATP and GMP, a large conformational change upon substrate binding occurs which is essential for enzymatic activity. With a DNA molecular spring stretching the molecule in distinct ways, we demonstrate that the enzymatic functions of substrate binding and phosphoryl transfer can be separately controlled mechanically. Three different attachment points of the DNA spring on the surface of the protein were tested, corresponding to stretching the protein along three different directions. Using activity measurements with titration over substrate concentration, the kinetic parameters (i.e., binding affinity of substrates and catalytic rate constant) based on Michaelis-Menten kinetics were obtained in the presence and absence of the three different mechanical perturbations.

  7. RNA capping by the vaccinia virus guanylyltransferase. Structure of enzyme-guanylate intermediate.

    PubMed

    Roth, M J; Hurwitz, J

    1984-11-10

    GTP:RNA guanylyltransferase isolated from vaccinia virus catalyzes the transfer of GMP from GTP to the 5' terminus of RNA via an enzyme-guanylate intermediate. Incubation of the purified vaccinia RNA guanylyltransferase with [alpha- 32P]GTP and MgCl2 yields [32P]GMP covalently linked to the Mr = 95,000 subunit. The bond involves the phosphate moiety of GMP and the Ne-amino group of lysine. This was verified by treatment of the isolated 95-kDa subunit-[32P]GMP complex with sodium periodate, followed by methylamine-catalyzed beta-elimination. The product was then hydrolyzed by alkali producing 32P-labeled lysine (Ne-P)phosphate.

  8. A study of the single SGC7901 and GES1 cell using synchrotron infrared microspectroscopy and imaging

    NASA Astrophysics Data System (ADS)

    Wang, Xin; Qi, Zeming; Wang, Shengyi; Hu, Meicong; Liu, Gang; Tian, Yangchao

    2011-05-01

    In order to obtain unique information of the single gastric cancer cell and investigate gastric cancer further at the cellular and subcellular level, synchrotron infrared (SR-IR) microspectroscopy and imaging was used to study human gastric adenocarcinoma cell line SGC7901 and normal gastric mucosal epithelial cell line GES1. When we compared the infrared micro-spectra of the SGC7901 cells with those of the GES1 cells, the peak height ratios of 1121 cm -1/1020 cm -1 (RNA/DNA) of the SGC7901 cells were significantly higher than those of the GES1 cells, which might be useful for distinguishing malignant cells from normal cells. Furthermore, chemical imaging reveals that the amide II/the amide I ratio of the SGC7901 cell is lower than that of the GES1 cell in the most intense absorption region of vPO2-. The result indicates the study of the single gastric cancer cell at subcellular level by synchrotron infrared microspectroscopy and imaging can provide more detailed information which is very helpful for the research and diagnosis of gastric cancer.

  9. Presence of Ras guanyl nucleotide-releasing protein in striosomes of the mature and developing rat.

    PubMed

    Pierret, P; Mechawar, N; Vallée, A; Patel, J; Priestley, J V; Dunn, R J; Dower, N A; Stone, J C; Richardson, P M

    2002-01-01

    Ras signal transduction pathways have been implicated as key regulators in neuroplasticity and synaptic transmission in the brain. These pathways can be modulated by Ras guanyl nucleotide exchange factors, (GEF) which activate Ras proteins by catalysing the exchange of GDP for GTP. Ras guanyl nucleotide-releasing protein (RasGRP), a recently discovered Ras GEF, that links diacylglycerol and probably calcium to Ras signaling pathways, is expressed in brain as well as in T-cells. Here, we have used a highly selective monoclonal antibody against RasGRP to localize this protein within the striatum and related forebrain structures of developing and adult rats. RasGRP immunolabeling was found to be widespread in the mature and developing rat forebrain. Most notably, it presented a prominent patchy distribution throughout the striatum at birth and at all postnatal ages examined. These patches were found to correspond with the striosomal compartment of the striatum, as identified by micro-opioid receptor labeling in the adult. RasGRP-immunoreactivity was also observed in the matrix-like compartment surrounding these patches/striosomes but appeared later in development and was always weaker than in the patches. In both striatal compartments, RasGRP was exclusively expressed by medium-sized spiny neurons and showed no preference for neurons that project either directly or indirectly to the substantia nigra. At the ultrastructural level, immunogold labeling of RasGRP was confined to the cell bodies and dendritic shafts of these output neurons. We conclude that the prominent expression of RasGRP in striosomes may be of significance for diacylglycerol signaling in the striatum, and could be of importance for the processing of limbic-related activity within the basal ganglia.

  10. Phylogenetic distribution of extracellular guanyl-preferring ribonucleases renews taxonomic status of two Bacillus strains.

    PubMed

    Ulyanova, Vera; Shah Mahmud, Raihan; Dudkina, Elena; Vershinina, Valentina; Domann, Eugen; Ilinskaya, Olga

    2016-09-12

    The potential of microbial ribonucleases as promising antitumor and antiviral agents, determines today's directions of their study. One direction is connected with biodiversity of RNases. We have analyzed completed and drafted Bacillus genomes deposited in GenBank for the presence of coding regions similar to the gene of an extracellular guanyl-preferring RNase of Bacillus amyloliquefaciens (barnase). Orthologues of the barnase gene were detected in 9 species out of 83. All of these belong to "B. subtilis" group within the genus. B. subtilis itself, as well as some other species within this group, lack such types of RNases. RNases similar to barnase were also found in species of "B. cereus" group as a part of plasmid-encoded S-layer toxins. It was also found that taxonomic states of culture collection strains, which were initially described based on a limited set of phenotypic characteristics, can be misleading and need to be confirmed. Using several approaches such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), sequencing of genes for 16S ribosomal RNA and RNA polymerase subunit beta followed by reconstruction of phylogenetic trees, we have re-identified two RNase-secreting Bacillus strains: B. thuringiensis B-388 which should be assigned as B. altitudinis B388 and B. intermedius 7P which should be renamed as B. pumilus 7P. Therefore, small secreted guanyl-preferring RNases are the feature of "B. subtilis" group only, which is characterized by distinctive lifestyle and adaptation strategies to environment. PMID:27373509

  11. Tris(pentafluorophenyl)borane as an efficient catalyst in the guanylation reaction of amines.

    PubMed

    Antiñolo, Antonio; Carrillo-Hermosilla, Fernando; Fernández-Galán, Rafael; Martínez-Ferrer, Jaime; Alonso-Moreno, Carlos; Bravo, Iván; Moreno-Blázquez, Sonia; Salgado, Manuel; Villaseñor, Elena; Albaladejo, José

    2016-06-28

    Tris(pentafluorophenyl)borane, [B(C6F5)3], has been used as an efficient catalyst in the guanylation reaction of amines with carbodiimide under mild conditions. A combined approach involving NMR spectroscopy and DFT calculations was employed to gain a better insight into the mechanistic features of this process. The results allowed us to propose a new Lewis acid-assisted Brønsted acidic pathway for the guanylation reaction. The process starts with the interaction of tris(pentafluorphenyl)borane and the amine to form the corresponding adduct, [(C6F5)3B-NRH2] , followed by a straightforward proton transfer to one of the nitrogen atoms of the carbodiimide, (i)PrN[double bond, length as m-dash]C[double bond, length as m-dash]N(i)Pr, to produce, in two consequent steps, a guanidine-borane adduct, [(C6F5)3B-NRC(N(i)PrH)2] . The rupture of this adduct liberates the guanidine product RNC(N(i)PrH)2 and interaction with additional amine restarts the catalytic cycle. DFT studies have been carried out in order to study the thermodynamic characteristics of the proposed pathway. Significant borane adducts with amines and guanidines have been isolated and characterized by multinuclear NMR in order to study the N-B interaction and to propose the existence of possible Frustrated Lewis Pairs. Additionally, the molecular structures of significant components of the catalytic cycle, namely 4-tert-butylaniline-[B(C6F5)3] adduct and both free and [B(C6F5)3]-bonded 1-(phenyl)-2,3-diisopropylguanidine, and respectively, have been established by X-ray diffraction. PMID:27278089

  12. Tris(pentafluorophenyl)borane as an efficient catalyst in the guanylation reaction of amines.

    PubMed

    Antiñolo, Antonio; Carrillo-Hermosilla, Fernando; Fernández-Galán, Rafael; Martínez-Ferrer, Jaime; Alonso-Moreno, Carlos; Bravo, Iván; Moreno-Blázquez, Sonia; Salgado, Manuel; Villaseñor, Elena; Albaladejo, José

    2016-06-28

    Tris(pentafluorophenyl)borane, [B(C6F5)3], has been used as an efficient catalyst in the guanylation reaction of amines with carbodiimide under mild conditions. A combined approach involving NMR spectroscopy and DFT calculations was employed to gain a better insight into the mechanistic features of this process. The results allowed us to propose a new Lewis acid-assisted Brønsted acidic pathway for the guanylation reaction. The process starts with the interaction of tris(pentafluorphenyl)borane and the amine to form the corresponding adduct, [(C6F5)3B-NRH2] , followed by a straightforward proton transfer to one of the nitrogen atoms of the carbodiimide, (i)PrN[double bond, length as m-dash]C[double bond, length as m-dash]N(i)Pr, to produce, in two consequent steps, a guanidine-borane adduct, [(C6F5)3B-NRC(N(i)PrH)2] . The rupture of this adduct liberates the guanidine product RNC(N(i)PrH)2 and interaction with additional amine restarts the catalytic cycle. DFT studies have been carried out in order to study the thermodynamic characteristics of the proposed pathway. Significant borane adducts with amines and guanidines have been isolated and characterized by multinuclear NMR in order to study the N-B interaction and to propose the existence of possible Frustrated Lewis Pairs. Additionally, the molecular structures of significant components of the catalytic cycle, namely 4-tert-butylaniline-[B(C6F5)3] adduct and both free and [B(C6F5)3]-bonded 1-(phenyl)-2,3-diisopropylguanidine, and respectively, have been established by X-ray diffraction.

  13. Protein kinase C sensitizes olfactory adenylate cyclase

    PubMed Central

    1993-01-01

    Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory

  14. Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

    PubMed

    Kuhn, Michaela

    2016-04-01

    cGMP controls many cellular functions ranging from growth, viability, and differentiation to contractility, secretion, and ion transport. The mammalian genome encodes seven transmembrane guanylyl cyclases (GCs), GC-A to GC-G, which mainly modulate submembrane cGMP microdomains. These GCs share a unique topology comprising an extracellular domain, a short transmembrane region, and an intracellular COOH-terminal catalytic (cGMP synthesizing) region. GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure/volume and energy balance. GC-B is activated by C-type natriuretic peptide, stimulating endochondral ossification in autocrine way. GC-C mediates the paracrine effects of guanylins on intestinal ion transport and epithelial turnover. GC-E and GC-F are expressed in photoreceptor cells of the retina, and their activation by intracellular Ca(2+)-regulated proteins is essential for vision. Finally, in the rodent system two olfactorial GCs, GC-D and GC-G, are activated by low concentrations of CO2and by peptidergic (guanylins) and nonpeptidergic odorants as well as by coolness, which has implications for social behaviors. In the past years advances in human and mouse genetics as well as the development of sensitive biosensors monitoring the spatiotemporal dynamics of cGMP in living cells have provided novel relevant information about this receptor family. This increased our understanding of the mechanisms of signal transduction, regulation, and (dys)function of the membrane GCs, clarified their relevance for genetic and acquired diseases and, importantly, has revealed novel targets for therapies. The present review aims to illustrate these different features of membrane GCs and the main open questions in this field. PMID:27030537

  15. Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.

    PubMed

    Siedenburg, Gabriele; Breuer, Michael; Jendrossek, Dieter

    2013-02-01

    Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst. PMID:22526778

  16. Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.

    PubMed

    Siedenburg, Gabriele; Breuer, Michael; Jendrossek, Dieter

    2013-02-01

    Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.

  17. Regulation and organization of adenylyl cyclases and cAMP.

    PubMed Central

    Cooper, Dermot M F

    2003-01-01

    Adenylyl cyclases are a critically important family of multiply regulated signalling molecules. Their susceptibility to many modes of regulation allows them to integrate the activities of a variety of signalling pathways. However, this property brings with it the problem of imparting specificity and discrimination. Recent studies are revealing the range of strategies utilized by the cyclases to solve this problem. Microdomains are a consequence of these solutions, in which cAMP dynamics may differ from the broad cytosol. Currently evolving methodologies are beginning to reveal cAMP fluctuations in these various compartments. PMID:12940771

  18. AKAPs and Adenylyl Cyclase in Cardiovascular Physiology and Pathology

    PubMed Central

    Efendiev, Riad; Dessauer, Carmen W.

    2011-01-01

    Cyclic AMP, generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins (AKAPs) localize the effect of cAMP in space and time by organizing receptors, adenylyl cyclase, protein kinase A and other components of the cAMP cascade into multiprotein complexes. In this review we discuss how interaction of AKAPs with distinct AC isoforms affects cardiovascular physiology. PMID:21978991

  19. Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.

    PubMed Central

    Gueirard, P; Guiso, N

    1993-01-01

    Bordetella bronchiseptica is a pathogen of laboratory, domestic, and wild animals and sometimes of humans. In the present study some characteristics of the virulence of B. bronchiseptica isolates of different origin were studied. All isolates had similar phenotypes, similar bacteriological characters, and synthesized adenylate cyclase-hemolysin, filamentous hemagglutinin and pertactin but not pertussis toxin. These isolates, however, differed in their ability to express dermonecrotic toxin and to cause a lethal infection, but no correlation was found with the human or animal origin of the isolates. The fact that the most virulent isolate did not express dermonecrotic toxin suggests that this toxin does not play an important role in the virulence of the bacteria in the murine model. After infection with virulent B. bronchiseptica a very early synthesis and a persistence of anti-adenylate cyclase-hemolysin and anti-filamentous hemagglutinin antibodies were observed in the sera of infected mice, suggesting a persistence of the bacteria or of its antigens. B. bronchiseptica adenylate cyclase-hemolysin was purified and was shown to be a major protective antigen against B. bronchiseptica infection. Furthermore, we showed that its immunological and protective properties were different from that of B. pertussis adenylate cyclase-hemolysin, confirming that Bordetella species are immunologically different. Images PMID:8406794

  20. Tocopherol Cyclases-Substrate Specificity and Phylogenetic Relations.

    PubMed

    Dłużewska, Jolanta; Szymańska, Renata; Gabruk, Michal; Kós, Peter B; Nowicka, Beatrycze; Kruk, Jerzy

    2016-01-01

    In the present studies, we focused on substrate specificity of tocopherol cyclase, the key enzyme in the biosynthesis of the tocopherols and plastochromanol-8, the main plant lipid antioxidants, with special emphasis on the preference for tocopherols and plastochromanol-8 precursors, taking advantage of the recombinant enzyme originating from Arabidopsis thaliana and isolated plastoglobules, thylakoids and various model systems like micelles and thylakoids. Plastoglobules and triacylglycerol micelles were the most efficient reaction environment for the cyclase. In various investigated systems, synthesis of γ-tocopherol proceeded considerably faster than that of plastochromanol-8, probably mainly due to different localization of the corresponding substrates in the analyzed lipid structures. Moreover, our study was complemented by bioinformatics analysis of the phylogenetic relations of the cyclases and sequence motifs, crucial for the enzyme activity, were proposed. The analysis revealed also a group of tocopherol cyclase-like proteins in a number of heterotrophic bacterial species, with a conserved region common with photosynthetic organisms, that might be engaged in the catalytic activity of both groups of organisms. PMID:27462710

  1. General base-general acid catalysis by terpenoid cyclases.

    PubMed

    Pemberton, Travis A; Christianson, David W

    2016-07-01

    Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains.

  2. Gamma Interferon-Induced Guanylate Binding Protein 1 Is a Novel Actin Cytoskeleton Remodeling Factor

    PubMed Central

    Ostler, Nicole; Britzen-Laurent, Nathalie; Liebl, Andrea; Naschberger, Elisabeth; Lochnit, Günter; Ostler, Markus; Forster, Florian; Kunzelmann, Peter; Ince, Semra; Supper, Verena; Praefcke, Gerrit J. K.; Schubert, Dirk W.; Stockinger, Hannes; Herrmann, Christian

    2014-01-01

    Gamma interferon (IFN-γ) regulates immune defenses against viruses, intracellular pathogens, and tumors by modulating cell proliferation, migration, invasion, and vesicle trafficking processes. The large GTPase guanylate binding protein 1 (GBP-1) is among the cellular proteins that is the most abundantly induced by IFN-γ and mediates its cell biologic effects. As yet, the molecular mechanisms of action of GBP-1 remain unknown. Applying an interaction proteomics approach, we identified actin as a strong and specific binding partner of GBP-1. Furthermore, GBP-1 colocalized with actin at the subcellular level and was both necessary and sufficient for the extensive remodeling of the fibrous actin structure observed in IFN-γ-exposed cells. These effects were dependent on the oligomerization and the GTPase activity of GBP-1. Purified GBP-1 and actin bound to each other, and this interaction was sufficient to impair the formation of actin filaments in vitro, as demonstrated by atomic force microscopy, dynamic light scattering, and fluorescence-monitored polymerization. Cosedimentation and band shift analyses demonstrated that GBP-1 binds robustly to globular actin and slightly to filamentous actin. This indicated that GBP-1 may induce actin remodeling via globular actin sequestering and/or filament capping. These results establish GBP-1 as a novel member within the family of actin-remodeling proteins specifically mediating IFN-γ-dependent defense strategies. PMID:24190970

  3. Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp.

    PubMed

    Liu, Kuanqing; Myers, Angela R; Pisithkul, Tippapha; Claas, Kathy R; Satyshur, Kenneth A; Amador-Noguez, Daniel; Keck, James L; Wang, Jue D

    2015-02-19

    The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here, we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK), revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria, where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria.

  4. Guanylate Binding Protein (GBP) 5 Is an Interferon-Inducible Inhibitor of HIV-1 Infectivity.

    PubMed

    Krapp, Christian; Hotter, Dominik; Gawanbacht, Ali; McLaren, Paul J; Kluge, Silvia F; Stürzel, Christina M; Mack, Katharina; Reith, Elisabeth; Engelhart, Susanne; Ciuffi, Angela; Hornung, Veit; Sauter, Daniel; Telenti, Amalio; Kirchhoff, Frank

    2016-04-13

    Guanylate binding proteins (GBPs) are an interferon (IFN)-inducible subfamily of guanosine triphosphatases (GTPases) with well-established activity against intracellular bacteria and parasites. Here we show that GBP5 potently restricts HIV-1 and other retroviruses. GBP5 is expressed in the primary target cells of HIV-1, where it impairs viral infectivity by interfering with the processing and virion incorporation of the viral envelope glycoprotein (Env). GBP5 levels in macrophages determine and inversely correlate with infectious HIV-1 yield over several orders of magnitude, which may explain the high donor variability in macrophage susceptibility to HIV. Antiviral activity requires Golgi localization of GBP5, but not its GTPase activity. Start codon mutations in the accessory vpu gene from macrophage-tropic HIV-1 strains conferred partial resistance to GBP5 inhibition by increasing Env expression. Our results identify GBP5 as an antiviral effector of the IFN response and may explain the increased frequency of defective vpu genes in primary HIV-1 strains. PMID:26996307

  5. Ubiquitin systems mark pathogen-containing vacuoles as targets for host defense by guanylate binding proteins

    PubMed Central

    Haldar, Arun K.; Foltz, Clémence; Finethy, Ryan; Piro, Anthony S.; Feeley, Eric M.; Pilla-Moffett, Danielle M.; Komatsu, Masaki; Frickel, Eva-Maria; Coers, Jörn

    2015-01-01

    Many microbes create and maintain pathogen-containing vacuoles (PVs) as an intracellular niche permissive for microbial growth and survival. The destruction of PVs by IFNγ-inducible guanylate binding protein (GBP) and immunity-related GTPase (IRG) host proteins is central to a successful immune response directed against numerous PV-resident pathogens. However, the mechanism by which IRGs and GBPs cooperatively detect and destroy PVs is unclear. We find that host cell priming with IFNγ prompts IRG-dependent association of Toxoplasma- and Chlamydia-containing vacuoles with ubiquitin through regulated translocation of the E3 ubiquitin ligase tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6). This initial ubiquitin labeling elicits p62-mediated escort and deposition of GBPs to PVs, thereby conferring cell-autonomous immunity. Hypervirulent strains of Toxoplasma gondii evade this process via specific rhoptry protein kinases that inhibit IRG function, resulting in blockage of downstream PV ubiquitination and GBP delivery. Our results define a ubiquitin-centered mechanism by which host cells deliver GBPs to PVs and explain how hypervirulent parasites evade GBP-mediated immunity. PMID:26417105

  6. Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain

    PubMed Central

    Dembowski, Jill A.; An, Ping; Scoulos-Hanson, Maritsa; Yeo, Gene; Han, Joonhee; Fu, Xiang-Dong; Grabowski, Paula J.

    2012-01-01

    Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide. PMID:23008758

  7. Molecular mechanism and evolution of guanylate kinase regulation by (p)ppGpp

    PubMed Central

    Liu, Kuanqing; Myers, Angela R.; Pisithkul, Tippapha; Claas, Kathy R.; Satyshur, Kenneth A.; Amador-Noguez, Daniel; Keck, James L.; Wang, Jue D.

    2015-01-01

    SUMMARY The nucleotide (p)ppGpp mediates bacterial stress responses, but its targets and underlying mechanisms of action vary among bacterial species and remain incompletely understood. Here we characterize the molecular interaction between (p)ppGpp and guanylate kinase (GMK) revealing the importance of this interaction in adaptation to starvation. Combining structural and kinetic analyses, we show that (p)ppGpp binds the GMK active site and competitively inhibits the enzyme. The (p)ppGpp-GMK interaction prevents the conversion of GMP to GDP, resulting in GMP accumulation upon amino acid downshift. Abolishing this interaction leads to excess (p)ppGpp and defective adaptation to amino acid starvation. A survey of GMKs from phylogenetically diverse bacteria shows that the (p)ppGpp-GMK interaction is conserved in members of Firmicutes, Actinobacteria, and Deinococcus-Thermus, but not in Proteobacteria where (p)ppGpp regulates RNA polymerase (RNAP). We propose that GMK is an ancestral (p)ppGpp target and RNAP evolved more recently as a direct target in Proteobacteria. PMID:25661490

  8. Structure of the GH1 domain of guanylate kinase-associated protein from Rattus norvegicus

    SciTech Connect

    Tong, Junsen; Yang, Huiseon; Eom, Soo Hyun; Chun, ChangJu; Im, Young Jun

    2014-09-12

    Graphical abstract: - Highlights: • The crystal structure of GKAP homology domain 1 (GH1) was determined. • GKAP GH1 is a three-helix bundle connected by short flexible loops. • The predicted helix α4 associates weakly with the helix α3, suggesting dynamic nature of the GH1 domain. - Abstract: Guanylate-kinase-associated protein (GKAP) is a scaffolding protein that links NMDA receptor-PSD-95 to Shank–Homer complexes by protein–protein interactions at the synaptic junction. GKAP family proteins are characterized by the presence of a C-terminal conserved GKAP homology domain 1 (GH1) of unknown structure and function. In this study, crystal structure of the GH1 domain of GKAP from Rattus norvegicus was determined in fusion with an N-terminal maltose-binding protein at 2.0 Å resolution. The structure of GKAP GH1 displays a three-helix bundle connected by short flexible loops. The predicted helix α4 which was not visible in the crystal structure associates weakly with the helix α3 suggesting dynamic nature of the GH1 domain. The strict conservation of GH1 domain across GKAP family members and the lack of a catalytic active site required for enzyme activity imply that the GH1 domain might serve as a protein–protein interaction module for the synaptic protein clustering.

  9. Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

    SciTech Connect

    Liang, B.T.

    1989-06-01

    Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).

  10. Strophalloside induces apoptosis of SGC-7901 cells through the mitochondrion-dependent caspase-3 pathway.

    PubMed

    Zhang, Xue-Jiao; Mei, Wen-Li; Tan, Guang-Hong; Wang, Cai-Chun; Zhou, Song-Lin; Huang, Feng-Ru; Chen, Bin; Dai, Hao-Fu; Huang, Feng-Ying

    2015-01-01

    Cardenolides with special chemical structures have been considered as effective anti-cancer drugs in clinic trials. Strophalloside is a cardenolide we recently isolated from Antiaris toxicaria obtained from Hainan, China. The aim of this study was to investigate the possible anticancer effects induced by strophalloside and the underlying molecular mechanism. Gastric carcinoma SGC-7901 cells were treated with strophalloside at various concentrations for different times, and resulting cell viability was determined by the MTT assay, and the motility and invasion of tumor cells were assessed by the Transwell chamber assay. Apoptosis were measured by Annexin V-FITC/PI and Hoechst staining. The changes of mitochondrial transmembrane potential were examined by a JC-1 kit. The expressions of pro-apoptotic protein cytochrome c, caspase-3 and caspase-9 were detected by western blotting analysis. The results showed that strophalloside was capable of reducing cell viability, inhibiting cell growth, and suppressing cell migration and invasion in a time- and dose-dependent manner. Mitochondrial membrane potential declined and the concentration of cytochrome c increased in cytoplasm and caspase-3 and caspase-9 were cleaved into activated states, suggesting that cytochrome c was released from the mitochondrion to cytoplasm and finally activated the caspase-dependent apoptosis pathway. Our results indicate that strophalloside is a potential anticancer drug.

  11. SKI-II reverses the chemoresistance of SGC7901/DDP gastric cancer cells.

    PubMed

    Liu, Ying; Zhu, Zuan; Cai, Hongxing; Liu, Qinghua; Zhou, Honglian; Zhu, Zhengqiu

    2014-07-01

    Cisplatin is frequently used in treating gastric cancers; however, acquired resistance to the drug often reduces the efficacy of therapy. The present study analyzed the efficacy of the combination of 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) and cisplatin [cis-diamminedichloroplatinum (II); DDP] on the gastric cancer SGC7901/DDP cell line. The results revealed that SKI-II and DDP had a clear synergistic effect. Glutathione (GSH) and glutathione S-transferase (GST) levels decreased significantly subsequent to the cells being treated with the combination of DDP and SKI-II compared with the cells that were treated with DDP or SKI-II alone. Phosphorylated extracellular-signal-regulated kinase (p-ERK) and phosphorylated c-Jun N-terminal kinase (p-JNK) expression levels also decreased following treatment with SKI-II. The results suggested that SKI-II is able to reverse the drug resistance in human gastric carcinoma cells and enhance the antitumor effect of DDP through the ras/mitogen-activated protein kinase (MAPK) proliferation pathway.

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

    PubMed

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

    2016-09-01

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

  13. A composite guanyl thiourea (GTU), dicyandiamide (DCD) inhibitor improves the efficacy of nitrification inhibition in soil.

    PubMed

    Duncan, Elliott G; O'Sullivan, Cathryn A; Simonsen, Anna K; Roper, Margaret M; Treble, Karen; Whisson, Kelley

    2016-11-01

    This study investigated whether applying dicyandiamide (DCD) and guanyl thiourea (GTU) in conjunction with urea improves the efficacy of nitrification inhibition relative to traditional fertiliser application of urea or urea + DCD. Urea at a rate of 100 mg N kg(-1) soil was applied to soil microcosms (high nutrient tenosol and low nutrient hydrosol) which were treated with either no inhibitor (urea-only); 15 mg DCD kg(-1) soil or 15 mg DCD kg(-1) soil plus 21 mg GTU kg soil(-1). Mineral N (NH4(+) & NO3(-)) concentrations, potential nitrification rates (PNR) and abundances of ammonia oxidising bacteria (AOB) were measured over time. After 100-days incubation, ∼73 mg N kg(-1) soil was found as NH4(+) when urea + DCD + GTU were applied to the tenosol. NH4(+) concentrations were lower (11-32 mg N kg(-1) soil) when urea or urea + DCD were applied. This suggests that the application of GTU in conjunction with DCD elongated the effects of nitrification inhibition. In both soils, PNRs were faster and AOB abundances (gene copies g(-1) soil) were higher when urea was applied without nitrification inhibitors. There were, however, no differences in PNR or AOB abundances in either soil type when 'urea + DCD' or 'urea + DCD + GTU' were applied. The results indicate that the application of GTU with DCD may extend nitrification inhibition in certain soil types. This finding has the potential to improve the efficacy of commercially available and widely used inhibitors such as DCD. PMID:27517126

  14. Guanylate binding proteins enable rapid activation of canonical and noncanonical inflammasomes in Chlamydia-infected macrophages.

    PubMed

    Finethy, Ryan; Jorgensen, Ine; Haldar, Arun K; de Zoete, Marcel R; Strowig, Till; Flavell, Richard A; Yamamoto, Masahiro; Nagarajan, Uma M; Miao, Edward A; Coers, Jörn

    2015-12-01

    Interferon (IFN)-inducible guanylate binding proteins (GBPs) mediate cell-autonomous host resistance to bacterial pathogens and promote inflammasome activation. The prevailing model postulates that these two GBP-controlled activities are directly linked through GBP-dependent vacuolar lysis. It was proposed that the rupture of pathogen-containing vacuoles (PVs) by GBPs destroyed the microbial refuge and simultaneously contaminated the host cell cytosol with microbial activators of inflammasomes. Here, we demonstrate that GBP-mediated host resistance and GBP-mediated inflammatory responses can be uncoupled. We show that PVs formed by the rodent pathogen Chlamydia muridarum, so-called inclusions, remain free of GBPs and that C. muridarum is impervious to GBP-mediated restrictions on bacterial growth. Although GBPs neither bind to C. muridarum inclusions nor restrict C. muridarum growth, we find that GBPs promote inflammasome activation in C. muridarum-infected macrophages. We demonstrate that C. muridarum infections induce GBP-dependent pyroptosis through both caspase-11-dependent noncanonical and caspase-1-dependent canonical inflammasomes. Among canonical inflammasomes, we find that C. muridarum and the human pathogen Chlamydia trachomatis activate not only NLRP3 but also AIM2. Our data show that GBPs support fast-kinetics processing and secretion of interleukin-1β (IL-1β) and IL-18 by the NLRP3 inflammasome but are dispensable for the secretion of the same cytokines at later times postinfection. Because IFN-γ fails to induce IL-1β transcription, GBP-dependent fast-kinetics inflammasome activation can drive the preferential processing of constitutively expressed IL-18 in IFN-γ-primed macrophages in the absence of prior Toll-like receptor stimulation. Together, our results reveal that GBPs control the kinetics of inflammasome activation and thereby shape macrophage responses to Chlamydia infections.

  15. Guanylate Binding Proteins Enable Rapid Activation of Canonical and Noncanonical Inflammasomes in Chlamydia-Infected Macrophages

    PubMed Central

    Finethy, Ryan; Jorgensen, Ine; Haldar, Arun K.; de Zoete, Marcel R.; Strowig, Till; Flavell, Richard A.; Yamamoto, Masahiro; Nagarajan, Uma M.; Miao, Edward A.

    2015-01-01

    Interferon (IFN)-inducible guanylate binding proteins (GBPs) mediate cell-autonomous host resistance to bacterial pathogens and promote inflammasome activation. The prevailing model postulates that these two GBP-controlled activities are directly linked through GBP-dependent vacuolar lysis. It was proposed that the rupture of pathogen-containing vacuoles (PVs) by GBPs destroyed the microbial refuge and simultaneously contaminated the host cell cytosol with microbial activators of inflammasomes. Here, we demonstrate that GBP-mediated host resistance and GBP-mediated inflammatory responses can be uncoupled. We show that PVs formed by the rodent pathogen Chlamydia muridarum, so-called inclusions, remain free of GBPs and that C. muridarum is impervious to GBP-mediated restrictions on bacterial growth. Although GBPs neither bind to C. muridarum inclusions nor restrict C. muridarum growth, we find that GBPs promote inflammasome activation in C. muridarum-infected macrophages. We demonstrate that C. muridarum infections induce GBP-dependent pyroptosis through both caspase-11-dependent noncanonical and caspase-1-dependent canonical inflammasomes. Among canonical inflammasomes, we find that C. muridarum and the human pathogen Chlamydia trachomatis activate not only NLRP3 but also AIM2. Our data show that GBPs support fast-kinetics processing and secretion of interleukin-1β (IL-1β) and IL-18 by the NLRP3 inflammasome but are dispensable for the secretion of the same cytokines at later times postinfection. Because IFN-γ fails to induce IL-1β transcription, GBP-dependent fast-kinetics inflammasome activation can drive the preferential processing of constitutively expressed IL-18 in IFN-γ-primed macrophages in the absence of prior Toll-like receptor stimulation. Together, our results reveal that GBPs control the kinetics of inflammasome activation and thereby shape macrophage responses to Chlamydia infections. PMID:26416908

  16. Autophagy restricts Chlamydia trachomatis growth in human macrophages via IFNG-inducible guanylate binding proteins

    PubMed Central

    Al-Zeer, Munir A.; Al-Younes, Hesham M.; Lauster, Daniel; Abu Lubad, Mohammad; Meyer, Thomas F.

    2013-01-01

    Interferon γ (IFNG) is a key host response regulator of intracellular pathogen replication, including that of Chlamydia spp The antichlamydial functions of IFNG manifest in a strictly host, cell-type and chlamydial strain dependent manner. It has been recently shown that the IFNG-inducible family of immunity-related GTPases (IRG) proteins plays a key role in the defense against nonhost adapted chlamydia strains in murine epithelial cells. In humans, IFN-inducible guanylate binding proteins (hGBPs) have been shown to potentiate the antichlamydial effect of IFNG; however, how hGBPs regulate this property of IFNG is unknown. In this study, we identified hGBP1/2 as important resistance factors against C. trachomatis infection in IFNG-stimulated human macrophages. Exogenous IFNG reduced chlamydial infectivity by 50 percent in wild-type cells, whereas shRNA hGBP1/2 knockdown macrophages fully supported chlamydial growth in the presence of exogenous IFNG. hGBP1/2 were recruited to bacterial inclusions in human macrophages upon stimulation with IFNG, which triggered rerouting of the typically nonfusogenic bacterial inclusions for lysosomal degradation. Inhibition of lysosomal activity and autophagy impaired the IFNG-mediated elimination of inclusions. Thus, hGBP1/2 are critical effectors of antichlamydial IFNG responses in human macrophages. Through their capacity to remodel classically nonfusogenic chlamydial inclusions and stimulate fusion with autophagosomes, hGBP1/2 disable a major chlamydial virulence mechanism and contribute to IFNG-mediated pathogen clearance. PMID:23086406

  17. Interaction of Trypanosoma cruzi adenylate cyclase with liver regulatory factors.

    PubMed Central

    Eisenschlos, C; Flawiá, M M; Torruella, M; Torres, H N

    1986-01-01

    Trypanosoma cruzi adenylate cyclase catalytic subunits may interact with regulatory factors from rat liver membranes, reconstituting heterologous systems which are catalytically active in assay mixtures containing MgATP. The systems show stimulatory responses to glucagon and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) or fluoride. Reconstitution was obtained by three different methods: fusion of rat liver membranes (pretreated with N-ethylmaleimide) to T. cruzi membranes; interaction of detergent extracts of rat liver membranes with T. cruzi membranes; or interaction of purified preparations of T. cruzi adenylate cyclase and of liver membrane factors in phospholipid vesicles. The liver factors responsible for the guanine nucleotide effect were characterized as the NS protein. Data also indicate that reconstitution requires the presence of a membrane substrate. PMID:2947568

  18. High skeletal muscle adenylate cyclase in malignant hyperthermia.

    PubMed Central

    Willner, J H; Cerri, C G; Wood, D S

    1981-01-01

    Malignant hyperthermia occurs in humans with several congenital myopathies, usually in response to general anesthesia. Commonly, individuals who develop this syndrome lack symptoms of muscle disease, and their muscle lacks specific pathological changes. A biochemical marker for this myopathy has not previously been available; we found activity of adenylate cyclase and content of cyclic AMP to be abnormally high in skeletal muscle. Secondary modification of protein phosphorylation could explain observed abnormalities of phosphorylase activation and sarcoplasmic reticulum function. PMID:6271806

  19. Engineering adenylate cyclases regulated by near-infrared window light

    PubMed Central

    Ryu, Min-Hyung; Kang, In-Hye; Nelson, Mathew D.; Jensen, Tricia M.; Lyuksyutova, Anna I.; Siltberg-Liberles, Jessica; Raizen, David M.; Gomelsky, Mark

    2014-01-01

    Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset. PMID:24982160

  20. Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells.

    PubMed

    Wu, Ping; Zhu, Xueping; Jin, Wei; Hao, Shumei; Liu, Qi; Zhang, Linjie

    2015-05-01

    Intrinsic apoptotic pathway is considered to be responsible for cell death induced by platinum anticancer drugs. While in this study, we found that, necrosis is an indispensable pathway besides apoptosis in oxaliplatin-treated gastric cancer SGC-7901 cells. Upon exposure to oxaliplatin, both apoptotic and necrotic features were observed. The majority of dead cells were double positive for Annexin V and propidium iodide (PI). Moreover, mitochondrial membrane potential collapsed and caspase cascades were activated. However, ultrastructural changes under transmission electron microscope, coupled with the release of cellular contents, demonstrated the rupture of the plasma membrane. Oxaliplatin administration did not stimulate reactive oxygen species (ROS) production and autophagy, but elevated the protein level of Bmf. In addition, receptor interacting protein 1 (RIP1), but not receptor interacting protein 3 (RIP3) and its downstream components participated in this death process. Necrostatin-1 (Nec-1) blocked oxaliplatin-induced cell death nearly completely, whereas z-VAD-fmk could partially suppress cell death. Oxaliplatin treatment resulted in poly(ADP-ribose) polymerase-1 (PARP-1) overactivation, as indicated by the increase of poly(ADP-ribose) (PAR), which led to NAD(+) and ATP depletion. PARP-1 inhibitor, olaparib, could significantly block oxaliplatin-induced cell death, thus confirming that PARP-1 activation is mainly responsible for the cytotoxicity of oxaliplatin. Phosphorylation of H2AX at Ser139 and translocalization of apoptosis-inducing factor (AIF) are critical for this death process. Taken together, these results indicate that oxaliplatin can bypass canonical cell death pathways to kill gastric cancer cells, which may be of therapeutic advantage in the treatment of gastric cancer.

  1. Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells

    SciTech Connect

    Wu, Ping; Zhu, Xueping; Jin, Wei; Hao, Shumei; Liu, Qi; Zhang, Linjie

    2015-05-01

    Intrinsic apoptotic pathway is considered to be responsible for cell death induced by platinum anticancer drugs. While in this study, we found that, necrosis is an indispensable pathway besides apoptosis in oxaliplatin-treated gastric cancer SGC-7901 cells. Upon exposure to oxaliplatin, both apoptotic and necrotic features were observed. The majority of dead cells were double positive for Annexin V and propidium iodide (PI). Moreover, mitochondrial membrane potential collapsed and caspase cascades were activated. However, ultrastructural changes under transmission electron microscope, coupled with the release of cellular contents, demonstrated the rupture of the plasma membrane. Oxaliplatin administration did not stimulate reactive oxygen species (ROS) production and autophagy, but elevated the protein level of Bmf. In addition, receptor interacting protein 1 (RIP1), but not receptor interacting protein 3 (RIP3) and its downstream components participated in this death process. Necrostatin-1 (Nec-1) blocked oxaliplatin-induced cell death nearly completely, whereas z-VAD-fmk could partially suppress cell death. Oxaliplatin treatment resulted in poly(ADP-ribose) polymerase-1 (PARP-1) overactivation, as indicated by the increase of poly(ADP-ribose) (PAR), which led to NAD{sup +} and ATP depletion. PARP-1 inhibitor, olaparib, could significantly block oxaliplatin-induced cell death, thus confirming that PARP-1 activation is mainly responsible for the cytotoxicity of oxaliplatin. Phosphorylation of H2AX at Ser139 and translocalization of apoptosis-inducing factor (AIF) are critical for this death process. Taken together, these results indicate that oxaliplatin can bypass canonical cell death pathways to kill gastric cancer cells, which may be of therapeutic advantage in the treatment of gastric cancer. - Highlights: • Oxaliplatin induces apoptotic and necrotic cell death. • Nec-1 can inhibit oxaliplatin-induced cell death nearly completely. • RIP3 and its

  2. Effects of PDE5 Inhibitors and sGC Stimulators in a Rat Model of Artificial Ureteral Calculosis

    PubMed Central

    Sandner, Peter; Tinel, Hanna; Affaitati, Giannapia; Costantini, Raffaele; Giamberardino, Maria Adele

    2015-01-01

    Urinary colics from calculosis are frequent and intense forms of pain whose current pharmacological treatment remains unsatisfactory. New and more effective drugs are needed to control symptoms and improve stone expulsion. Recent evidence suggested that the Nitric Oxide (NO) / cyclic guanosine monophosphate (cGMP) / phosphodiesterase type 5 (PDE5) system may contribute to ureteral motility influencing stone expulsion. We investigated if PDE5 inhibitors and sGC stimulators influence ureteral contractility, pain behaviour and stone expulsion in a rat model of ureteral calculosis. We investigated: a)the sex-specific PDE5 distribution in the rat ureter; b)the functional in vitro effects of vardenafil and sildenafil (PDE5 inhibitors) and BAY41-2272 (sGC stimulator) on induced ureteral contractility in rats and c)the in vivo effectiveness of vardenafil and BAY41-2272, alone and combined with ketoprofen, vs hyoscine-N-butylbromide alone or combined with ketoprofen, on behavioural pain indicators and stone expulsion in rats with artificial calculosis in one ureter. PDE5 was abundantly expressed in male and female rats’ ureter. In vitro, both vardenafil and BAY41-2272 significantly relaxed pre-contracted ureteral strips. In vivo, all compounds significantly reduced number and global duration of “ureteral crises” and post-stone lumbar muscle hyperalgesia in calculosis rats. The highest level of reduction of the pain behaviour was observed with BAY41-2272 among all spasmolytics administered alone, and with the combination of ketoprofen with BAY41-2272. The percentage of stone expulsion was maximal in the ketoprofen+BAY41-2272 group. The NO/cGMP/PDE5 pathway is involved in the regulation of ureteral contractility and pain behaviour in urinary calculosis. PDE5 inhibitors and sGC stimulators could become a potent new option for treatment of urinary colic pain. PMID:26509272

  3. Effects of PDE5 Inhibitors and sGC Stimulators in a Rat Model of Artificial Ureteral Calculosis.

    PubMed

    Sandner, Peter; Tinel, Hanna; Affaitati, Giannapia; Costantini, Raffaele; Giamberardino, Maria Adele

    2015-01-01

    Urinary colics from calculosis are frequent and intense forms of pain whose current pharmacological treatment remains unsatisfactory. New and more effective drugs are needed to control symptoms and improve stone expulsion. Recent evidence suggested that the Nitric Oxide (NO) / cyclic guanosine monophosphate (cGMP)/phosphodiesterase type 5 (PDE5) system may contribute to ureteral motility influencing stone expulsion. We investigated if PDE5 inhibitors and sGC stimulators influence ureteral contractility, pain behaviour and stone expulsion in a rat model of ureteral calculosis. We investigated: a) the sex-specific PDE5 distribution in the rat ureter; b) the functional in vitro effects of vardenafil and sildenafil (PDE5 inhibitors) and BAY41-2272 (sGC stimulator) on induced ureteral contractility in rats and c) the in vivo effectiveness of vardenafil and BAY41-2272, alone and combined with ketoprofen, vs hyoscine-N-butylbromide alone or combined with ketoprofen, on behavioural pain indicators and stone expulsion in rats with artificial calculosis in one ureter. PDE5 was abundantly expressed in male and female rats' ureter. In vitro, both vardenafil and BAY41-2272 significantly relaxed pre-contracted ureteral strips. In vivo, all compounds significantly reduced number and global duration of "ureteral crises" and post-stone lumbar muscle hyperalgesia in calculosis rats. The highest level of reduction of the pain behaviour was observed with BAY41-2272 among all spasmolytics administered alone, and with the combination of ketoprofen with BAY41-2272. The percentage of stone expulsion was maximal in the ketoprofen+BAY41-2272 group. The NO/cGMP/PDE5 pathway is involved in the regulation of ureteral contractility and pain behaviour in urinary calculosis. PDE5 inhibitors and sGC stimulators could become a potent new option for treatment of urinary colic pain.

  4. Crystal structure of papaya glutaminyl cyclase, an archetype for plant and bacterial glutaminyl cyclases.

    PubMed

    Wintjens, René; Belrhali, Hassan; Clantin, Bernard; Azarkan, Mohamed; Bompard, Coralie; Baeyens-Volant, Danielle; Looze, Yvan; Villeret, Vincent

    2006-03-24

    Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the intramolecular cyclization of protein N-terminal glutamine residues into pyroglutamic acid with the concomitant liberation of ammonia. QCs may be classified in two groups containing, respectively, the mammalian enzymes, and the enzymes from plants, bacteria, and parasites. The crystal structure of the QC from the latex of Carica papaya (PQC) has been determined at 1.7A resolution. The structure was solved by the single wavelength anomalous diffraction technique using sulfur and zinc as anomalous scatterers. The enzyme folds into a five-bladed beta-propeller, with two additional alpha-helices and one beta hairpin. The propeller closure is achieved via an original molecular velcro, which links the last two blades into a large eight stranded beta-sheet. The zinc ion present in the PQC is bound via an octahedral coordination into an elongated cavity located along the pseudo 5-fold axis of the beta-propeller fold. This zinc ion presumably plays a structural role and may contribute to the exceptional stability of PQC, along with an extended hydrophobic packing, the absence of long loops, the three-joint molecular velcro and the overall folding itself. Multiple sequence alignments combined with structural analyses have allowed us to tentatively locate the active site, which is filled in the crystal structure either by a Tris molecule or an acetate ion. These analyses are further supported by the experimental evidence that Tris is a competitive inhibitor of PQC. The active site is located at the C-terminal entrance of the PQC central tunnel. W83, W110, W169, Q24, E69, N155, K225, F22 and F67 are highly conserved residues in the C-terminal entrance, and their putative role in catalysis is discussed. The PQC structure is representative of the plants, bacterial and parasite enzymes and contrasts with that of mammalian enzymes, that may possibly share a conserved scaffold of the bacterial aminopeptidase.

  5. Expression of a fungal sesquiterpene cyclase gene in transgenic tobacco.

    PubMed

    Hohn, T M; Ohlrogge, J B

    1991-09-01

    The complete coding sequence for the trichodiene synthase gene from Fusarium sporotrichioides was introduced into tobacco (Nicotiana tabacum) under the regulation of the cauliflower mosiac virus 35S promoter. Expression of trichodiene synthase was demonstrated in the leaves of transformed plants. Leaf homogenates incubated with [(3)H]farnesyl pyrophosphate produced trichodiene as a major product. Trichodiene was detected in the leaves of a transformed plant at a level of 5 to 10 nanograms per gram fresh weight. The introduction of a fungal sesquiterpene cyclase gene into tobacco has resulted in the expression of an active enzyme and the accumulation of low levels of its sesquiterpenoid product. PMID:16668409

  6. A Twist on Heme Signaling

    PubMed Central

    Spiro, Thomas G.

    2009-01-01

    Proteins in the H-NOX family act as sensors of NO or O2. This family includes soluble guanylate cyclase, sGC, the NO sensor that is responsible for vasodilation and neurotransmission in mammals. The crystal structures of bacterial H-NOX domains have revealed a highly distorted heme cofactor. This distortion has now been shown, via replacement of a single sterically critical residue, to be associated with a concerted displacement of the entire N-terminal half of the protein. This displacement likely provides the mechanism for transducing the ligand binding event into signaling. PMID:19032089

  7. Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c.

    PubMed

    Sinha, Sangita C; Wetterer, Martina; Sprang, Stephen R; Schultz, Joachim E; Linder, Jürgen U

    2005-02-23

    Rv1900c, a Mycobacterium tuberculosis adenylyl cyclase, is composed of an N-terminal alpha/beta-hydrolase domain and a C-terminal cyclase homology domain. It has an unusual 7% guanylyl cyclase side-activity. A canonical substrate-defining lysine and a catalytic asparagine indispensable for mammalian adenylyl cyclase activity correspond to N342 and H402 in Rv1900c. Mutagenic analysis indicates that these residues are dispensable for activity of Rv1900c. Structures of the cyclase homology domain, solved to 2.4 A both with and without an ATP analog, form isologous, but asymmetric homodimers. The noncanonical N342 and H402 do not interact with the substrate. Subunits of the unliganded open dimer move substantially upon binding substrate, forming a closed dimer similar to the mammalian cyclase heterodimers, in which one interfacial active site is occupied and the quasi-dyad-related active site is occluded. This asymmetry indicates that both active sites cannot simultaneously be catalytically active. Such a mechanism of half-of-sites-reactivity suggests that mammalian heterodimeric adenylyl cyclases may have evolved from gene duplication of a primitive prokaryote-type cyclase, followed by loss of function in one active site. PMID:15678099

  8. Crystallization of the class IV adenylyl cyclase from Yersinia pestis

    SciTech Connect

    Smith, Natasha; Kim, Sook-Kyung; Reddy, Prasad T.; Gallagher, D. Travis

    2006-03-01

    The class IV adenylyl cyclase from Y. pestis has been crystallized in an orthorhombic form suitable for structure determination. The class IV adenylyl cyclase from Yersinia pestis has been cloned and crystallized in both a triclinic and an orthorhombic form. An amino-terminal His-tagged construct, from which the tag was removed by thrombin, crystallized in a triclinic form diffracting to 1.9 Å, with one dimer per asymmetric unit and unit-cell parameters a = 33.5, b = 35.5, c = 71.8 Å, α = 88.7, β = 82.5, γ = 65.5°. Several mutants of this construct crystallized but diffracted poorly. A non-His-tagged native construct (179 amino acids, MW = 20.5 kDa) was purified by conventional chromatography and crystallized in space group P2{sub 1}2{sub 1}2{sub 1}. These crystals have unit-cell parameters a = 56.8, b = 118.6, c = 144.5 Å, diffract to 3 Å and probably have two dimers per asymmetric unit and V{sub M} = 3.0 Å{sup 3} Da{sup −1}. Both crystal forms appear to require pH below 5, complicating attempts to incorporate nucleotide ligands into the structure. The native construct has been produced as a selenomethionine derivative and crystallized for phasing and structure determination.

  9. Adenylate cyclase in Arthrospira platensis responds to light through transcription.

    PubMed

    Kashith, M; Keerthana, B; Sriram, S; Ramamurthy, V

    2016-08-19

    Cyclic 3',5' adenosine monophosphate (cAMP) is a ubiquitous signaling molecule, but its role in higher plants was in doubt due to its very low concentration. In this study we wanted to look at the flux of cAMP in response to light in algae, considered to be the more primitive form of photosynthetic organisms. While it did not fluctuate very much in the tested green algae, in the cyanobacterium Arthrospira platensis its level was closely linked to exposure to light. The expression from cyaC, the major isoform of adenylate cyclase was strongly influenced by exposure of the cells to light. There was about 300 fold enhancement of cyaC transcripts in cells exposed to light compared to the transcripts in cells in the dark. Although post-translational regulation of adenylate cyclase activity has been widely known, our studies suggest that transcriptional control could also be an important aspect of its regulation in A. platensis. PMID:27311855

  10. Structural analysis of an oxygen-regulated diguanylate cyclase.

    PubMed

    Tarnawski, Miroslaw; Barends, Thomas R M; Schlichting, Ilme

    2015-11-01

    Cyclic di-GMP is a bacterial second messenger that is involved in switching between motile and sessile lifestyles. Given the medical importance of biofilm formation, there has been increasing interest in understanding the synthesis and degradation of cyclic di-GMPs and their regulation in various bacterial pathogens. Environmental cues are detected by sensing domains coupled to GGDEF and EAL or HD-GYP domains that have diguanylate cyclase and phosphodiesterase activities, respectively, producing and degrading cyclic di-GMP. The Escherichia coli protein DosC (also known as YddV) consists of an oxygen-sensing domain belonging to the class of globin sensors that is coupled to a C-terminal GGDEF domain via a previously uncharacterized middle domain. DosC is one of the most strongly expressed GGDEF proteins in E. coli, but to date structural information on this and related proteins is scarce. Here, the high-resolution structural characterization of the oxygen-sensing globin domain, the middle domain and the catalytic GGDEF domain in apo and substrate-bound forms is described. The structural changes between the iron(III) and iron(II) forms of the sensor globin domain suggest a mechanism for oxygen-dependent regulation. The structural information on the individual domains is combined into a model of the dimeric DosC holoprotein. These findings have direct implications for the oxygen-dependent regulation of the activity of the cyclase domain.

  11. Guanylate-Binding Protein-1 protects ovarian cancer cell lines but not breast cancer cell lines from killing by paclitaxel.

    PubMed

    Tipton, Aaron R; Nyabuto, Geoffrey O; Trendel, Jill A; Mazur, Travis M; Wilson, John P; Wadi, Suzan; Justinger, Jacob S; Moore, Garret L; Nguyen, Peter T; Vestal, Deborah J

    2016-09-30

    Forced expression of the cytokine-induced large GTPase, human Guanylate-Binding Protein-1 (hGBP-1), in ovarian cancer cell lines increases resistance to paclitaxel. Elevated hGBP-1 RNA in ovarian tumors correlates with shorter recurrence-free survival. In contract, hGBP-1 is part of a gene signature predicting improved prognosis in all subtypes of breast cancers. hGBP-1 does not confer paclitaxel resistance on MCF-7 and TMX2-28 breast cancer cells. Expression of the isotype of the hGBP-1-interacting protein, PIM1, which may contribute to paclitaxel resistance when associated with hGBP-1, is different in breast and ovarian cancer cell lines. Breast cancer cell lines express the 44 kDa isoform of PIM-1, and ovarian cancer cell lines express the 33 kDa isoform. PMID:27590579

  12. Complete genomic organization of the human erythroid p55 gene (MPP1), a membrane-associated guanylate kinase homologue

    SciTech Connect

    Kim, A.C.; Metzenberg, A.B.; Sahr, K.E.

    1996-01-15

    Human p55 is an abundantly palmitoylated phosphoprotein of the erythroid membrane. It is the prototype of a newly discovered family of membrane-associated proteins termed MAGUKs (membrane-associated guanylate kinase homologues). The MAGUKs interact with the cytoskeleton and regulate cell proliferation, signaling pathways, and intercellular junctions. Here, we report the complete intron-exon map of the human erythroid p55 gene (HGMW-approved symbol MPP1). The structure of the p55 gene was determined from cosmid clones isolated from a cosmid library specific for the human X chromosome. There is a single copy of the p55 gene, composed of 12 exons and spanning approximately 28 kb in the q28 region of the human X chromosome. The exon sizes range from 69 (exon 5) to 203 bp (intron 2) to {approximately}14 kb (intron 1). The intron-exon boundaries conform to the donor/acceptor consensus sequence, GT-AG, for splice junctions. Several of the exon boundaries correspond to the boundaries of functional domains in the p55 protein. These domains include a SH3 motif and a region that binds to cytoskeletal protein 4.1. In addition, a comparison of the genomic and the primary structures of p55 reveals a highly conserved phosphotyrosine domain located between the protein 4.1 binding domain and the guanylate kinase domain. Finally, promoter activity measurements of the region immediately upstream of the p55 gene, which contains several cis-elements commonly found in housekeeping genes, suggest that a CpG island may be associated with the p55 gene expression in vivo. 42 refs., 5 figs., 1 tab.

  13. Fangchinoline targets PI3K and suppresses PI3K/AKT signaling pathway in SGC7901 cells

    PubMed Central

    TIAN, FENG; DING, DING; LI, DANDAN

    2015-01-01

    Fangchinoline, an important compound in Stephania tetrandra S. Moore, as a novel antitumor agent, has been implicated in several types of cancers cells except gastric cancer. To investigate whether fangchinoline affects gastric cancer cells, we detected the signaling pathway by which fangchinoline plays a role in different human gastric cancer cells lines. We found that fangchinoline effectively suppressed proliferation and invasion of SGC7901 cell lines, but not MKN45 cell lines by inhibiting the expression of PI3K and its downstream pathway. All of the Akt/MMP2/MMP9 pathway, Akt/Bad pathway, and Akt/Gsk3β/CDK2 pathway could be inhibited by fangchinoline through inhibition of PI3K. Taken together, these results suggest that fangchinoline targets PI3K in tumor cells that express PI3K abundantly and inhibits the growth and invasive ability of the tumor cells. PMID:25872479

  14. Fangchinoline targets PI3K and suppresses PI3K/AKT signaling pathway in SGC7901 cells.

    PubMed

    Tian, Feng; Ding, Ding; Li, Dandan

    2015-01-01

    Fangchinoline, an important compound in Stephania tetrandra S. Moore, as a novel antitumor agent, has been implicated in several types of cancers cells except gastric cancer. To investigate whether fangchinoline affects gastric cancer cells, we detected the signaling pathway by which fangchinoline plays a role in different human gastric cancer cells lines. We found that fangchinoline effectively suppressed proliferation and invasion of SGC7901 cell lines, but not MKN45 cell lines by inhibiting the expression of PI3K and its downstream pathway. All of the Akt/MMP2/MMP9 pathway, Akt/Bad pathway, and Akt/Gsk3β/CDK2 pathway could be inhibited by fangchinoline through inhibition of PI3K. Taken together, these results suggest that fangchinoline targets PI3K in tumor cells that express PI3K abundantly and inhibits the growth and invasive ability of the tumor cells. PMID:25872479

  15. Inhibition of heat shock protein 90 attenuates adenylate cyclase sensitization after chronic morphine treatment.

    PubMed

    Koshimizu, Taka-aki; Tsuchiya, Hiroyoshi; Tsuda, Hidetoshi; Fujiwara, Yoko; Shibata, Katsushi; Hirasawa, Akira; Tsujimoto, Gozoh; Fujimura, Akio

    2010-02-19

    Cellular adaptations to chronic opioid treatment result in enhanced responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether chaperone molecules such as heat shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to effective attenuation of morphine-induced adenylate cyclase sensitization. In SK-N-SH human neuroblastoma cells, morphine significantly increased RNA transcript and protein levels of type I adenylate cyclase, leading to sensitization. Whole-genome tiling array analysis revealed that cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human embryonic kidney cells. Hsp90AB1 transcript and protein levels increased significantly during morphine treatment, and co-application of geldanamycin (0.1-10 nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate cyclase, but not Hsp90AB1, underwent significant degradation during geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization induced by chronic morphine treatment.

  16. The Effects of Thrombin on Adenyl Cyclase Activity and a Membrane Protein from Human Platelets

    PubMed Central

    Brodie, G. N.; Baenziger, Nancy Lewis; Chase, Lewis R.; Majerus, Philip W.

    1972-01-01

    Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The T½ for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin E1, or theophylline protected adenyl cyclase from inhibition by thrombin. Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (T½ < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin E1, and theophylline at varying concentrations of thrombin. Images PMID:4331802

  17. Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum.

    PubMed

    Witters, Erwin; Quanten, Lieve; Bloemen, Jo; Valcke, Roland; Van Onckelen, Harry

    2004-01-01

    In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled (15)N-ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method was developed to measure de novo formed (15)N-adenosine 3',5'-cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv. Petit Havana and the kinetic parameters and influence of various metabolic effectors are discussed in their context.

  18. Monosodium glutamate, disodium inosinate, disodium guanylate, lysine and taurine improve the sensory quality of fermented cooked sausages with 50% and 75% replacement of NaCl with KCl.

    PubMed

    dos Santos, Bibiana Alves; Campagnol, Paulo Cezar Bastianello; Morgano, Marcelo Antônio; Pollonio, Marise Aparecida Rodrigues

    2014-01-01

    Fermented cooked sausages were produced by replacing 50% and 75% of NaCl with KCl and adding monosodium glutamate, disodium inosinate, disodium guanylate, lysine and taurine. The manufacturing process was monitored by pH and water activity measurements. The sodium and potassium contents of the resulting products were measured. The color values (L*, a* and b*), texture profiles and sensory profiles were also examined. Replacing 50% and 75% NaCl with KCl depreciated the sensory quality of the products. The reformulated sausages containing monosodium glutamate combined with lysine, taurine, disodium inosinate and disodium guanylate masked the undesirable sensory attributes associated with the replacement of 50% and 75% NaCl with KCl, allowing the production of fermented cooked sausages with good sensory acceptance and approximately 68% sodium reduction. PMID:24008059

  19. Dopaminergic modulation of adenylate cyclase stimulation by vasoactive intestinal peptide in anterior pituitary.

    PubMed Central

    Onali, P; Schwartz, J P; Costa, E

    1981-01-01

    The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by vasoactive intestinal peptide (VIP) was used as a model to investigate the molecular mechanisms triggered by the occupancy of dopamine recognition sites in rat anterior pituitary. Dopamine failed to change the basal enzyme activity, but it inhibited the stimulation of adenylate cyclase elicited by VIP. Apomorphine, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, and 2-bromo-alpha-ergocryptine mimicked the effect of dopamine, whereas (-)-sulpiride and and classical neuroleptics antagonized it. Dopamine failed to modulate the activation of pituitary adenylate cyclase by prostaglandin E1, which does not increase prolactin secretion. From these results we infer that stimulation of D-2 (dopamine) receptors may affect pituitary secretion by inhibiting the activation of anterior pituitary adenylate cyclase by VIP or other secretagogues. PMID:6171819

  20. Structural and Biochemical Analysis of the Essential Diadenylate Cyclase CdaA from Listeria monocytogenes*

    PubMed Central

    Rosenberg, Jonathan; Dickmanns, Achim; Neumann, Piotr; Gunka, Katrin; Arens, Johannes; Kaever, Volkhard; Stülke, Jörg; Ficner, Ralf; Commichau, Fabian M.

    2015-01-01

    The recently identified second messenger cyclic di-AMP (c-di-AMP) is involved in several important cellular processes, such as cell wall metabolism, maintenance of DNA integrity, ion transport, transcription regulation, and allosteric regulation of enzyme function. Interestingly, c-di-AMP is essential for growth of the Gram-positive model bacterium Bacillus subtilis. Although the genome of B. subtilis encodes three c-di-AMP-producing diadenlyate cyclases that can functionally replace each other, the phylogenetically related human pathogens like Listeria monocytogenes and Staphylococcus aureus possess only one enzyme, the diadenlyate cyclase CdaA. Because CdaA is also essential for growth of these bacteria, the enzyme is a promising target for the development of novel antibiotics. Here we present the first crystal structure of the L. monocytogenes CdaA diadenylate cyclase domain that is conserved in many human pathogens. Moreover, biochemical characterization of the cyclase revealed an unusual metal cofactor requirement. PMID:25605729

  1. Interactions between lysergic acid diethylamide and dopamine-sensitive adenylate cyclase systems in rat brain.

    PubMed

    Hungen, K V; Roberts, S; Hill, D F

    1975-08-22

    Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-brono-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation of adenylate cyclase activity of equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10 muM concentration, D-LSD not only completely eradicated the response to 10 muM dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80 muM) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1 muM D-LSD. Activation of striatal adenylate cyclase of either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the beta-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and

  2. Functional classification of cNMP-binding proteins and nucleotide cyclases with implications for novel regulatory pathways in Mycobacterium tuberculosis.

    PubMed

    McCue, L A; McDonough, K A; Lawrence, C E

    2000-02-01

    We have analyzed the cyclic nucleotide (cNMP)-binding protein and nucleotide cyclase superfamilies using Bayesian computational methods of protein family identification and classification. In addition to the known cNMP-binding proteins (cNMP-dependent kinases, cNMP-gated channels, cAMP-guanine nucleotide exchange factors, and bacterial cAMP-dependent transcription factors), new functional groups of cNMP-binding proteins were identified, including putative ABC-transporter subunits, translocases, and esterases. Classification of the nucleotide cyclases revealed subtle differences in sequence conservation of the active site that distinguish the five classes of cyclases: the multicellular eukaryotic adenylyl cyclases, the eukaryotic receptor-type guanylyl cyclases, the eukaryotic soluble guanylyl cyclases, the unicellular eukaryotic and prokaryotic adenylyl cyclases, and the putative prokaryotic guanylyl cyclases. Phylogenetic distribution of the cNMP-binding proteins and cyclases was analyzed, with particular attention to the 22 complete archaeal and eubacterial genome sequences. Mycobacterium tuberculosis H37Rv and Synechocystis PCC6803 were each found to encode several more putative cNMP-binding proteins than other prokaryotes; many of these proteins are of unknown function. M. tuberculosis also encodes several more putative nucleotide cyclases than other prokaryotic species. PMID:10673278

  3. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    PubMed Central

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions. PMID:21602811

  4. 3',5'-cyclic adenosine monophosphate and adenylate cyclase in phototransduction by limulus ventral photoreceptors.

    PubMed Central

    Brown, J E; Kaupp, U B; Malbon, C C

    1984-01-01

    Biochemical and electrophysiological measurements were made on photoreceptor cells from Limulus ventral eyes to investigate the possible role of cyclic AMP and adenylate cyclase in the visual transduction mechanism. Cyclic AMP content in a photoreceptor-enriched fraction (the end organs) of Limulus ventral eyes was approximately 15 pmol/mg protein. The cyclic AMP content was increased by bathing eyes in 1-methyl-3-isobutyl xanthine or forskolin and was increased almost 100-fold when bathed in both. Illumination did not change cyclic AMP content significantly in any of these conditions. Discrete events that can be recorded electrophysiologically occur spontaneously in darkness. An increase in the frequency of discrete events is evoked by dim illumination. The discrete events are a sign of excitation of Limulus photoreceptor cells. Drug-induced changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness were not correlated with changes in cyclic AMP content. Adenylate cyclase activity measured from a small number of pooled photoreceptor clusters was stimulated by fluoride and vanadate ions, hydrolysis-resistant analogues of GTP, cholera toxin and forskolin. The Limulus enzyme is similar pharmacologically to mammalian and avian adenylate cyclases. Activation of adenylate cyclase by drugs was not correlated with changes in the rate of occurrence of discrete events recorded electrophysiologically in darkness. A heat-treated Lubrol extract of membranes from Limulus ventral eyes reconstituted the adenylate cyclase activity of membranes from S49 mouse lymphoma cyc- mutant cells which lack a functional regulatory protein. These findings suggest that Limulus ventral eye photoreceptors contain a regulatory protein that mediates the activation of adenylate cyclase by guanine nucleotides, fluoride or cholera toxin. This regulatory protein is homologous with that found in mammalian and avian adenylate cyclases. Our findings suggest that

  5. Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface.

    PubMed

    Peshenko, Igor V; Olshevskaya, Elena V; Dizhoor, Alexander M

    2015-08-01

    The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met(823) was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg(822). The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met(823) or Arg(822) was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg(822) and Met(823).

  6. Cloning, chromosomal mapping, and expression of human fetal brain type I adenylyl cyclase

    SciTech Connect

    Villacres, E.C.; Xia, Z.; Bookbinder, L.H.; Edelhoff, S.; Disteche, C.M.; Storm, D.R.

    1993-05-01

    The neural-specific calmodulin-sensitive adenylyl cyclase (type I), which was first cloned from bovine brain, has been implicated in learning and memory. The objective of this study was to clone and determine the chromosomal localization of human fetal brain type I adenylyl cyclase. A 3.8-kb cDNA clone was isolated that contained sequence coinciding with the 3{prime} end 2553 nucleotides of the bovine open reading frame. This clone shows 87% nucleotide and 92% translated amino acid sequence identity to the bovine clone. The most significant sequence differences were in the carboxy-terminal 100 amino acid residues. This region contains one of several possible calmodulin binding domains and the only putative cAMP-dependent protein kinase A phosphorylation site. A chimera was constructed that contained the 5{prime} half of the bovine type I adenylyl cyclase and the 3{prime} half of the human type I adenylyl cyclase. The activity of the chimeric gene product and its sensitivity to calmodulin and calcium were indistinguishable from those of the bovine type I adenylyl cyclase. In situ hybridization was used to localize the human type I adenylyl cyclase gene to the proximal portion of the short arm of chromosome 7. 36 refs., 4 figs.

  7. Guanylate binding protein-1 mediates EGFRvIII and promotes glioblastoma growth in vivo but not in vitro

    PubMed Central

    Cheng, Yanwei; Mukasa, Akitaki; Ma, Jiawei; Hong, Lei; Yu, Shuye; Sun, Lili; Huang, Qiang; Purow, Benjamin; Li, Ming

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common and deadly primary brain tumor in adults. Epidermal growth factor receptor (EGFR) is frequently amplified and mutated in GBM. We previously reported that Guanylate binding protein-1 (GBP1) is a novel transcriptional target gene of EGFR and plays a role in GBM invasion. Here we demonstrate that GBP1 can also be induced by EGFRvIII at the transcriptional level through the p38 MAPK/Yin Yang 1 (YY1) signaling pathway. Silencing of GBP1 by RNA interference significantly inhibits EGFRvIII-mediated GBM cell proliferation in vitro and in a mouse model. Overexpression of GBP1 has no obvious effect on glioblastoma cell proliferation in vitro. In contrast, in an orthotopic glioma mouse model GBP1 overexpression significantly promotes glioma growth and reduces survival rate of glioma-bearing mice by increasing cell proliferation and decreasing cell apoptosis in tumor. Clinically, GBP1 expression is elevated in human GBM tumors and positively correlates with EGFRvIII status in GBM specimens, and its expression is inversely correlated with the survival rate of GBM patients. Taken together, these results reveal that GBP1 may serve as a potential therapeutic target for GBMs with EGFRvIII mutation. PMID:26848767

  8. Nonstructural Protein 1 of Influenza A Virus Interacts with Human Guanylate-Binding Protein 1 to Antagonize Antiviral Activity

    PubMed Central

    Yan, Wenjun; Wei, Jianchao; Shao, Donghua; Deng, Xufang; Wang, Shaohui; Li, Beibei; Tong, Guangzhi; Ma, Zhiyong

    2013-01-01

    Human guanylate-binding protein 1 (hGBP1) is an interferon-inducible protein involved in the host immune response against viral infection. In response to infection by influenza A virus (IAV), hGBP1 transcript and protein were significantly upregulated. Overexpression of hGBP1 inhibited IAV replication in a dose-dependent manner in vitro. The lysine residue at position 51 (K51) of hGBP1 was essential for inhibition of IAV replication. Mutation of K51 resulted in an hGBP1 that was unable to inhibit IAV replication. The viral nonstructural protein 1 (NS1) was found to interact directly with hGBP1. K51 of hGBP1 and a region between residues 123 and 144 in NS1 were demonstrated to be essential for the interaction between NS1 and hGBP1. Binding of NS1 to hGBP1 resulted in a significant reduction in both GTPase activity and the anti-IAV activity of hGBP1. These findings indicated that hGBP1 contributed to the host immune response against IAV replication and that hGBP1-mediated antiviral activity was antagonized by NS1 via binding to hGBP1. PMID:23405236

  9. Nonstructural protein 1 of influenza A virus interacts with human guanylate-binding protein 1 to antagonize antiviral activity.

    PubMed

    Zhu, Zixiang; Shi, Zixue; Yan, Wenjun; Wei, Jianchao; Shao, Donghua; Deng, Xufang; Wang, Shaohui; Li, Beibei; Tong, Guangzhi; Ma, Zhiyong

    2013-01-01

    Human guanylate-binding protein 1 (hGBP1) is an interferon-inducible protein involved in the host immune response against viral infection. In response to infection by influenza A virus (IAV), hGBP1 transcript and protein were significantly upregulated. Overexpression of hGBP1 inhibited IAV replication in a dose-dependent manner in vitro. The lysine residue at position 51 (K51) of hGBP1 was essential for inhibition of IAV replication. Mutation of K51 resulted in an hGBP1 that was unable to inhibit IAV replication. The viral nonstructural protein 1 (NS1) was found to interact directly with hGBP1. K51 of hGBP1 and a region between residues 123 and 144 in NS1 were demonstrated to be essential for the interaction between NS1 and hGBP1. Binding of NS1 to hGBP1 resulted in a significant reduction in both GTPase activity and the anti-IAV activity of hGBP1. These findings indicated that hGBP1 contributed to the host immune response against IAV replication and that hGBP1-mediated antiviral activity was antagonized by NS1 via binding to hGBP1.

  10. cRGD conjugated mPEG-PLGA-PLL nanoparticles for SGC-7901 gastric cancer cells-targeted Delivery of fluorouracil.

    PubMed

    Liu, Peifeng; Wang, Hongbin; Wang, Qi; Sun, Ying; Shen, Ming; Zhu, Mingjie; Wan, Zhiyong; Duan, Yourong

    2012-06-01

    The main purpose of this study was to evaluate the targeting effect of cyclic arginine-glycine-aspartic peptide (cRGD)-modified monomethoxy (polyethylene glycol)-poly (D, L-lactide-co-glycolide)-poly (L-lysine) nanoparticles (mPEG-PLGA-PLL-cRGD NPs) for gastric cancer SGC-7901 cells. We prepared the 5-Fulorouracil (5Fu)-loaded mPEG-PLGA-PLL-cRGD (5Fu/mPEG-PLGA-PLL-cRGD) NPs that had an average particle size of 180 nm and a zeta potential 2.77 mV. The results of cytotoxicity demonstrated the mPEG-PLGA-PLL-cRGD NPs showed the ignorable cytotoxicity and the 5Fu/mPEG-PLGA-PLL-cRGD NPs could significantly enhance the cytotoxicity of 5Fu. In vitro drug release experiments showed that the release of drug was effectively prolonged and sustained. The results of confocal laser scanning microscope (CLSM) and flow cytometer analysis demonstrated that the fluorescence intensity of the SGC-7901 gastric cancer cells treated with Rb/mPEG-PLGA-PLL-cRGD NPs was significantly higher than that treated with Rb, this suggested that Rb/mPEG-PLGA-PLL-cRGD NPs could effectively be internalized by SGC-7901 gastric cancer cells. In summary, the above experimental results illustrate that mPEG-PLGA-PLL-cRGD NPs have great potential to be used as an effective delivery carriers.

  11. Control of outflow resistance by soluble adenylyl cyclase.

    PubMed

    Lee, Yong Suk; Marmorstein, Alan D

    2014-01-01

    Abstract Glaucoma is a leading cause of blindness in the United States affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure, even in patients with normal tension glaucoma. Typically, this is accomplished by reducing the rate of aqueous flow by limiting aqueous production or enhancing drainage using drugs and surgery. Whereas these strategies are effective in diminishing vision loss, some patients continue to lose vision and many discontinue use of their medications because of undesirable side effects. Drugs known to be effective in altering conventional outflow have for the most part been abandoned from modern clinical practice due to undesirable side effects. Identification of new drugs that could enhance conventional outflow, would offer additional options in the treatment of glaucoma and ocular hypertension. To this end, our laboratory has recently uncovered a novel pathway for regulation of conventional outflow by the ciliary body. This pathway is dependent on soluble adenylyl cyclase, an enzyme that catalyzes the generation of cyclic adenosine 3',5' monophosphate (cAMP) in response to bicarbonate.

  12. Guanylyl cyclase C signaling axis and colon cancer prevention

    PubMed Central

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-01-01

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C’s role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention. PMID:27688649

  13. Structure of RNA 3'-phosphate cyclase bound to substrate RNA.

    PubMed

    Desai, Kevin K; Bingman, Craig A; Cheng, Chin L; Phillips, George N; Raines, Ronald T

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme.

  14. Guanylyl cyclase C signaling axis and colon cancer prevention.

    PubMed

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-09-28

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C's role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention. PMID:27688649

  15. Soluble adenylyl cyclase is essential for proper lysosomal acidification.

    PubMed

    Rahman, Nawreen; Ramos-Espiritu, Lavoisier; Milner, Teresa A; Buck, Jochen; Levin, Lonny R

    2016-10-01

    Lysosomes, the degradative organelles of the endocytic and autophagic pathways, function at an acidic pH. Lysosomes are acidified by the proton-pumping vacuolar ATPase (V-ATPase), but the molecular processes that set the organelle's pH are not completely understood. In particular, pH-sensitive signaling enzymes that can regulate lysosomal acidification in steady-state physiological conditions have yet to be identified. Soluble adenylyl cyclase (sAC) is a widely expressed source of cAMP that serves as a physiological pH sensor in cells. For example, in proton-secreting epithelial cells, sAC is responsible for pH-dependent translocation of V-ATPase to the luminal surface. Here we show genetically and pharmacologically that sAC is also essential for lysosomal acidification. In the absence of sAC, V-ATPase does not properly localize to lysosomes, lysosomes fail to fully acidify, lysosomal degradative capacity is diminished, and autophagolysosomes accumulate. PMID:27670898

  16. Guanylyl cyclase C signaling axis and colon cancer prevention

    PubMed Central

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-01-01

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C’s role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.

  17. The Growth of SGC-7901 Tumor Xenografts Was Suppressed by Chinese Bayberry Anthocyanin Extract through Upregulating KLF6 Gene Expression

    PubMed Central

    Wang, Yue; Zhang, Xia-nan; Xie, Wen-hua; Zheng, Yi-xiong; Cao, Jin-ping; Cao, Pei-rang; Chen, Qing-jun; Li, Xian; Sun, Chong-de

    2016-01-01

    To investigate the antitumor effect of anthocyanins extracted from Chinese bayberry fruit (Myrica rubra Sieb. et Zucc.), a nude mouse tumor xenograft model was established. Treatments with C3G (cyanidin-3-glucoside, an anthocyanin) significantly suppressed the growth of SGC-7901 tumor xenografts in a dose-dependent manner. Immunohistochemical staining showed a significant increase in p21 expression, indicating that the cell cycle of tumor xenografts was inhibited. qPCR screening showed that C3G treatment up-regulated the expression of the KLF6 gene, which is an important tumor suppressor gene inactivated in many human cancers. Western blot showed that C3G treatments markedly increased KLF6 and p21 protein levels, inhibited CDK4 and Cyclin D1 expression, but did not notably change the expression of p53. These results indicated that KLF6 up-regulates p21 in a p53-independent manner and significantly reduces tumor proliferation. This study provides important information for the possible mechanism of C3G-induced antitumor activity against gastric adenocarcinoma in vivo. PMID:27690088

  18. Alterations in adipocyte adenylate cyclase activity in morbidly obese and formerly morbidly obese humans.

    PubMed

    Martin, L F; Klim, C M; Vannucci, S J; Dixon, L B; Landis, J R; LaNoue, K F

    1990-08-01

    Studies examining animal models of genetic obesity have identified defects in adipocyte hormone-stimulated lipolysis that involve the adenylate cyclase transmembrane signaling system, specifically those components that decrease adenylate cyclase activity. To determine whether obese people demonstrate alterations in adenylate cyclase activity that could contribute to the maintenance of obesity by inhibiting lipolysis, we examined human adipocytes from patients who were lean, obese, or formerly obese. Fat samples were obtained from the lower abdomen of 14 women who were morbidly obese (obese group), from 10 women who were formerly morbidly obese and had lost weight after gastric stapling (postobese group), and from 10 similarly aged women of normal weight (controls). Adipocyte adenylate cyclase activity was determined under ligand-free (no stimulatory or inhibitory influences present), hormone-stimulated (isoproterenol, 10(-6) mmol/L), and maximal (cells stimulated with 10 mumol/L forskolin) conditions by measuring cyclic adenosine monophosphate (cAMP) levels by radioimmunoassay. The activity of adenylate cyclase was significantly different (p less than 0.01) in the three groups. Adipocytes from obese women had lower levels of cyclase activity under both ligand-free (5% vs 16% of maximal) and hormone-stimulated conditions (76% vs 100% of maximal) than adipocytes from normal women. Postobese women had levels of hormone-stimulated cAMP identical to those of normal women but still had abnormal ligand-free levels (under 5%). These results suggest the presence of an alteration in adipocyte adenylate cyclase regulation in morbidly obese women that is not entirely corrected when weight is lost after food intake is reduced by gastric stapling. This alteration in ligand-free cAMP activity may contribute to the development and maintenance of obesity. PMID:2166354

  19. Protective Macroautophagy Is Involved in Vitamin E Succinate Effects on Human Gastric Carcinoma Cell Line SGC-7901 by Inhibiting mTOR Axis Phosphorylation.

    PubMed

    Hou, Liying; Li, Yuze; Song, Huacui; Zhang, Zhihong; Sun, Yanpei; Zhang, Xuguang; Wu, Kun

    2015-01-01

    Vitamin E succinate (VES), a potential cancer therapeutic agent, potently induces apoptosis and inhibits the growth of various cancer cells. Autophagy has been supposed to promote cancer cell survival or trigger cell death, depending on particular cancer types and tumor microenvironments. The role of autophagy in the growth suppressive effect of VES on gastric cancer cell is basically unknown. We aimed to determine whether and how autophagy affected the VES-induced inhibition of SGC-7901 human gastric carcinoma cell growth. SGC-7901 cells were treated with VES or pre-treated with autophagy inhibitor, chloroquine (CQ) and 3-methyladenine (3-MA). Electron microscopy, fluorescence microscopy and Western blot were used to study whether VES induced autophagy reaction in SGC-7901 cells. Western blot evaluated the activities of the mammalian target of rapamycin (mTOR) axis. Then we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry to detect the level of cell viability and apoptosis. Collectively, our data indeed strongly support our hypothesis that VES treatment produced cytological variations that depict autophagy, increased the amount of intracellular green fluorescent protein-microtubule associated protein 1 light chain 3 (GFP-LC3) punctate fluorescence and the number of autophagic vacuoles. It altered the expression of endogenous autophagy marker LC3. VES activated the suppression of mTOR through inhibiting upstream regulators p38 MAPK and Akt. mTOR suppression consequently inhibited the activation of mTOR downstream targets p70S6K and 4E-BP-1. The activation of the upstream mTOR inhibitor AMPK had been up-regulated by VES. The results showed that pre-treatment SGC-7901 with autophagy inhibitors before VES treatment could increase the capacity of VES to reduce cell viability and to provoke apoptosis. In conclusion, VES-induced autophagy participates in SGC-7901 cell protection by inhibiting mTOR axis phosphorylation. Our

  20. A lycopene β-cyclase/lycopene ε-cyclase/light-harvesting complex-fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α-carotene and β-carotene at different ratios.

    PubMed

    Blatt, Andreas; Bauch, Matthias E; Pörschke, Yvonne; Lohr, Martin

    2015-05-01

    Biosynthesis of asymmetric carotenoids such as α-carotene and lutein in plants and green algae involves the two enzymes lycopene β-cyclase (LCYB) and lycopene ε-cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C-terminal light-harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α-carotene and β-carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C-terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α-carotene-to-β-carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α-carotene or lutein in bacteria or fungi.

  1. Stimulation of hormone-responsive adenylate cyclase activity by a factor present in the cell cytosol.

    PubMed Central

    MacNeil, S; Crawford, A; Amirrasooli, H; Johnson, S; Pollock, A; Ollis, C; Tomlinson, S

    1980-01-01

    1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E. PMID:7396869

  2. Overexpression and characterization of lycopene cyclase (CrtY) from marine bacterium Paracoccus haeundaensis.

    PubMed

    Jeong, Tae Hyug; Ji, Keunho; Kim, Young Tae

    2013-02-01

    Lycopene cyclase converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into beta-carotene rings via the monocyclic gamma-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The Km values for lycopene and NADPH were 3.5 microM and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.

  3. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides

    PubMed Central

    Gagne, Steve J.; Stout, Jake M.; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M.; Page, Jonathan E.

    2012-01-01

    Δ9-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2–C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity. PMID:22802619

  4. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

    PubMed

    Gagne, Steve J; Stout, Jake M; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M; Page, Jonathan E

    2012-07-31

    Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

  5. Sweet tastants stimulate adenylate cyclase coupled to GTP-binding protein in rat tongue membranes.

    PubMed

    Striem, B J; Pace, U; Zehavi, U; Naim, M; Lancet, D

    1989-05-15

    Sucrose and other saccharides, which produce an appealing taste in rats, were found to significantly stimulate the activity of adenylate cyclase in membranes derived from the anterior-dorsal region of rat tongue. In control membranes derived from either tongue muscle or tongue non-sensory epithelium, the effect of sugars on adenylate cyclase activity was either much smaller or absent. Sucrose enhanced adenylate cyclase activity in a dose-related manner, and this activation was dependent on the presence of guanine nucleotides, suggesting the involvement of a GTP-binding protein ('G-protein'). The activation of adenylate cyclase by various mono- and di-saccharides correlated with their electrophysiological potency. Among non-sugar sweeteners, sodium saccharin activated the enzyme, whereas aspartame and neohesperidin dihydrochalcone did not, in correlation with their sweet-taste effectiveness in the rat. Sucrose activation of the enzyme was partly inhibited by Cu2+ and Zn2+, in agreement with their effect on electrophysiological sweet-taste responses. Our results are consistent with a sweet-taste transduction mechanism involving specific receptors, a guanine-nucleotide-binding protein and the cyclic AMP-generating enzyme adenylate cyclase.

  6. Biochemistry and physiology of the natriuretic peptide receptor guanylyl cyclases.

    PubMed

    Tremblay, Johanne; Desjardins, Richard; Hum, David; Gutkowska, Jolanta; Hamet, Pavel

    2002-01-01

    Guanylyl cyclases (GC) exist as soluble and particulate, membrane-associated enzymes which catalyse the conversion of GTP to cGMP, an intracellular signalling molecule. Several membrane forms of the enzyme have been identified up to now. Some of them serve as receptors for the natriuretic peptides, a family of peptides which includes atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP), three peptides known to play important roles in renal and cardiovascular physiology. These are transmembrane proteins composed of a single transmembrane domain, a variable extracellular natriuretic peptide-binding domain, and a more conserved intracellular kinase homology domain (KHD) and catalytic domain. GC-A, the receptor for ANP and BNP, also named natriuretic peptide receptor-A or -1 (NPR-A or NPR- 1), has been studied widely. Its mode of activation by peptide ligands and mechanisms of regulation serve as prototypes for understanding the function of other particulate GC. Activation of this enzyme by its ligand is a complex process requiring oligomerization, ligand binding, KHD phosphorylation and ATP binding. Gene knockout and genetic segregation studies have provided strong evidence for the importance of GC-A in the regulation of blood pressure and heart and renal functions. GC-B is the main receptor for CNP, the latter having a more paracrine role at the vascular and venous levels. The structure and regulation of GC-B is similar to that of GC-A. This chapter reviews the structure and roles of GC-A and GC-B in blood pressure regulation and cardiac and renal pathophysiology. PMID:11952095

  7. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-10-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  8. Dynamics of adenylate cyclase regulation via heterotrimeric G-proteins.

    PubMed

    Milde, Markus; Werthmann, Ruth C; von Hayn, Kathrin; Bünemann, Moritz

    2014-04-01

    A wide variety of G-protein-coupled receptors either activate or inhibit ACs (adenylate cyclases), thereby regulating cellular cAMP levels and consequently inducing proper physiological responses. Stimulatory and inhibitory G-proteins interact directly with ACs, whereas G(q)-coupled receptors exert their effects primarily via Ca2+. Using the FRET-based cAMP sensor Epac1 (exchange protein directly activated by cAMP 1)-cAMPS (adenosine 3',5'-cyclic monophosphorothioate), we studied cAMP levels in single living VSMCs (vascular smooth muscle cells) or HUVECs (human umbilical vein endothelial cells) with subsecond temporal resolution. Stimulation of purinergic (VSMCs) or thrombin (HUVECs) receptors rapidly decreased cAMP levels in the presence of the β-adrenergic agonist isoprenaline via a rise in Ca2+ and subsequent inhibition of AC5 and AC6. Specifically in HUVECs, we observed that, in the continuous presence of thrombin, cAMP levels climbed slowly after the initial decline with a delay of a little less than 1 min. The underlying mechanism includes phospholipase A2 activity and cyclo-oxygenase-mediated synthesis of prostaglandins. We studied further the dynamics of the inhibition of ACs via G(i)-proteins utilizing FRET imaging to resolve interactions between fluorescently labelled G(i)-proteins and AC5. FRET between Gα(i1) and AC5 developed at much lower concentration of agonist compared with the overall G(i)-protein activity. We found the dissociation of Gα(i1) subunits and AC5 to occur slower than the G(i)-protein deactivation. This led us to the conclusion that AC5, by binding active Gα(i1), interferes with G-protein deactivation and reassembly and thereby might sensitize its own regulation. PMID:24646224

  9. Molecular characterization of tick salivary gland glutaminyl cyclase.

    PubMed

    Adamson, Steven W; Browning, Rebecca E; Chao, Chien-Chung; Bateman, Robert C; Ching, Wei-Mei; Karim, Shahid

    2013-09-01

    Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood feeding of the black-legged tick (Ixodes scapularis) and the gulf-coast tick (Amblyomma maculatum). QC sequences from I. scapularis and A. maculatum showed a high degree of amino acid identity to each other and other arthropods and residues critical for zinc binding/catalysis (D159, E202, and H330) or intermediate stabilization (E201, W207, D248, D305, F325, and W329) are conserved. Analysis of QC transcriptional gene expression kinetics depicts an upregulation during the bloodmeal of adult female ticks prior to fast-feeding phases in both I. scapularis and A. maculatum suggesting a functional link with bloodmeal uptake. QC enzymatic activity was detected in saliva and extracts of tick salivary glands and midguts. Recombinant QC was shown to be catalytically active. Furthermore, knockdown of QC transcript by RNA interference resulted in lower enzymatic activity, and small, unviable egg masses in both studied tick species as well as lower engorged tick weights for I. scapularis. These results suggest that the post-translational modification of neurotransmitters and other bioactive peptides by QC is critical to oviposition and potentially other physiological processes. Moreover, these data suggest that tick-specific QC-modified neurotransmitters/hormones or other relevant parts of this system could potentially be used as novel physiological targets for tick control. PMID:23770496

  10. Renal Phosphate Wasting in the Absence of Adenylyl Cyclase 6

    PubMed Central

    Fenton, Robert A.; Murray, Fiona; Dominguez Rieg, Jessica A.; Tang, Tong; Levi, Moshe

    2014-01-01

    Parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23) enhance phosphate excretion by the proximal tubule of the kidney by retrieval of the sodium-dependent phosphate transporters (Npt2a and Npt2c) from the apical plasma membrane. PTH activates adenylyl cyclase (AC) through PTH 1 receptors and stimulates the cAMP/PKA signaling pathway. However, the precise role and isoform(s) of AC in phosphate homeostasis are not known. We report here that mice lacking AC6 (AC6−/−) have increased plasma PTH and FGF-23 levels compared with wild-type (WT) mice but comparable plasma phosphate concentrations. Acute activation of the calcium-sensing receptor or feeding a zero phosphate diet almost completely suppressed plasma PTH levels in both AC6−/− and WT mice, indicating a secondary cause for hyperparathyroidism. Pharmacologic blockade of FGF receptors resulted in a comparable increase in plasma phosphate between genotypes, whereas urinary phosphate remained significantly higher in AC6−/− mice. Compared with WT mice, AC6−/− mice had reduced renal Npt2a and Npt2c protein abundance, with approximately 80% of Npt2a residing in lysosomes. WT mice responded to exogenous PTH with redistribution of Npt2a from proximal tubule microvilli to intracellular compartments and lysosomes alongside a PTH-induced dose–response relationship for fractional phosphate excretion and urinary cAMP excretion. These responses were absent in AC6−/− mice. In conclusion, AC6 in the proximal tubule modulates cAMP formation, Npt2a trafficking, and urinary phosphate excretion, which are highlighted by renal phosphate wasting in AC6−/− mice. PMID:24854272

  11. Role of Adenylate Cyclase 1 in Retinofugal Map Development

    PubMed Central

    Dhande, Onkar S.; Bhatt, Shivani; Anishchenko, Anastacia; Elstrott, Justin; Iwasato, Takuji; Swindell, Eric C.; Xu, Hong-Ping; Jamrich, Milan; Itohara, Shigeyoshi; Feller, Marla B.; Crair, Michael C.

    2013-01-01

    The development of topographic maps of the sensory periphery is sensitive to the disruption of adenylate cyclase 1 (AC1) signaling. AC1 catalyzes the production of cAMP in a Ca2+/calmodulin-dependent manner, and AC1 mutant mice (AC1−/−) have disordered visual and somatotopic maps. However, the broad expression of AC1 in the brain and the promiscuous nature of cAMP signaling have frustrated attempts to determine the underlying mechanism of AC1-dependent map development. In the mammalian visual system, the initial coarse targeting of retinal ganglion cell (RGC) projections to the superior colliculus (SC) and lateral geniculate nucleus (LGN) is guided by molecular cues, and the subsequent refinement of these crude projections occurs via an activity-dependent process that depends on spontaneous retinal waves. Here, we show that AC1−/− mice have normal retinal waves but disrupted map refinement. We demonstrate that AC1 is required for the emergence of dense and focused termination zones and elimination of inaccurately targeted collaterals at the level of individual retinofugal arbors. Conditional deletion of AC1 in the retina recapitulates map defects, indicating that the locus of map disruptions in the SC and dorsal LGN of AC1−/− mice is presynaptic. Finally, map defects in mice without AC1 and disrupted retinal waves (AC1−/−;β2−/− double KO mice) are no worse than those in mice lacking only β2−/−, but loss of AC1 occludes map recovery in β2−/− mice during the second postnatal week. These results suggest that AC1 in RGC axons mediates the development of retinotopy and eye-specific segregation in the SC and dorsal LGN. PMID:22102330

  12. Involvement of Brain-Enriched Guanylate Kinase-Associated Protein (BEGAIN) in Chronic Pain after Peripheral Nerve Injury

    PubMed Central

    Fukuda, Masafumi; Furue, Hidemasa; Abe, Manabu; Nishida, Kazuhiko; Yao, Ikuko; Yamada, Akihiro; Okumura, Nobuaki; Nakazawa, Takanobu; Yamamoto, Tadashi; Sakimura, Kenji; Takao, Toshifumi; Ito, Seiji

    2016-01-01

    Maintenance of neuropathic pain caused by peripheral nerve injury crucially depends on the phosphorylation of GluN2B, a subunit of the N-methyl-d-aspartate (NMDA) receptor, at Tyr1472 (Y1472) and subsequent formation of a postsynaptic density (PSD) complex of superficial spinal dorsal horn neurons. Here we took advantage of comparative proteomic analysis based on isobaric stable isotope tags (iTRAQ) between wild-type and knock-in mice with a mutation of Y1472 to Phe of GluN2B (Y1472F-KI) to search for PSD proteins in the spinal dorsal horn that mediate the signaling downstream of phosphorylated Y1472 GluN2B. Among several candidate proteins, we focused on brain-enriched guanylate kinase-associated protein (BEGAIN), which was specifically up-regulated in wild-type mice after spared nerve injury (SNI). Immunohistochemical analysis using the generated antibody demonstrated that BEGAIN was highly localized at the synapse of inner lamina II in the spinal dorsal horn and that its expression was up-regulated after SNI in wild-type, but not in Y1472F-KI, mice. In addition, alteration of the kinetics of evoked excitatory postsynaptic currents for NMDA but not those for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in spinal lamina II was demonstrated by BEGAIN deletion. We demonstrated that mechanical allodynia, a condition of abnormal pain induced by innocuous stimuli, in the SNI model was significantly attenuated in BEGAIN-deficient mice. However, there was no significant difference between naive wild-type and BEGAIN-knockout mice in terms of physiological threshold for mechanical stimuli. These results suggest that BEGAIN was involved in pathological pain transmission through NMDA receptor activation by the phosphorylation of GluN2B at Y1472 in spinal inner lamina II. PMID:27785460

  13. Diversity in guanosine 3',5'-bisdiphosphate (ppGpp) sensitivity among guanylate kinases of bacteria and plants.

    PubMed

    Nomura, Yuhta; Izumi, Atsushi; Fukunaga, Yoshinori; Kusumi, Kensuke; Iba, Koh; Watanabe, Seiya; Nakahira, Yoichi; Weber, Andreas P M; Nozawa, Akira; Tozawa, Yuzuru

    2014-05-30

    The guanosine 3',5'-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a Ki of 2.8 μM relative to the substrate GMP, whereas the Km of this enzyme for GMP was 73 μM. The IC50 of ppGpp for GKpm was ∼10 μM. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers' yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp.

  14. ADAM22, a Kv1 channel-interacting protein, recruits membrane-associated guanylate kinases to juxtaparanodes of myelinated axons.

    PubMed

    Ogawa, Yasuhiro; Oses-Prieto, Juan; Kim, Moon Young; Horresh, Ido; Peles, Elior; Burlingame, Alma L; Trimmer, James S; Meijer, Dies; Rasband, Matthew N

    2010-01-20

    Clustered Kv1 K(+) channels regulate neuronal excitability at juxtaparanodes of myelinated axons, axon initial segments, and cerebellar basket cell terminals (BCTs). These channels are part of a larger protein complex that includes cell adhesion molecules and scaffolding proteins. To identify proteins that regulate assembly, clustering, and/or maintenance of axonal Kv1 channel protein complexes, we immunoprecipitated Kv1.2 alpha subunits, and then used mass spectrometry to identify interacting proteins. We found that a disintegrin and metalloproteinase 22 (ADAM22) is a component of the Kv1 channel complex and that ADAM22 coimmunoprecipitates Kv1.2 and the membrane-associated guanylate kinases (MAGUKs) PSD-93 and PSD-95. When coexpressed with MAGUKs in heterologous cells, ADAM22 and Kv1 channels are recruited into membrane surface clusters. However, coexpression of Kv1.2 with ADAM22 and MAGUKs does not alter channel properties. Among all the known Kv1 channel-interacting proteins, only ADAM22 is found at every site where Kv1 channels are clustered. Analysis of Caspr-null mice showed that, like other previously described juxtaparanodal proteins, disruption of the paranodal junction resulted in redistribution of ADAM22 into paranodal zones. Analysis of Caspr2-, PSD-93-, PSD-95-, and double PSD-93/PSD-95-null mice showed ADAM22 clustering at BCTs requires PSD-95, but ADAM22 clustering at juxtaparanodes requires neither PSD-93 nor PSD-95. In direct contrast, analysis of ADAM22-null mice demonstrated juxtaparanodal clustering of PSD-93 and PSD-95 requires ADAM22, whereas Kv1.2 and Caspr2 clustering is normal in ADAM22-null mice. Thus, ADAM22 is an axonal component of the Kv1 K(+) channel complex that recruits MAGUKs to juxtaparanodes. PMID:20089912

  15. Ontogeny of regulatory mechanisms for beta-adrenoceptor control of rat cardiac adenylyl cyclase: targeting of G-proteins and the cyclase catalytic subunit.

    PubMed

    Zeiders, J L; Seidler, F J; Slotkin, T A

    1997-02-01

    Fetal and neonatal tissues are resistant to catecholamine-induced desensitization of essential physiological responses. We examined the mechanisms underlying the ontogeny of desensitization in neonatal rat heart for the beta-adrenergic receptor/adenylyl cyclase signaling cascade. Animals of different ages received isoproterenol daily or 4 days and cardiac membrane preparations were evaluated on the 5th day (6, 15, 25 days old and adults). Measurements were made of basal activity, activity stimulated by two agonists (isoproterenol or glucagon) that operate at different receptors but that share Gs as the transduction intermediate, or by forskolin-Mn' to assess total catalytic capacity of the cyclase subunit; we also assessed inhibition of activity by carbachol which acts via muscarinic cholinergic receptors and G. Adult rats exhibited robust desensitization of the adenylyl cyclase response but the effect was heterologous in that equivalent loss of activity was seen for basal, isoproterenol- and glucagon-stimulated activity forskolin-Mn(2+)-stimulated activity was also decreased. Two factors contributed to desensitization; generalized reduction in membrane protein concentrations caused by cell enlargement (reduced surface-to-volume ratio), and specific interference with the G-protein component that couples receptors to the cyclase. Thus, after adjustment for changes in membrane protein, the desensitization of the forskolin-Mn2, response was no longer evident, but the effects on the other measures were still present. In addition, isoproterenol treatment produced crosstalk with the carbachol/Gi signaling pathway, with significant reductions in the ability of carbachol to inhibit adenylyl cyclase activity. Heterologous desensitization by isoproterenol was also present in 15 and 25 day old rats, but involved only selective components of the effects seen in adults. At 25 days, uncoupling of signals operating through Gs and Gi was obtained without a reduction in forskolin

  16. Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability*

    PubMed Central

    Ochoa, Cristhiaan D.; Alexeyev, Mikhail; Pastukh, Viktoriya; Balczon, Ron; Stevens, Troy

    2012-01-01

    Exotoxin Y (ExoY) is a type III secretion system effector found in ∼ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases. PMID:22637478

  17. Inhibition of hormonally regulated adenylate cyclase by the beta gamma subunit of transducin.

    PubMed Central

    Bockaert, J; Deterre, P; Pfister, C; Guillon, G; Chabre, M

    1985-01-01

    Transducin (T), the GTP-binding protein of the retina activates the cGMP phosphodiesterase system, and presents analogies with the proteins GS and Gi which respectively mediate adenylate cyclase activation and inhibition by hormone receptors. These proteins are all comprised of an alpha subunit carrying the GTP-binding site and a beta gamma subunit made of two peptides. The beta peptide (35 kd) appears similar in the three proteins. We demonstrate here that purified T beta gamma inhibits adenylate cyclase from human platelet membranes. This inhibition was observed when adenylate cyclase was stimulated by GTP, prostaglandin E1 (PGE1), NaF and forskolin, but not when stimulated by GTP(gamma)S. In the presence of GTP and forskolin, the T beta gamma-induced maximal inhibition was not additive with the alpha 2-receptor-induced adenylate cyclase inhibition mediated by Gi. Both inhibitions were suppressed at high Mg2+ concentrations, which as also known to dissociate T beta gamma from T alpha-GDP. This suggests that these adenylate cyclase inhibitions are due to the formation of inactive complexes of GS alpha-GDP with T beta gamma or Gi beta gamma. T beta gamma-induced inhibition did not require detergent and could be suppressed by simple washing. T beta gamma effects are dependent on its concentration rather than on its total amount. This suggests that T beta gamma can operate in solution with no integration into the membrane. Similar inhibitory effects of T beta gamma are observed on adenylate cyclase from anterior pituitary and lymphoma S49 cell lines. PMID:3861319

  18. Changed sensitivity of adenylate cyclase signaling system to biogenic amines and peptide hormones in tissues of starving rats.

    PubMed

    Shpakov, A O; Kuznetsova, L A; Plesneva, S A; Pertseva, M N

    2007-07-01

    In the myocardium and skeletal muscles of rats deprived of food for 2 days, basal activity of adenylate cyclase decreased, while the sensitivity of adenylate cyclase signaling system to the stimulating effects of non-hormonal agents (guanine nucleotides and NaF) and beta-agonist isoproterinol modulating adenylate cyclase through stimulating G proteins increased. In starving organism, the regulatory effects of hormones realizing their effects through inhibitory G proteins (somatostatin in the myocardium and bromocryptin in the brain) weakened. Their inhibitory effects on forskolin-stimulated adenylate cyclase activity and stimulating effects on binding of guanosine triphosphate decreased. In the brain of starving rats, the differences in the sensitivity of the adenylate cyclase signaling system to hormones and nonhormonal agents were less pronounced than in the muscle tissues, which attested to tissue-specific changes in the functional state of this system under conditions of 2-day starvation.

  19. A Novel Mechanism for Adenylyl Cyclase Inhibition from the Crystal Structure of its Complex with Catechol Estrogen

    SciTech Connect

    Steegborn,C.; Litvin, T.; Hess, K.; Capper, A.; Taussig, R.; Buck, J.; Levin, L.; Wu, H.

    2005-01-01

    Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.

  20. Effects of 5‑fluorouracil and class III phosphoinositide 3‑kinase small interfering RNA combination therapy on SGC7901 human gastric cancer cells.

    PubMed

    Zhu, Bao-Song; Sun, Jia-Lei; Gong, Wei; Zhang, Xing-Ding; Wu, Yong-You; Xing, Chun-Gen

    2015-03-01

    The aim of the present study was to investigate the effects of small interfering RNA‑mediated inhibition of Class III phosphoinositide 3‑kinase (PI3K) signal transduction on the proliferation, apoptosis and autophagy of SGC7901 gastric cancer cells. The present study also aimed to examine the contribution of autophagic inhibition to the antitumor effects of 5‑fluorouracil (5‑FU). A PI3K(III)‑RNA interference (i)‑green fluorescent protein (GFP) recombinant replication adenovirus (AD) and the negative control (NC)‑RNAi‑GFP control AD were constructed and infected into SGC7901 cells. A methyl thiazolyl tetrazolium assay was used to determine the growth rate of the SGC7901 cells. Immunofluorescent staining was used to detect microtubule‑associated protein 1 light chain 3 expression. The mitochondrial membrane potential was measured using the JC‑1 fluorescent probe. Autophagic expression was monitored with MDC staining and transmission electron microscopy. The results revealed that following combination treatment of the SGC7901 gastric cancer cells with 5‑FU + PI3K(III)‑RNAi‑AD, the optical density absorbance values at 24, 48 and 72 h were 0.17 ± 1.64, 0.13 ± 4.64 and 0.11 ± 3.56%, respectively, with cell viability inhibition ratios of 45.89 ± 6.67, 72.57 ± 9.48 and 87.51 ± 4.65%, respectively. As compared with the other treatment groups, the inhibition rate in the combined treatment group was significantly higher (P<0.05). The percentages of the cells with green fluorescence in the combined treatment group were 74.4 ± 3.86 (24 h), 82.3 ± 1.84 (48 h) and 92.5 ± 1.1% (72 h), which were larger than those of the other groups. The percentage of cells with green fluorescence became larger, which indicated that the mitochondrion membrane potential had been reduced to a greater extent. MDC staining revealed that the number of autophagic vacuoles in the cells (measured at 24, 48 and 72 h) decreased gradually with time, with more autophagic

  1. [Reactivity of the adenylyl cyclase system in rat tissues to biogenic amines and peptide hormones under starvation condition].

    PubMed

    Shpakov, A O; Kuznetsova, L A; Plesneva, S A; Pertseva, M N

    2007-04-01

    Under starvation condition, sensitivity of the adenylyl cyclase system to regulatory action of biogenic amines and peptide hormones in rat tissues are changed. In the myocardium and skeletal muscles, after 2 and 4 days of starvation, the regulatory effects of isoproterenol and relaxin acting via G,-proteins on the adenylyl cyclase activity and the G-protein GTP-binding are significantly increased compared with control. At the same time, regulatory effects ofsomatostatin which are realized via Gi-proteins, on adenylyl cyclase system in the myocardium are decreased. Under prolonged starvation consisting of two consecutive 4-days periods, the effects of hormones acting via Gs-proteins on the adenylyl cyclase activity in muscle tissues are decreased to control value levels. The effects of hormones acting via Gi-proteins are largely reduced. In the brain, intensification of adenylyl cyclase stimulating hormonal effects was late and only observed after a 4-day starvation. Unlike muscle tissues, the increase of adenylyl cyclase stimulating effects in the brain is preserved after two-period starvation. The weakening of adenylyl cyclase inhibiting hormonal signals both in the brain and muscles is observed after a 2-day starvation and then the weakening is intensified. Possible role of glucose level and basal adenylyl cyclase activity in determination of the sensitivity of the adenylyl cyclase system to hormones under study is discussed. It is suggested that one of the key causes of physiological changes in animal organism under starvation involves alteration of hormonal signalling systems sensitivity, in particular that of the adenylyl cyclase system, to hormone regulatory action.

  2. Oncogenic NanogP8 expression regulates cell proliferation and migration through the Akt/mTOR signaling pathway in human gastric cancer – SGC-7901cell line

    PubMed Central

    Jiang, Zheng; Liu, Yao; Wang, Chuan

    2016-01-01

    Background Although elevated expression of NanogP8 has been detected in many human tumor tissues, its role in gastric tumorigenesis remains unclear. Therefore, this study aimed to investigate the function and regulatory mechanism of NanogP8 in gastric cancer. Methods In this study, NanogP8 cDNA was amplified by real time polymerase chain reaction from the human gastric cancer cell line SGC-7901. The shRNA for RNA interference was established. The NanogP8, pAkt, Akt, pERK, ERK, p-mTOR, and mTOR proteins were detected by using the Western blot assay. Cell viability was evaluated by using the CCK-8 assay. Cell migration and invasion were also examined by using the transwell assay. Results The results indicated that the NanogP8 overexpression promoted proliferation and migration of SGC-7901 cell line, whereas its ablation exerted opposite effects. Interestingly, NanogP8 activated Akt, a key mediator of survival signals, and without affecting total Akt protein level. The NanogP8-increased gastric cell proliferation was downregulated by Akt inhibition. Our results further showed that increasing NanogP8 expression in human gastric cancer cells promoted cell proliferation by activating the AKT/mTOR pathway and further maintained gastric cell survival. Conclusion Our findings extend the knowledge regarding the oncogenic functions and proved that the NanogP8 regulates cell proliferation and migration by Akt/mTOR signaling pathway in human gastric cancer SGC-7901cell line. PMID:27563247

  3. Characterization of cancer stem-like cells derived from a side population of a human gallbladder carcinoma cell line, SGC-996

    SciTech Connect

    Li, Xin-xing; Wang, Jian; Wang, Hao-lu; Wang, Wei; Yin, Xiao-bin; Li, Qi-wei; Chen, Yu-ying; Yi, Jing

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer We sorted SP cells from a human gallbladder carcinoma cell lines, SGC-996. Black-Right-Pointing-Pointer SP cells displayed higher proliferation and stronger clonal-generating capability. Black-Right-Pointing-Pointer SP cells showed more migratory and invasive abilities. Black-Right-Pointing-Pointer SP cells were more resistant and tumorigenic than non-SP counterparts. Black-Right-Pointing-Pointer ABCG2 might be a candidate as a marker for SP cells. -- Abstract: The cancer stem cell (CSC) hypothesis proposes that CSCs, which can renew themselves proliferate infinitely, and escape chemotherapy, become the root of recurrence and metastasis. Previous studies have verified that side population (SP) cells, characterized by their ability to efflux lipophilic substrate Hoechst 33342, to share many characteristics of CSCs in multiplying solid tumors. The purpose of this study was to sort SP cells from a human gallbladder carcinoma cell line, SGC-996 and to preliminarily identify the biological characteristics of SP cells from the cell line. Using flow cytometry we effectively sorted SP cells from the cell line SGC-996. SP cells not only displayed higher proliferative, stronger clonal-generating, more migratory and more invasive capacities, but showed stronger resistance. Furthermore, our experiments demonstrated that SP cells were more tumorigenic than non-SP counterparts in vivo. Real-time PCR analysis and immunocytochemistry showed that the expression of ATP-binding cassette subfamily G member 2 (ABCG2) was significantly higher in SP cells. Hence, these results collectively suggest that SP cells are progenitor/stem-like cells and ABCG2 might be a candidate marker for SP cells in human gallbladder cancer.

  4. Cytidylyl- and Uridylyl Cyclase Activity of Bacillus anthracis Edema Factor and Bordetella pertussis CyaA

    PubMed Central

    Göttle, Martin; Dove, Stefan; Kees, Frieder; Schlossmann, Jens; Geduhn, Jens; König, Burkhard; Shen, Yuequan; Tang, Wei-Jen; Kaever, Volkhard; Seifert, Roland

    2010-01-01

    Cyclic adenosine 3′:5′-monophosphate (cAMP) and cyclic guanosine 3′:5′-monophosphate (cGMP) are second messengers for a numerous mammalian cell functions. The natural occurrence and synthesis of a third cyclic nucleotide (cNMP), cyclic cytidine 3′:5′-monophosphate (cCMP) is discussed controversially, and almost nothing is known about cyclic uridine 3′:5′-monophosphate (cUMP). Bacillus anthracis and Bordetella pertussis secrete the adenylyl cyclase (AC) toxins edema factor (EF) and CyaA, respectively, weakening immune responses and facilitating bacterial proliferation. A cell-permeable cCMP analog inhibits human neutrophil superoxide production. Here, we report that EF and CyaA also possess cytidylyl cyclase (CC) and uridylyl cyclase (UC) activity. CC- and UC activity was determined by a radiometric assay, using [α-32P]CTP and [α-32P]UTP as substrates, respectively, and by an HPLC method. The identity of cNMPs was confirmed by mass spectrometry. Based on available crystal structures, we developed a model illustrating conversion of CTP to cCMP by bacterial toxins. In conclusion, we have shown both EF and CyaA have a rather broad substrate-specificity and exhibit cytidylyl- and uridylyl cyclase activity. Both cCMP and cUMP may contribute to toxin actions. PMID:20521845

  5. Soluble guanylyl cyclase is involved in PDT-induced injury of crayfish glial cells

    NASA Astrophysics Data System (ADS)

    Kovaleva, V. D.; Uzdensky, A. B.

    2016-04-01

    Photodynamic therapy (PDT) is a potential tool for selective destruction of malignant brain tumors. However, not only malignant but also healthy neurons and glial cells may be damaged during PDT. Nitric oxide is an important modulator of cell viability and intercellular neuroglial communications. NO have been already shown to participate in PDT-induced injury of neurons and glial cells. As soluble guanylyl cyclase is the only known receptor for NO, we have studied the possible role of soluble guanylyl cyclase in the regulation of survival and death of neurons and surrounding glial cells under photo-oxidative stress induced by photodynamic treatment (PDT). The crayfish stretch receptor consisting of a single identified sensory neuron enveloped by glial cells is a simple but informative model object. It was photosensitized with alumophthalocyanine photosens (10 nM) and irradiated with a laser diode (670 nm, 0.4 W/cm2). Using inhibitory analysis we have shown that during PDT soluble guanylyl cyclase, probably, has proapoptotic and antinecrotic effect on the glial cells of the isolated crayfish stretch receptor. Proapoptotic effect of soluble guanylyl cyclase could be mediated by protein kinase G (PKG). Thus, the involvement of NO/sGC/cGMP/PKG signaling pathway in PDT-induced apoptosis of glial cells was indirectly demonstrated.

  6. Defective responsiveness of adenylate cyclase to forskolin in the Drosophila memory mutant rutabaga.

    PubMed

    Dudai, Y; Sher, B; Segal, D; Yovell, Y

    1985-12-01

    The Drosophila memory mutant rutabaga (rut) has been previously shown to have a defective subpopulation (or functional state) of the enzyme adenylate cyclase. We report here that the reduced adenylate cyclase activity is also associated with a defective responsiveness of the enzyme to forskolin. Forskolin activation isotherms of the enzyme in normal membranes reveal low- and high-affinity forskolin-interacting components; the residual enzyme in the mutant shows a smaller proportion of the high-affinity response. In addition, in mutant membrane preparations, forskolin fails to shift the Km of the enzyme for free Mg2+ and for MgATP, in contrast to the situation in the normal tissue. The defect in the responsiveness to forskolin in rut is even more pronounced in a Lubrol-solubilized enzyme preparation, and is due to intrinsic properties of the cyclase system rather than to the absence (or presence) of a soluble, or detergent solubilized, factor in rut. The reduced forskolin responsiveness maps to the X chromosomal segment 12F5-6 to 13A1-5, within the region previously reported to span the locus that controls both the abortive memory and the lack of Ca2+-stimulation of adenylate cyclase in rut17. The possible relevance of the findings to postulated molecular mechanisms of short-term memory formation is discussed. PMID:3935769

  7. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    SciTech Connect

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. Univ. of California, Los Angeles )

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the {beta}-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the {beta}-adrenergic pathway, adenylate cyclase activity and {beta}-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. {beta}-Adrenergic receptors were identified in BAT using ({sup 125}I)iodocyanopindolol. Binding sites had the characteristics of mixed {beta}{sub 1}- and {beta}{sub 2}-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in {beta}-adrenergic receptor density due to a loss of the {beta}{sub 1}-adrenergic subtype. This BAT {beta}-adrenergic receptor downregulation was tissue specific, since myocardial {beta}-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of {beta}-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

  8. Mode of coupling between the beta-adrenergic receptor and adenylate cyclase in turkey erythrocytes.

    PubMed

    Tolkovsky, A M; Levitzki, A

    1978-09-01

    The mode of coupling of the beta-adrenergic receptor to the enzyme adenylate cyclase in turkey erythrocyte membranes was analyzed in detail. A number of experimental techniques have been used: (1) measurement of the kinetics of cyclase activation to its permanetly active state in the presence of guanylyl imidodiphosphate, as a function of hormone concentrations; (2) measurement of antagonist and agoinst binding to the beta-adrenergic receptor prior and subsequent to the enzyme activation by hormone and guanylyl imidodiphosphate. On the bases of these two approaches, all the models of receptor to enzyme coupling which involve an equilibrium between the enzyme and the receptor can be rejected. The binding and the kinetic data, however, can be fitted by two diametrically opposed models of receptor to enzyme coupling: (a) the precouped enzyme-receptor model where activation of the enzyme occurs, according to the following scheme: formula (see text) where H is the hormone, RE is the precoupled respetor-enzyme complex, k1 and k2 are the rate constants describing hormone binding, and k is the rate constant characterizing the formation of HRE' from the intermediate HRE. According to this model, the activated complex is composed of all of the interacting species. (b) The other model is the collision coupling mechanism: formula (see test) wheere KH is the horome-receptor dissociation constant, k1 is the bimolecular rate constant governing the formation of HRE, and k3 the rate constant governing the activation of the enzyme. In this case the intermediate never accumulates and constitutes only a small fraction of the total receptor and adenylate cyclase concentrations. In order to establish which of the two mechanisms governs the mode of adenylate cyclase activation by its receptor, a diagnostic experiment was performed: Progressive inactivation of the beta receptor by a specific affinity label was found to cause a decrease in the maximal binding capacity of the receptor and a

  9. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure.

    PubMed

    Scarpace, P J; Baresi, L A; Morley, J E

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the beta-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the beta-adrenergic pathway, adenylate cyclase activity and beta-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. beta-Adrenergic receptors were identified in BAT using [125I]iodocyanopindolol. Binding sites had the characteristics of mixed beta 1- and beta 2-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in beta-adrenergic receptor density due to a loss of the beta 1-adrenergic subtype. This BAT beta-adrenergic receptor downregulation was tissue specific, since myocardial beta-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. In contrast, food deprivation did not alter BAT beta-adrenergic receptor density. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. The ratio of isoproterenol-stimulated to forskolin-stimulated adenylate cyclase activity decreased in the sucrose-fed and cold-exposed rats but not in the food-deprived rats. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of beta-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability. PMID:2827501

  10. Regional distribution of somatostatin receptor binding and modulation of adenylyl cyclase activity in Alzheimer's disease brain.

    PubMed

    Bergström, L; Garlind, A; Nilsson, L; Alafuzoff, I; Fowler, C J; Winblad, B; Cowburn, R F

    1991-10-01

    We have previously reported a reduction in the inhibitory effect of somatostatin on adenylyl cyclase activity in the superior temporal cortex of a group of Alzheimer's disease cases, compared to a group of matched controls. In the present study, the levels of high affinity 125I-Tyr11-somatostatin-14 binding, its modulation by guanine nucleotides and the effects of somatostatin on adenylyl cyclase activity have been measured in preparations of frontal cortex, hippocampus, caudate nucleus and cerebellum from the same patient and control groups. A significant reduction in 125I-Tyr11-somatostatin-14 binding was observed in the frontal cortex, but not other regions, of the Alzheimer's disease group, compared with control values. The profiles of inhibition of specific 125I-Tyr11-somatostatin-14 binding by Gpp(NH)p were similar in all regions in both groups. No significant differences in basal, forskolin-stimulated, or somatostatin and neuropeptide Y inhibitions of adenylyl cyclase activity were found between the two groups. The pattern of change of somatostatin binding in the Alzheimer's disease cases observed in the present study differs from the reported pattern of loss of somatostatin neurons and may be secondary to the degeneration of somatostatin receptor-bearing cholinergic afferents arising from the nucleus basalis. The results of this study indicate that impaired somatostatin modulation of adenylyl cyclase is not a global phenomenon in Alzheimer's disease brain and also that there are no major disruptions of somatostatin receptor-G-protein coupling or of adenylyl cyclase catalytic activity in this disorder. PMID:1684616

  11. Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

    PubMed Central

    Cunningham, F X; Sun, Z; Chamovitz, D; Hirschberg, J; Gantt, E

    1994-01-01

    A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme. PMID:7919981

  12. Mechanisms of nonhormonal activation of adenylate cyclase based on target analysis

    SciTech Connect

    Verkman, A.S.; Ausiello, D.A.; Jung, C.Y.; Skorecki, K.L.

    1986-08-12

    Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK1 with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 degrees C (0-14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The ln (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin- (2 microM) stimulated activity and concave downward for unstimulated (10 mM Mn/sup 2 +/), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 microM vasopressin did not alter the ln activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the ln activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously (Verkman, A. S., Skorecki, K. L., and Ausiello, D. A. (1986) Am. J. Physiol. 260, C103-C123) and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between alpha beta and alpha + beta favors alpha beta; dissociated alpha binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to alpha. GTP and its analogues accelerate the rate of alpha GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without alpha beta dissociation or GTP addition through activation of C by an alpha beta-F complex.

  13. Properties of Adenyl Cyclase from Human Jejunal Mucosa during Naturally Acquired Cholera and Convalescence

    PubMed Central

    Chen, Lincoln C.; Rohde, Jon E.; Sharp, Geoffrey W. G.

    1972-01-01

    The enterotoxin of Vibrio cholerae causes copious fluid production throughout the lenght of the small intestine. As this is thought to be mediated by stimulation of adenyl cyclase, a study has been made of the activity and properties of this enzyme in jejunal biopsy tissue taken from patients during the diarrheal phase of cholera and after recovery. Adenyl cyclase activity during cholera was increased more than twofold relative to the enzyme in convalescence. Under both conditions stimulation by prostaglandin E1 (PGE1) and by fluoride was observed. The responsiveness to PGE1 was not altered in cholera; the total activity of the fluoride-stimulated enzyme was similar, a finding that suggests cholera toxin stimulates pre-existing enzyme in the intestinal cell. The enzymes during cholera and convalescence were similar in all other properties examined. Optimal Mg++ concentration was 10 mM; Mn++ at 5 mM stimulated the enzyme but could not replace Mg++ except in the presence of 10 mM fluoride. Calcium was markedly inhibitory at concentrations greater than 10-4 M. The pH optimum was 7.5 and the Michaelis constant (Km) for ATP concentration approximated 10-4 M. Thus the interaction of cholera toxin with human intestinal adenyl cyclase does not alter the basic properties of the enzyme. When biopsy specimens were maintained intact in oxygenated Ringer's solution at 0°C, no loss of activity was observed at 1½ and 3 hr. In contrast, when the cells were homogenized, rapid loss of activity, with a half-life of 90 min was seen even at 0°C. Consequently for comparative assays of human jejunal adenyl cyclase, strict control of the experimental conditions is required. It was under such conditions that a twofold increase in basal adenyl cyclase activity during cholera was observed. Images PMID:4335441

  14. Inhibition of PRL-3 gene expression in gastric cancer cell line SGC7901 via microRNA suppressed reduces peritoneal metastasis

    SciTech Connect

    Li Zhengrong; Zhan Wenhua . E-mail: wcywk@hotmail.com; Wang Zhao; Zhu Baohe; He Yulong; Peng Junsheng; Cai Shirong; Ma Jinping

    2006-09-15

    High expression of PRL-3, a protein tyrosine phosphatase, is proved to be associated with lymph node metastasis in gastric carcinoma from previous studies. In this paper, we examined the relationship between PRL-3 expression and peritoneal metastasis in gastric carcinoma. We applied the artificial miRNA (pCMV-PRL3miRNA), which is based on the murine miR-155 sequence, to efficiently silence the target gene expression of PRL-3 in SGC7901 gastric cancer cells at both mRNA and protein levels. Then we observed that, in vitro, pCMV-PRL3miRNA significantly depressed the SGC7901 cell invasion and migration independent of cellular proliferation. In vivo, PRL-3 knockdown effectively suppressed the growth of peritoneal metastases and improved the prognosis in nude mice. Therefore, we concluded that artificial miRNA can depress the expression of PRL-3, and that PRL-3 might be a potential therapeutic target for gastric cancer peritoneal metastasis.

  15. Inactivation of Akt by arsenic trioxide induces cell death via mitochondrial-mediated apoptotic signaling in SGC-7901 human gastric cancer cells.

    PubMed

    Gao, Yan-Hui; Zhang, Hao-Peng; Yang, Shu-Meng; Yang, Yue; Ma, Yu-Yan; Zhang, Xin-Yu; Yang, Yan-Mei

    2014-04-01

    Arsenic trioxide (As2O3) has been recognized as a potential chemotherapeutic agent, yet the details concerning its mechanism of action in solid cancers remain undetermined. The present study assessed the role of Akt in the cell death induced by As2O3. The MTT assay showed that As2O3 suppressed the proliferation of SGC-7901 cells in a dose- and time-dependent manner. Characteristic apoptotic changes were observed in the As2O3‑treated cells by Hoechst 33342 staining, and FACS analysis showed that As2O3 caused dose-dependent apoptotic cell death. As2O3 activated caspase-3 and -9, and PARP cleavage in a dose-dependent manner. Compromised mitochondrial membrane potential and an increased protein level of Bax indicated involvement of mitochondia. As2O3 decreased the levels of p-Akt (Ser473), p-Akt (Thr308) and p-GSK-3β (Ser9), suggesting that As2O3 inactivated Akt kinase. In addition, LY294002 (a PI3 kinase inhibitor) augmented the apoptosis induced by As2O3. These results demonstrated that inhibition of PI3K/Akt signaling was involved in As2O3-induced apoptosis of gastric cancer SGC-7901 cells. PMID:24482137

  16. Characterization of sphere‑forming cells with stem‑like properties from the gastric cancer cell lines MKN45 and SGC7901.

    PubMed

    Wang, Xuming; Zou, Feng; Deng, Hao; Fu, Zhengqi; Li, Yan; Wu, Lixia; Wang, Zhaoyi; Liu, Lijiang

    2014-12-01

    Traditionally, it was presumed that gastric cancer was derived from tumor cells with stem‑like properties. In the present study, stem‑like cells from the gastric cancer cell lines MKN45 and SGC7901 were enriched by growing them as spheres in a defined serum‑free medium. Following enrichment for stem‑like cells, cluster of differentiation (CD)24 and CD44 were applied as candidate stem cell markers to examine the expression profile. It was revealed that the sphere‑derived cells contained a higher proportion of cells expressing the stem cell surface markers CD24 and CD44 when compared with the parental cells. It was also identified that the expression of cytokeratin 18 in sphere‑derived cells was decreased and the expression of vimentin and aldehyde dehydrogenase 1 (ALDH1) was increased compared with the parental cells. This finding supports the existence of a population of tumor sphere‑forming cells with stem cell properties in the MKN45 and SGC7901 cell lines. Furthermore, the stem cell population was enriched in cells expressing CD24, CD44, vimentin and ALDH1 cell surface markers. These results support the existence of gastric cancer stem cells and provide an alternative approach to the diagnosis and treatment of gastric cancer. PMID:25270642

  17. Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and prostaglandins

    PubMed Central

    Kimberg, Daniel V.; Field, Michael; Johnson, Judith; Henderson, Antonia; Gershon, Elaine

    1971-01-01

    The effects of several prostaglandins (PG) and a highly purified preparation of cholera enterotoxin (CT) on intestinal mucosal adenyl cyclase activity and the effect of CT on intestinal mucosal cyclic 3′,5′-adenosine monophosphate concentration were determined in guinea pig and rabbit small intestine and were correlated with the effects of the same agents on ion transport. Adenyl cyclase activity, measured in a crude membrane fraction of the mucosa, was found at all levels of the small intestine with the highest activity per milligram protein in the duodenum. The prostaglandins, when added directly to the assay, increased adenyl cyclase activity; the greatest effect (2-fold increase) was obtained with PGE1 (maximal effect at 0.03 mM) and PGE2. The prostaglandins also increased short-circuit current (SCC) in isolated guinea pig ileal mucosa, with PGE1 and PGE2 again giving the greatest effects. The prior addition of theophylline (10 mM) reduced the subsequent SCC response to PGE1 and vice versa. It was concluded, therefore, that the SCC response to PGE1, like the response to theophylline, represented active Cl secretion. CT increased adenyl cyclase activity in guinea pig and rabbit ileal mucosa when preincubated with the mucosa from 1 to 2.5 hr in vitro or for 2.5 hr in vivo but not when added directly to the assay. The increments in activity caused by PGE1 and NaF were the same in CT-treated and control mucosa. Cyclic 3′,5′-AMP concentration in rabbit ileal mucosa was increased 3.5-fold after a 2 hr preincubation with CT in vitro. Phosphodiesterase activity in the crude membrane fraction of the mucosa was unaffected by either CT or PGE1. A variety of other agents including insulin, glucagon, parathormone, thyroid-stimulating hormone, L-thyroxine, thyrocalcitonin, vasopressin, and epinephrine all failed to change adenyl cyclase activity. It is concluded that CT and certain prostaglandins produce small intestinal fluid secretion by increasing mucosal adenyl

  18. Opposing effects of ethanol on pig ovarian adenylyl cyclase desensitized by human choriogonadotropin or isoproterenol.

    PubMed

    Ekstrom, R C; Hunzicker-Dunn, M

    1990-11-01

    Pig ovarian follicular membranes contain a gonadotropin-responsive adenylyl cyclase, which becomes partially desensitized (approximately 40%) upon a 40-min incubation with a saturating concentration of human (h) CG. This in vitro desensitization is time and hormone dependent and also requires the presence of micromolar concentrations of GTP. In this report we show that 10% ethanol present during the desensitization phase of the incubation increases the extent of hCG-induced desensitization of adenylyl cyclase by 2-fold. Ethanol shortened the time necessary to reach maximal hCG-induced desensitization from 20 to 10 min, but had no effect on the dose dependency for GTP. In addition, ethanol had no effect on the affinity of the LH/hCG receptor for 125I-hCG but did cause an increase in the ED50 of hCG for inducing desensitization from 0.25 to 0.75 nM. Interestingly, ethanol decreased the apparent number of LH/hCG-receptor sites by 55%, yet the control hCG-sensitive adenylyl cyclase activity was not reduced. The "hyperdesensitized" state achieved in the presence of ethanol could not be reversed by washing the membranes and incubating them in ethanol-free medium. NaF-sensitive adenylyl cyclase was also not impaired in hCG-desensitized membranes from control or ethanol-treated samples. Thus, hCG-induced desensitization was not due to a defect in the functioning of the stimulatory guanine nucleotide-binding regulatory protein (G8) or catalytic subunits, but rather was caused by an impairment of the coupling of the lutropin (LH)/hCG receptor with G8, which was exacerbated further by ethanol. In spite of the effect of ethanol on hCG-induced desensitization, this agent had an inhibitory effect on isoproterenol-induced desensitization of isoproterenol-responsive luteal adenylyl cyclase. These results indicate that membrane fluidity is important in modulating the structure and functional interaction of the LH/hCG receptor with G8 because ethanol is a well known lipid

  19. Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria.

    PubMed

    Yamanashi, Kaori; Minamizaki, Kei; Fujita, Yuichi

    2015-08-01

    The fifth ring (E-ring) of chlorophyll (Chl) a is produced by Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. There are two evolutionarily unrelated MPE cyclases: oxygen-independent (BchE) and oxygen-dependent (ChlA/AcsF) MPE cyclases. Although ChlA is the sole MPE cyclase in Synechocystis PCC 6803, it is yet unclear whether BchE exists in cyanobacteria. A BLAST search suggests that only few cyanobacteria possess bchE. Here, we report that two bchE candidate genes from Cyanothece strains PCC 7425 and PCC 7822 restore the photosynthetic growth and bacteriochlorophyll production in a bchE-lacking mutant of Rhodobacter capsulatus. We termed these cyanobacterial bchE orthologs "chlE."

  20. Effects of adenylate cyclase toxin from Bordetella pertussis on human neutrophil interactions with Coccidioides immitis and Staphylococcus aureus.

    PubMed Central

    Galgiani, J N; Hewlett, E L; Friedman, R L

    1988-01-01

    Bordetella pertussis extract that contained adenylate cyclase toxin produced large increases in human neutrophil cyclic AMP levels and inhibited their oxidative burst, as reflected by luminol-enhanced chemiluminescence and superoxide release. The adenylate cyclase toxin-containing extract blocked neutrophil-mediated inhibition of N-acetylglucosamine incorporation by arthroconidia of Coccidioides immitis in a dose-dependent fashion but had no effect on neutrophil phagocytosis of Candida glabrata and only a slight inhibitory effect on arthroconidial attachment. Neither purified pertussis toxin nor extracts from Bordetella mutants lacking the adenylate cyclase toxin affected neutrophil-mediated inhibition of arthroconidial N-acetylglucosamine incorporation. These studies indicate that adenylate cyclase toxin, alone or in concert with other B. pertussis-elaborated toxins, blocks neutrophil inhibition of arthroconidia, primarily by affecting neutrophil responses other than attachment or phagocytosis. PMID:2894360

  1. S-benzyl-cysteine-mediated cell cycle arrest and apoptosis involving activation of mitochondrial-dependent caspase cascade through the p53 pathway in human gastric cancer SGC-7901 cells.

    PubMed

    Sun, Hua-Jun; Meng, Lin-Yi; Shen, Yang; Zhu, Yi-Zhun; Liu, Hong-Rui

    2013-01-01

    S-benzyl-cysteine (SBC) is a structural analog of S-allylcysteine (SAC), which is one of the major water- soluble compounds in aged garlic extract. In this study, anticancer activities and the underlying mechanisms of SBC action were investigated and compared these with those of SAC using human gastric cancer SGC-7901 cells. SBC significantly suppressed the survival rate of SGC-7901 cells in a concentration- and time-dependent manner, and the inhibitory activities of SBC were stronger than those of SAC. Flow cytometry revealed that SBC induced G2-phase arrest and apoptosis in SGC-7901 cells. Typical apoptotic morphological changes were observed by Hoechst 33258 dye assay. SBC-treatment dramatically induced the dissipation of mitochondrial membrane potential (Δψm), and enhanced the enzymatic activities of caspase-9 and caspase-3 whilst hardly affecting caspase-8 activity. Furthermore, Western blotting indicated that SBC-induced apoptosis was accompanied by up-regulation of the expression of p53, Bax and the down-regulation of Bcl-2. Taken together, this study suggested that SBC exerts cytotoxic activity involving activation of mitochondrial-dependent apoptosis through p53 and Bax/Bcl-2 pathways in human gastric cancer SGC-7901 cells. PMID:24377536

  2. Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes

    SciTech Connect

    Stadel, J.M.; Rebar, R.; Crooke, S.T.

    1987-05-01

    Desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes results in 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of ..beta..-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoproterenol- and cAMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37/sup 0/C, pH = 8.0. In both cases alkaline phosphatase treatment significantly reduced desensitization of agonist-stimulated adenylate cyclase activity by 40-60%. Similar results were obtained following alkaline phosphatase treatment of membranes from isoproterenol- and cAMP-desensitized duck erythrocytes. In addition, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with phorbol 12-mystrate 13-acetate returned adenylate cyclase activity to near control values. In all experiments inclusion of 20 mM NaPO/sub 4/ to inhibit alkaline phosphatase during treatment of membranes blocked the enzyme's effect on agonist-stimulated adenylate cyclase activity. These results demonstrate a role for phosphorylation in desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes.

  3. Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.

    PubMed

    Green, J P; Johnson, C L; Weinstein, H; Maayani, S

    1977-12-01

    D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide.

  4. Overexpression of the Type 1 Adenylyl Cyclase in the Forebrain Leads to Deficits of Behavioral Inhibition

    PubMed Central

    Cao, Hong; Saraf, Amit; Zweifel, Larry S.

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition. PMID:25568126

  5. Hypothesis: glutaminyl cyclase inhibitors decrease risks of Alzheimer's disease and related dementias.

    PubMed

    Hennekens, Charles H; Bensadon, Benjamin A; Zivin, Robert; Gaziano, J Michael

    2015-01-01

    Alzheimer's disease and related dementias (ADRD) comprise several progressive and incurable neurodegenerative disorders that some have classified as amyloidosis. With increased aging of the world's population, the prevalence of the sporadic form of ADRD, which comprises over 99% of cases, continues to rise at an alarming rate. The enormous societal burdens of ADRD already rival those of the many other major chronic diseases causing premature morbidity and mortality in the USA and worldwide such as cardiovascular disease and cancer. At present, there is an insufficient totality of evidence concerning the efficacy and safety of any pharmacologic agents to delay slow progression or reduce complications of ADRD. In this context, glutaminyl cyclase (QC) inhibitors have shown some early possible evidence of efficacy with a reassuring safety profile. To reliably test the glutaminyl cyclase (QC) and any other promising hypotheses will require cogent data from large-scale randomized trials of sufficient size and duration. PMID:26450764

  6. Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

    PubMed

    Burkhardt, Immo; Siemon, Thomas; Henrot, Matthias; Studt, Lena; Rösler, Sarah; Tudzynski, Bettina; Christmann, Mathias; Dickschat, Jeroen S

    2016-07-18

    Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers.

  7. Mechanistic Characterisation of Two Sesquiterpene Cyclases from the Plant Pathogenic Fungus Fusarium fujikuroi.

    PubMed

    Burkhardt, Immo; Siemon, Thomas; Henrot, Matthias; Studt, Lena; Rösler, Sarah; Tudzynski, Bettina; Christmann, Mathias; Dickschat, Jeroen S

    2016-07-18

    Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers. PMID:27294564

  8. Overexpression of the type 1 adenylyl cyclase in the forebrain leads to deficits of behavioral inhibition.

    PubMed

    Chen, Xuanmao; Cao, Hong; Saraf, Amit; Zweifel, Larry S; Storm, Daniel R

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition.

  9. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

    PubMed Central

    Eggerickx, D; Denef, J F; Labbe, O; Hayashi, Y; Refetoff, S; Vassart, G; Parmentier, M; Libert, F

    1995-01-01

    A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed. Images Figure 4 Figure 5 PMID:7639700

  10. Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation

    PubMed Central

    Peshenko, Igor V.; Olshevskaya, Elena V.; Dizhoor, Alexander M.

    2012-01-01

    Guanylyl cyclase activating proteins (GCAPs) are calcium/magnesium binding proteins within neuronal calcium sensor proteins group (NCS) of the EF-hand proteins superfamily. GCAPs activate retinal guanylyl cyclase (RetGC) in vertebrate photoreceptors in response to light-dependent fall of the intracellular free Ca2+ concentrations. GCAPs consist of four EF-hand domains and contain N-terminal fatty acylated glycine, which in GCAP1 is required for the normal activation of RetGC. We analyzed the effects of a substitution prohibiting N-myristoylation (Gly2 → Ala) on the ability of the recombinant GCAP1 to co-localize with its target enzyme when heterologously expressed in HEK293 cells. We also compared Ca2+ binding and RetGC-activating properties of the purified non-acylated G2A mutant and C14:0 acylated GCAP1 in vitro. The G2A GCAP1 expressed with a C-terminal GFP tag was able to co-localize with the cyclase, albeit less efficiently than the wild type, but much less effectively stimulated cyclase activity in vitro. Ca2+ binding isotherm of the G2A GCAP1 was slightly shifted toward higher free Ca2+ concentrations and so was Ca2+ sensitivity of RetGC reconstituted with the G2A mutant. At the same time, myristoylation had little effect on the high-affinity Ca2+-binding in the EF-hand proximal to the myristoyl residue in three-dimensional GCAP1 structure. These data indicate that the N-terminal fatty acyl group may alter the activity of EF-hands in the distal portion of the GCAP1 molecule via presently unknown intramolecular mechanism. PMID:22371697

  11. Non-co-ordinate development of beta-adrenergic receptors and adenylate cyclase in chick heart.

    PubMed Central

    Alexander, R W; Galper, J B; Neer, E J; Smith, T W

    1982-01-01

    We have studied the properties of beta-adrenergic receptors and of their interaction with adenylate cyclase in the chick myocardium during embryogenesis. Between 4.5 and 7.5 days in ovo the number of receptors determined by (-)-[3H]dihydroalprenolol ([3H]DHA) binding is constant at approx. 0.36 pmol of receptor/mg of protein. By day 9 the density decreases significantly to 0.22 pmol of receptor/mg of protein. At day 12.5--13.5 the number was 0.14--0.18 pmol of receptor/mg of protein. This number did not change further up to day 16. The same results were obtained with guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) added to the assay mixtures. There was no significant change in receptor affinity for the antagonist [3H]DHA between days 5.5 and 13. Despite the decrease in numbers of beta-adrenergic receptors, there was no change in basal, p[NH]ppG-, isoprenaline- or isoprenaline-plus-p[NH]ppG-stimulated adenylate cyclase activity between days 3 and 12 of development. We conclude that beta-adrenergic receptors and adenylate cyclase are not co-ordinately regulated during early embryonic development of the chick heart. Some of the beta-adrenergic receptors present very early in the ontogeny of cardiac tissue appear not to be coupled to adenylate cyclase since their loss is not reflected in decreased activation of the enzyme. PMID:6289805

  12. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  13. Elevation of lutein content in tomato: a biochemical tug-of-war between lycopene cyclases.

    PubMed

    Giorio, Giovanni; Yildirim, Arzu; Stigliani, Adriana Lucia; D'Ambrosio, Caterina

    2013-11-01

    Lutein is becoming increasingly important in preventive medicine due to its possible role in maintaining good vision and in preventing age-related maculopathy. Average daily lutein intake in developed countries is often below suggested daily consumption levels, and lutein supplementation could be beneficial. Lutein is also valuable in the food and feed industries and is emerging in nutraceutical and pharmaceutical markets. Currently, lutein is obtained at high cost from marigold petals, and synthesis alternatives are thus desirable. Tomato constitutes a promising starting system for production as it naturally accumulates high levels of lycopene. To develop tomato for lutein synthesis, the tomato Red Setter cultivar was transformed with the tomato lycopene ε-cyclase-encoding gene under the control of a constitutive promoter, and the HighDelta (HD) line, characterised by elevated lutein and δ-carotene content in ripe fruits, was selected. HD was crossed to the transgenic HC line and to RS(B) with the aim of converting all residual fruit δ-carotene to lutein. Fruits of both crosses were enriched in lutein and presented unusual carotenoid profiles. The unique genetic background of the crosses used in this study permitted an unprecedented analysis of the role and regulation of the lycopene cyclase enzymes in tomato. A new defined biochemical index, the relative cyclase activity ratio, was used to discern post-transcriptional regulation of cyclases, and will help in the study of carotenoid biosynthesis in photosynthetic plant species and particularly in those, like tomato, that have been domesticated for the production of food, feed or useful by-products.

  14. Control of the Diadenylate Cyclase CdaS in Bacillus subtilis

    PubMed Central

    Mehne, Felix M. P.; Schröder-Tittmann, Kathrin; Eijlander, Robyn T.; Herzberg, Christina; Hewitt, Lorraine; Kaever, Volkhard; Lewis, Richard J.; Kuipers, Oscar P.; Tittmann, Kai; Stülke, Jörg

    2014-01-01

    The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination. PMID:24939848

  15. The sGC activator inhibits the proliferation and migration, promotes the apoptosis of human pulmonary arterial smooth muscle cells via the up regulation of plasminogen activator inhibitor-2

    SciTech Connect

    Zhang, Shuai; Zou, Lihui; Yang, Ting; Yang, Yuanhua; Zhai, Zhenguo; Xiao, Fei; Wang, Chen

    2015-03-15

    Background: Different types of pulmonary hypertension (PH) share the same process of pulmonary vascular remodeling, the molecular mechanism of which is not entirely clarified by far. The abnormal biological behaviors of pulmonary arterial smooth muscle cells (PASMCs) play an important role in this process. Objectives: We investigated the regulation of plasminogen activator inhibitor-2 (PAI-2) by the sGC activator, and explored the effect of PAI-2 on PASMCs proliferation, apoptosis and migration. Methods: After the transfection with PAI-2 overexpression vector and specific siRNAs or treatment with BAY 41-2272 (an activator of sGC), the mRNA and protein levels of PAI-2 in cultured human PASMCs were detected, and the proliferation, apoptosis and migration of PASMCs were investigated. Results: BAY 41-2272 up regulated the endogenous PAI-2 in PASMCs, on the mRNA and protein level. In PAI-2 overexpression group, the proliferation and migration of PASMCs were inhibited significantly, and the apoptosis of PASMCs was increased. In contrast, PAI-2 knockdown with siRNA increased PASMCs proliferation and migration, inhibited the apoptosis. Conclusions: PAI-2 overexpression inhibits the proliferation and migration and promotes the apoptosis of human PASMCs. Therefore, sGC activator might alleviate or reverse vascular remodeling in PH through the up-regulation of PAI-2. - Highlights: • sGC activator BAY41-2272 up regulated PAI-2 in PASMCs, on the mRNA and protein level. • PAI-2 overexpression inhibits the proliferation and migration of human PASMCs. • PAI-2 overexpression promotes the apoptosis of human PASMCs. • sGC activator might alleviate the vascular remodeling in pulmonary hypertension.

  16. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    PubMed

    Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

    2013-01-01

    Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light. PMID:24201148

  17. Evidence for an essential histidine residue in 4S-limonene synthase and other terpene cyclases.

    PubMed

    Rajaonarivony, J I; Gershenzon, J; Miyazaki, J; Croteau, R

    1992-11-15

    (4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha x piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidine-directed reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn2+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn2+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes. PMID:1444454

  18. Dcsbis (PA2771) from Pseudomonas aeruginosa is a highly active diguanylate cyclase with unique activity regulation

    PubMed Central

    Chen, Ying; Liu, Shiheng; Liu, Cuilan; Huang, Yan; Chi, Kaikai; Su, Tiantian; Zhu, Deyu; Peng, Jin; Xia, Zhijie; He, Jing; Xu, Sujuan; Hu, Wei; Gu, Lichuan

    2016-01-01

    C-di-GMP (3’,5’ -Cyclic diguanylic acid) is an important second messenger in bacteria that influences virulence, motility, biofilm formation, and cell division. The level of c-di-GMP in cells is controlled by diguanyl cyclases (DGCs) and phosphodiesterases (PDEs). Here, we report the biochemical functions and crystal structure of the potential diguanylase Dcsbis (PA2771, a diguanylate cyclase with a self-blocked I-site) from Pseudomonas aeruginosa PAO1. The full-length Dcsbis protein contains an N-terminal GAF domain and a C-terminal GGDEF domain. We showed that Dcsbis tightly coordinates cell motility without markedly affecting biofilm formation and is a diguanylate cyclase with a catalytic activity much higher than those of many other DGCs. Unexpectedly, we found that a peptide loop (protecting loop) extending from the GAF domain occupies the conserved inhibition site, thereby largely relieving the product-inhibition effect. A large hydrophobic pocket was observed in the GAF domain, thus suggesting that an unknown upstream signaling molecule may bind to the GAF domain, moving the protecting loop from the I-site and thereby turning off the enzymatic activity. PMID:27388857

  19. The first structure of a bacterial diterpene cyclase: CotB2.

    PubMed

    Janke, Ronja; Görner, Christian; Hirte, Max; Brück, Thomas; Loll, Bernhard

    2014-06-01

    Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacterium Streptomyces melanosporofaciens, at 1.64 Å resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclooctatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2(W288G) produced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistant Staphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound.

  20. Evolutionary Divergence of Sedoheptulose 7-phosphate Cyclases Leads to Several Distinct Cyclic Products

    PubMed Central

    Asamizu, Shumpei; Xie, Pengfei; Brumsted, Corey J.; Flatt, Patricia M.; Mahmud, Taifo

    2012-01-01

    Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway, and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3–1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules. PMID:22741921

  1. HAMP domain-mediated signal transduction probed with a mycobacterial adenylyl cyclase as a reporter.

    PubMed

    Mondéjar, Laura García; Lupas, Andrei; Schultz, Anita; Schultz, Joachim E

    2012-01-01

    HAMP domains, ∼55 amino acid motifs first identified in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases, operate as signal mediators in two-component signal transduction proteins. A bioinformatics study identified a coevolving signal-accepting network of 10 amino acids in membrane-delimited HAMP proteins. To probe the functionality of this network we used a HAMP containing mycobacterial adenylyl cyclase, Rv3645, as a reporter enzyme in which the membrane anchor was substituted by the Escherichia coli chemotaxis receptor for serine (Tsr receptor) and the HAMP domain alternately with that from the protein Af1503 of the archaeon Archaeoglobus fulgidus or the Tsr receptor. In a construct with the Tsr-HAMP, cyclase activity was inhibited by serine, whereas in a construct with the HAMP domain from A. fulgidus, enzyme activity was not responsive to serine. Amino acids of the signal-accepting network were mutually swapped between both HAMP domains, and serine signaling was examined. The data biochemically tentatively established the functionality of the signal-accepting network. Based on a two-state gearbox model of rotation in HAMP domain-mediated signal propagation, we characterized the interaction between permanent and transient core residues in a coiled coil HAMP structure. The data are compatible with HAMP rotation in signal propagation but do not exclude alternative models for HAMP signaling. Finally, we present data indicating that the connector, which links the α-helices of HAMP domains, plays an important structural role in HAMP function.

  2. Cyanobacteriochrome SesA Is a Diguanylate Cyclase That Induces Cell Aggregation in Thermosynechococcus*♦

    PubMed Central

    Enomoto, Gen; Nomura, Ryouhei; Shimada, Takashi; Ni-Ni-Win; Narikawa, Rei; Ikeuchi, Masahiko

    2014-01-01

    Cyanobacteria have unique photoreceptors, cyanobacteriochromes, that show diverse spectral properties to sense near-UV/visible lights. Certain cyanobacteriochromes have been shown to regulate cellular phototaxis or chromatic acclimation of photosynthetic pigments. Some cyanobacteriochromes have output domains involved in bacterial signaling using a second messenger cyclic dimeric GMP (c-di-GMP), but its role in cyanobacteria remains elusive. Here, we characterize the recombinant Tlr0924 from a thermophilic cyanobacterium Thermosynechococcus elongatus, which was expressed in a cyanobacterial system. The protein reversibly photoconverts between blue- and green-absorbing forms, which is consistent with the protein prepared from Escherichia coli, and has diguanylate cyclase activity, which is enhanced 38-fold by blue light compared with green light. Therefore, Tlr0924 is a blue light-activated diguanylate cyclase. The protein's relatively low affinity (10.5 mm) for Mg2+, which is essential for diguanylate cyclase activity, suggests that Mg2+ might also regulate c-di-GMP signaling. Finally, we show that blue light irradiation under low temperature is responsible for Thermosynechococcus vulcanus cell aggregation, which is abolished when tlr0924 is disrupted, suggesting that Tlr0924 mediates blue light-induced cell aggregation by producing c-di-GMP. Given our results, we propose the name “sesA (sessility-A)” for tlr0924. This is the first report for cyanobacteriochrome-dependent regulation of a sessile/planktonic lifestyle in cyanobacteria via c-di-GMP. PMID:25059661

  3. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F. )

    1990-12-01

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.

  4. Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

    SciTech Connect

    Azarkan, Mohamed; Clantin, Bernard; Bompard, Coralie; Belrhali, Hassan; Baeyens-Volant, Danielle; Looze, Yvan; Wintjens, René

    2005-01-01

    The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution. In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å.

  5. Two members of a widely expressed subfamily of hormone-stimulated adenylyl cyclases.

    PubMed Central

    Premont, R T; Chen, J; Ma, H W; Ponnapalli, M; Iyengar, R

    1992-01-01

    cDNA encoding a hormone- and guanine nucleotide-stimulated adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (type 6) from rat liver and kidney has been cloned and expressed. This enzyme is stimulated by forskolin, guanosine 5'-[gamma-thio]triphosphate, and isoproterenol plus GTP but is not stimulated by beta gamma subunits of guanine nucleotide-binding proteins. A second form (type 5), which is 75% similar to type 6, has also been cloned. Both types 5 and 6 cDNAs have multiple messages. PCR-based detection of the mRNA for the type 5 and 6 enzymes indicates that both are widely distributed. Homology analyses indicate at least four distinct subfamilies of guanine nucleotide stimulatory protein-regulated adenylyl cyclases. Types 5 and 6 enzymes define one distinct subfamily of mammalian adenylyl cyclases. Diversity of one guanine nucleotide-binding protein-regulated effector may allow different modes of regulation of cell-surface signal transmission. Images PMID:1409703

  6. H2S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway.

    PubMed

    Li, Sen; Ping, Na-Na; Cao, Lei; Mi, Yan-Ni; Cao, Yong-Xiao

    2015-12-15

    Hydrogen sulfide (H2S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H2S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H2S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca(2+) channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H2S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway.

  7. The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin

    PubMed Central

    Ravichandran, Akshaya; Gu, Ganyu; Escano, Jerome; Lu, Shi-En; Smith, Leif

    2014-01-01

    Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and β-hydroxyasparagine variants coordinate the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides. PMID:23394257

  8. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    PubMed

    Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

    2013-01-01

    Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

  9. Persistent stimulation of adenylate cyclase and urea transport by an AVP photolabel

    SciTech Connect

    Eggena, P.; Ma, C.L.; Fahrenholz, F.; Schwartz, I.L.

    1985-07-01

    The effects of a photoaffinity label for arginine vasopressin receptors, (Phe2, Phe(p-N3)3)AVP (N3-AVP), on urea permeability and adenylate cyclase activity have been investigated in the toad urinary bladder. This compound, when activated by ultraviolet light, induced a maximal and persistent increase in the urea permeability of the intact bladder and a persistent increase in the adenylate cyclase activity of toad bladder epithelial cell homogenates. Covalent attachment of the analogue to target tissue during photolysis was equivalent at 4 and 20 degrees C. Bladders exposed to N3-AVP in the presence of AVP during photolysis were substantially less permeable to urea than controls that had been exposed to N3-AVP alone. These findings constitute further evidence in support of the previous suggestion that N3-AVP binds covalently to AVP receptors and, in addition, demonstrates that N3-AVP evokes a persistent increase in adenylate cyclase activity which, in turn, triggers a persistent increase in bladder permeability to urea.

  10. Central role of soluble adenylyl cyclase and cAMP in sperm physiology

    PubMed Central

    Buffone, Mariano G.; Wertheimer, Eva V.; Visconti, Pablo E.; Krapf, Dario

    2014-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cylases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. PMID:25066614

  11. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  12. Guanine-nucleotide-dependent inhibition of adenylate cyclase of rabbit heart by glucagon.

    PubMed

    Kiss, Z; Tkachuk, V A

    1984-07-16

    The present study demonstrates an inhibitory effect of glucagon on the adenylate cyclase system of rabbit heart. Inhibition was maximal (22-40%) at 0.1-0.01 microM glucagon and required the presence of 0.01-0.1 mM GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GuoPP[NH]P). Reduced or no inhibitor effect of glucagon was observed: (a) after limited proteolysis of plasma membrane proteins by trypsin, (b) in the presence of 1 mM Mn2+, (c) in the absence of Na+, and (d) during the first 10 min of incubation if GuoPP[NH]P was the activating ligand. With GTP as the activating ligand, inhibition of cyclase by glucagon occurred without delay. These data are consistent with a mediation of glucagon inhibition by a guanine-nucleotide-binding protein. In the presence of ethanol (0.2 M) or benzyl alcohol (0.05 M), agents which are known to increase the fluidity of biological membranes, glucagon increased the enzyme activity in a guanine-nucleotide-dependent manner. Activation of cyclase in the presence of alcohols was maximal (30-60%) at 0.1-1.0 microM glucagon and 0.01 mM guanine nucleotides. Data suggest that glucagon receptors can interact with both the activatory and inhibitory guanine-nucleotide-binding proteins and the physical state of membranes may play a role in determining which interaction will be preferential.

  13. Developmental changes in ANP-stimulated guanylyl cyclase activity enhanced by ATP in rat lung membrane fractions.

    PubMed Central

    Charoonroje, P; Tokumitsu, Y; Nomura, Y

    1994-01-01

    1. ANP (atrial natriuretic peptides)- or ANP/ATP-stimulated guanylyl cyclase activities were compared in adult (2 month old) and neonatal (5-7 day old) rat lung membrane fractions. 2. The enzyme activities of both membranes depended on the incubation time and ATP concentration: although the activities of both membranes were similar after a short incubation time (4 min), those in adult membranes were lower than those of neonatal membranes after longer incubation times (10 and 30 min) or at lower concentrations of ATP. 3. ANP/ATP gamma S-stimulated guanylyl cyclase activities, which were much higher than ANP/ATP-stimulated activities, were similar in both membranes. 4. ATPase activity of adult membranes was higher than that of neonatal membranes, suggesting that hydrolysis of ATP leads to a decrease of ANP/ATP-guanylyl cyclase activity in adult membranes. Triton X-100 enhanced and diminished ANP/ATP-stimulated guanylyl cyclase activities of adult and neonatal membranes, respectively, and thereby abolished the adult/neonatal difference in the membrane response to ATP. 5. ANP-stimulated activities of both membranes were much more activated by pre-incubation with ATP gamma S than those induced by simultaneous addition of ATP gamma S. The former activities were decreased to levels of the latter by Triton X-100. The latter activities were not affected by Triton X-100. 6. The present results suggested that conformation of lung plasma membranes is related to activation of the ANP receptor/guanylyl cyclase system. PMID:7834209

  14. Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

    PubMed Central

    Li, Lian-Feng; Yu, Jiahui; Li, Yongfeng; Wang, Jinghan; Li, Su; Zhang, Lingkai; Xia, Shui-Li; Yang, Qian; Wang, Xiao; Yu, Shaoxiong; Luo, Yuzi; Sun, Yuan; Zhu, Yan; Munir, Muhammad

    2016-01-01

    ABSTRACT Many viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity. IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV

  15. Riociguat: first global approval.

    PubMed

    Conole, Daniel; Scott, Lesley J

    2013-11-01

    Riociguat (Adempas(®)), an oral first-in-class soluble guanylate cyclase (sGC) stimulator, is under global development by Bayer Healthcare Pharmaceuticals Inc. for the treatment of adult patients with inoperable or chronic/persistent chronic thromboembolic pulmonary hypertension (CTEPH) and for the treatment of adult patients with pulmonary arterial hypertension (PAH). The drug directly stimulates sGC in a nitric oxide independent manner, thereby increasing the sensitivity of sGC to nitric oxide, leading to increased cyclic guanosine monophosphate generation (a key signalling molecule involved in regulating vascular tone, proliferation, fibrosis and inflammation). Riociguat is the world's first approved pharmacotherapy for CTEPH, with its first global approval in this indication occurring in Canada. It has subsequently been approved in the USA for the treatment of patients with CTEPH and also received its first global approval in patients with PAH in the USA. It is undergoing regulatory review for these indications in Europe and for use in patients with CTEPH in Japan. This article summarizes the milestones in the development of riociguat, leading to its first global approvals in patients with CTEPH and PAH.

  16. The cyclic GMP/protein kinase G pathway as a therapeutic target in head and neck squamous cell carcinoma.

    PubMed

    Tuttle, Traci R; Mierzwa, Michelle L; Wells, Susanne I; Fox, Sejal R; Ben-Jonathan, Nira

    2016-01-28

    Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease with high mortality. Treatments, which can result in significant morbidity, have not substantially changed in three decades. The second messenger cyclic GMP (cGMP), which targets protein kinase G (PKG), is generated by guanylate cyclases (GCs), and is rapidly hydrolyzed by phosphodiesterases (PDEs). Activation of the cGMP/PKG pathway is antineoplastic in several cancer types, but its impact on HNSCC has not been fully exploited. We found differential expression of critical components of this pathway in four HNSCC cell lines. Several activators of soluble GC (sGC), as well as inhibitors of PDE5, increased intracellular cGMP, reduced cell viability, and induced apoptosis in HNSCC cells. The apoptotic effects of the sGC activator BAY 41-2272 and the PDE5 inhibitor Tadalafil (Cialis) were mediated by PKG. Furthermore, Tadalafil substantially reduced the growth of CAL27-derived tumors in athymic mice. Several drugs which either activate sGC or inhibit PDE5 are approved for treatment of nonmalignant conditions. These drugs could be repurposed as novel and effective therapeutics in patients with head and neck cancer.

  17. Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells.

    PubMed

    Abraham, Nader G; Quan, Shuo; Mieyal, Paul A; Yang, Liming; Burke-Wolin, Theresa; Mingone, Christopher J; Goodman, Alvin I; Nasjletti, Alberto; Wolin, Michael S

    2002-11-01

    Carbon monoxide (CO) stimulates guanylate cyclase (GC) and increases guanosine 3',5'-cyclic monophosphate (cGMP) levels. We transfected rat-lung pulmonary endothelial cells with a retrovirus-mediated human heme oxygenase (hHO)-1 gene. Pulmonary cells that expressed hHO-1 exhibited a fourfold increase in HO activity associated with decreases in the steady-state levels of heme and cGMP without changes in soluble GC (sGC) and endothelial nitric oxide synthase (NOS) proteins or basal nitrite production. Heme elicited significant increases in CO production and intracellular cGMP levels in both pulmonary endothelial and pulmonary hHO-1-expressing cells. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly decreased cGMP levels in heme-treated pulmonary endothelial cells but not heme-treated hHO-1-expressing cells. In the presence of exogenous heme, CO and cGMP levels in hHO-1-expressing cells exceeded the corresponding levels in pulmonary endothelial cells. Acute exposure of endothelial cells to SnCl2, which is an inducer of HO-1, increased cGMP levels, whereas chronic exposure decreased heme and cGMP levels. These results indicate that prolonged overexpression of HO-1 ultimately decreases sGC activity by limiting the availability of cellular heme. Heme activates sGC and enhances cGMP levels via a mechanism that is largely insensitive to NOS inhibition.

  18. Investigation of the role of the NO-cGMP pathway on YC-1 and DEA/NO effects on thoracic aorta smooth muscle responses in a rat preeclampsia model.

    PubMed

    Turgut, Nergiz Hacer; Temiz, Tijen Kaya; Turgut, Bülent; Karadas, Baris; Parlak, Mesut; Bagcivan, Ihsan

    2013-10-01

    The present study was designed to investigate the effects of YC-1, a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO, a NO donor, on smooth muscle responses in the preeclampsia model with suramin-treated rats and on the levels of cyclic guanosine monophosphate (cGMP) of thoracic aorta rings isolated from term-pregnant rats. Rats of 2 groups, control group and suramin group, were given intraperitoneal injection of saline or suramin, respectively. Suramin injection caused increased blood pressure, protein in urine, and fetal growth retardation. Thoracic aorta rings were exposed to contractile and relaxant agents. KCl contraction and papaverine relaxation responses were similar. Relaxation responses of YC-1 and DEA/NO decreased in suramin group. In both groups in the presence of ODQ, a sGC inhibitor, the relaxation responses of YC-1 and DEA/NO decreased. The cGMP content was determined by radioimmunoassay technique. The content of cGMP in the suramin group decreased. In the presence of YC-1 and DEA/NO in both groups, cGMP content increased, but in ODQ-added groups, there was a significant decrease. We conclude that in preeclampsia, the decrease of relaxation responses and the decrease of cGMP content could be due to the reduction in stimulation of sGC and the decrease in cGMP levels.

  19. [Octarphin--Nonopioid Peptide of the Opioid Origin].

    PubMed

    Navolotskaya, E V

    2015-01-01

    The data on the properties and mechanism of action of the peptide octarphin (TPLVTLFK, the fragment 12-19 of β-endorphin)--a selective agonist of nonopioid (insensitive to the action of the opioid antagonist naloxone) β-endorphin receptor found on n immune cells (peritoneal macrophages, T and B lymphocytes of spleen and blood), endocrine (adrenal cortex, hypothalamus), cardiovascular (cardiomyocytes) systems are analyzed and systematized. Binding to the receptor octarphin increases increases the mitogen-induced pro- liferation of human and mouse T and B lymphocytes in vitro, activates murine peritoneal macrophages in vitro and in vivo, stimulates growth of human T-lymphoblast cell lines Jurkat and MT-4, inhibits adenylate cyclase activity of rat adrenal cortex membranes and suppresses the secretion of glucocorticoids from the adrenal gland into the blood. It was shown that in a concentration range of 1-1000 nM the peptide increases the activity of inducible NO-synthase (iNOS), and the content of NO and cGMP in lipopolysaccharide-activated murine peritoneal macrophages. Taking into account that NO acts as a primary activator of soluble guanylate cyclase (sGC), it can be assumed that the activating effect of octarphin on macrophages is realized in the following way: increase in th iNOS expression --> increase in the NO production --> increase in the sGC activity --> increase in intracellular levels of cGMP. PMID:26762089

  20. Intracellular role of adenylyl cyclase in regulation of lateral pseudopod formation during Dictyostelium chemotaxis.

    PubMed

    Stepanovic, Vesna; Wessels, Deborah; Daniels, Karla; Loomis, William F; Soll, David R

    2005-04-01

    Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA- cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA- cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA- cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.

  1. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    PubMed

    Andre, Christelle M; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; Cooney, Janine M; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; Laing, William A

    2016-09-01

    Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis. PMID:27214242

  2. Structure of a Sedoheptulose 7-Phosphate Cyclase: ValA from Streptomyces hygroscopicus

    PubMed Central

    2015-01-01

    Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp → Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis but is accomplished by selective binding of either the α or β pyranose anomer of the substrate. PMID:24832673

  3. Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes

    PubMed Central

    Lopez, Miguel A.; Saada, Edwin A.

    2014-01-01

    Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed “social motility,” based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies. PMID:25416239

  4. Ancient conserved domains shared by animal soluble guanylyl cyclases and bacterial signaling proteins

    PubMed Central

    Iyer, Lakshminarayan M; Anantharaman, Vivek; Aravind, L

    2003-01-01

    Background Soluble guanylyl cyclases (SGCs) are dimeric enzymes that transduce signals downstream of nitric oxide (NO) in animals. They sense NO by means of a heme moiety that is bound to their N-terminal extensions. Results Using sequence profile searches we show that the N-terminal extensions of the SGCs contain two globular domains. The first of these, the HNOB (Heme NO Binding) domain, is a predominantly α-helical domain and binds heme via a covalent linkage to histidine. Versions lacking this conserved histidine and are likely to interact with heme non-covalently. We detected HNOB domains in several bacterial lineages, where they occur fused to methyl accepting domains of chemotaxis receptors or as standalone proteins. The standalone forms are encoded by predicted operons that also contain genes for two component signaling systems and GGDEF-type nucleotide cyclases. The second domain, the HNOB associated (HNOBA) domain occurs between the HNOB and the cyclase domains in the animal SGCs. The HNOBA domain is also detected in bacteria and is always encoded by a gene, which occurs in the neighborhood of a gene for a HNOB domain. Conclusion The HNOB domain is predicted to function as a heme-dependent sensor for gaseous ligands, and transduce diverse downstream signals, in both bacteria and animals. The HNOBA domain functionally interacts with the HNOB domain, and possibly binds a ligand, either in cooperation, or independently of the latter domain. Phyletic profiles and phylogenetic analysis suggest that the HNOB and HNOBA domains were acquired by the animal lineage via lateral transfer from a bacterial source. PMID:12590654

  5. Structure of a sedoheptulose 7-phosphate cyclase: ValA from Streptomyces hygroscopicus.

    PubMed

    Kean, Kelsey M; Codding, Sara J; Asamizu, Shumpei; Mahmud, Taifo; Karplus, P Andrew

    2014-07-01

    Sedoheptulose 7-phosphate cyclases (SH7PCs) encompass three enzymes involved in producing the core cyclitol structures of pseudoglycosides and similar bioactive natural products. One such enzyme is ValA from Streptomyces hygroscopicus subsp. jinggangensis 5008, which makes 2-epi-5-epi-valiolone as part of the biosynthesis of the agricultural antifungal agent validamycin A. We present, as the first SH7PC structure, the 2.1 Å resolution crystal structure of ValA in complex with NAD+ and Zn2+ cofactors. ValA has a fold and active site organization resembling those of the sugar phosphate cyclase dehydroquinate synthase (DHQS) and contains two notable, previously unrecognized interactions between NAD+ and Asp side chains conserved in all sugar phosphate cyclases that may influence catalysis. Because the domains of ValA adopt a nearly closed conformation even though no sugar substrate is present, comparisons with a ligand-bound DHQS provide a model for aspects of substrate binding. One striking active site difference is a loop that adopts a distinct conformation as a result of an Asp→Asn change with respect to DHQS and alters the identity and orientation of a key Arg residue. This and other active site differences in ValA are mostly localized to areas where the ValA substrate differs from that of DHQS. Sequence comparisons with a second SH7PC making a product with distinct stereochemistry lead us to postulate that the product stereochemistry of a given SH7PC is not the result of events taking place during catalysis but is accomplished by selective binding of either the α or β pyranose anomer of the substrate.

  6. Insect stage-specific adenylate cyclases regulate social motility in African trypanosomes.

    PubMed

    Lopez, Miguel A; Saada, Edwin A; Hill, Kent L

    2015-01-01

    Sophisticated systems for cell-cell communication enable unicellular microbes to act as multicellular entities capable of group-level behaviors that are not evident in individuals. These group behaviors influence microbe physiology, and the underlying signaling pathways are considered potential drug targets in microbial pathogens. Trypanosoma brucei is a protozoan parasite that causes substantial human suffering and economic hardship in some of the most impoverished regions of the world. T. brucei lives on host tissue surfaces during transmission through its tsetse fly vector, and cultivation on surfaces causes the parasites to assemble into multicellular communities in which individual cells coordinate their movements in response to external signals. This behavior is termed "social motility," based on its similarities with surface-induced social motility in bacteria, and it demonstrates that trypanosomes are capable of group-level behavior. Mechanisms governing T. brucei social motility are unknown. Here we report that a subset of receptor-type adenylate cyclases (ACs) in the trypanosome flagellum regulate social motility. RNA interference-mediated knockdown of adenylate cyclase 6 (AC6), or dual knockdown of AC1 and AC2, causes a hypersocial phenotype but has no discernible effect on individual cells in suspension culture. Mutation of the AC6 catalytic domain phenocopies AC6 knockdown, demonstrating that loss of adenylate cyclase activity is responsible for the phenotype. Notably, knockdown of other ACs did not affect social motility, indicating segregation of AC functions. These studies reveal interesting parallels in systems that control social behavior in trypanosomes and bacteria and provide insight into a feature of parasite biology that may be exploited for novel intervention strategies. PMID:25416239

  7. Effects of Ca++ and Prostaglandin E1 on Vasopressin Activation of Renal Adenyl Cyclase

    PubMed Central

    Marumo, Fumiaki; Edelman, Isidore S.

    1971-01-01

    Adenyl cyclase activity was assayed in crude homogenates of the renal cortex, medulla, and papilla of the golden hamster. The specific activity (moles C-AMP/unit of time per mg protein of tissue) of the enzyme under basal conditions, was greatest in papilla, somewhat lower in medulla, and least in cortex. On an absolute scale, the sensitivity to vasopressin was greater in the medullary and papillary than in the cortical homogenates. In addition, at concentrations of 0.1-1.0 mm, CaCl2 inhibited the enzyme in the order papilla > medulla > cortex. These results imply the existence of distinct differences in the composition of the adenyl cyclase-receptor complex in various parts of the kidney. We proposed that Ca++ inhibits the core enzyme directly since at the minimally inhibitory concentration (0.1 mm), CaCl2 reduced to an equivalent extent (a) basal activity, (b) the response to graded doses of vasopressin (0.5 to 50.0 mU/ml) and (c) the response to maximal stimulatory concentrations of NaF (10 mm). Prostaglandin E1 (PGE1 = 10−7m) had no effect on either basal adenyl-cyclase activity or the response to 10 mm NaF in medullary and papillary homogenates. 7-Oxa-13-prostynoic acid (10−4m) similarly had no effect under basal conditions or on stimulation with NaF in medullary homogenates. Both fatty acids, however, inhibited the enzymic response to vasopressin, particularly at low concentrations of the peptide. The straight-chain fatty acid, 11-eicosanoic acid (10−7m), was inactive on basal activity or on the response to vasopressin. The possibility that PGE1 modifies the coupling mechanism between the core enzyme and the hormone-specific receptor is discussed. PMID:4329002

  8. Investigation into the mechanism(s) of antithrombotic effects of carbon monoxide releasing molecule-3 (CORM-3).

    PubMed

    Soni, Hitesh; Jain, Mukul; Mehta, Anita A

    2011-06-01

    Carbon monoxide (CO) like nitric oxide (NO) has been recognized as activator of soluble guanylate cyclase (sGC) in many physiological functions. Studies, which demonstrate the mechanisms by which CO inhibits platelet aggregation in in vivo models, are few. Here we investigated the possible involvement of sGC, NO, plasminogen activator inhibitor (PAI-1) and p38 MAP Kinase in antithrombotic effects of CO released by a novel, water-soluble, CO releasing molecule-3 (CORM-3) using rat. The effects of CORM-3 on in vitro and ex vivo platelet aggregation induced by thrombin as well as in in vivo thrombosis models were studied. When added to rat washed platelets in in vitro study, CORM-3 (100 and 200 μM) inhibited thrombin-induced platelet aggregation. Similarly, antiplatelet effect was also observed when 3mg/kg i.v. infusion of CORM-3 administered for 10 minutes in ex vivo study using rat. Interestingly, in presence of inhibitor of sGC (ODQ, 10mg/kg,i.p.) and inhibitor of nitric oxide synthase (L-NAME, 30 mg/kg,i.p.), inhibition of thrombin-induced aggregation by CORM-3 was significantly blocked. Notably, in presence of inhibitor of K(ATP) channel (glibenclamide, 10mg/kg,i.p.) and p38 MAP Kinase (SCIO-469, 1mg/kg, i.p.), inhibition of aggregation by CORM-3 was not blocked. In in vivo studies using animal models of thrombosis, we found that CORM-3-mediated antithrombotic effect was dependent on activation of sGC, NO and suppression of PAI-1 in arterial thrombosis and Arterio-Venous (A-V) shunt models. Therefore, we concluded that antithrombotic activity of CORM-3 may be mediated by activation of sGC, NO and inhibition of PAI-1.

  9. Role of adenylyl cyclase in reduced β-adrenoceptor-mediated vasorelaxation during maturation

    PubMed Central

    López-Canales, O.A.; Castillo-Hernandez, M.C.; Vargas-Robles, H.; Rios, A.; López-Canales, J.S.; Escalante, B.

    2016-01-01

    Beta-adrenergic receptor (βAR)-dependent blood vessel relaxation is impaired in older animals and G protein activation has been suggested as the causative mechanism. Here, we investigated the role of βAR subtypes (β1AR, β2AR, and β3AR) and cAMP in maturation-dependent vasorelaxation impairment. Aortic rings from 15 Sprague-Dawley male rats (3 or 9 weeks old) were harvested and left intact or denuded of the endothelium. Vascular relaxation in aortic rings from younger and older groups was compared in the presence of βAR subtype agonists and antagonists along with cAMP and cGMP antagonists. Isolated aortic rings were used to evaluate relaxation responses, protein expression was evaluated by western blot or real time PCR, and metabolites were measured by ELISA. Expression of βAR subtypes and adenylyl cyclase was assessed, and cAMP activity was measured in vascular tissue from both groups. Isoproterenol- and BRL744-dependent relaxation in aortic rings with and without endothelium from 9-week-old rats was impaired compared with younger rats. The β1AR antagonist CGP20712A (10-7 M) did not affect isoproterenol or BRL744-dependent relaxation in arteries from either group. The β2AR antagonist ICI-118,551 (10-7 M) inhibited isoproterenol-dependent aortic relaxation in both groups. The β3AR antagonist SR59230A (10-7 M) inhibited isoproterenol- and BRL744-dependent aortic ring relaxation in younger but not in older rats. All βAR subtypes were expressed in both groups, although β3AR expression was lower in the older group. Adenylyl cyclase (SQ 22536) or protein kinase A (H89) inhibitors prevented isoproterenol-induced relaxation in younger but not in older rats. Production of cAMP was reduced in the older group. Adenylyl cyclase III and RyR3 protein expression was higher in the younger group. In conclusion, altered expression of β3AR and adenylyl cyclase III may be responsible for reduced cAMP production in the older group. PMID:27383122

  10. A kinase-anchoring proteins and adenylyl cyclase in cardiovascular physiology and pathology.

    PubMed

    Efendiev, Riad; Dessauer, Carmen W

    2011-10-01

    3'-5'-Cyclic adenosine monophosphate (cAMP), generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology, including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins localize the effect of cAMP in space and time by organizing receptors, AC, protein kinase A, and other components of the cAMP cascade into multiprotein complexes. In this review, we discuss how the interaction of A kinase-anchoring proteins with distinct AC isoforms affects cardiovascular physiology.

  11. Aluminum: a requirement for activation of the regulatory component of adenylate cyclase by fluoride.

    PubMed Central

    Sternweis, P C; Gilman, A G

    1982-01-01

    Activation of the purified guanine nucleotide-binding regulatory component (G/F) of adenylate cyclase by F- requires the presence of Mg2+ and another factor. This factor, which contaminates commercial preparations of various nucleotides and disposable glass test tubes, has been identified as Al3+. In the presence of 10 mM Mg2+ and 5 mM F-, AlCl3 causes activation of G/F with an apparent activation constant of approximately 1-5 muM. The requirement for Al3+ is highly specific; of 28 other metals tested, only Be2+ promoted activation of G/F by F-. PMID:6289322

  12. Isolated neuronal growth cones from developing rat forebrain possess adenylate cyclase activity which can be augmented by various receptor agonists.

    PubMed

    Lockerbie, R O; Hervé, D; Blanc, G; Tassin, J P; Glowinski, J

    1988-01-01

    Isolated neuronal growth cones from neonatal rat forebrain were found to contain a high specific activity of adenylate cyclase (61 pmol cyclic AMP/min/mg protein) compared to the pelleted starting homogenate (5 pmol cyclic AMP/min/mg protein). Forskolin at 10(-4) M increased adenylate cyclase activity in both the pelleted homogenate and growth cone fraction by 70 and 217 pmol cyclic AMP/min/mg protein, respectively, over basal levels. The incremental effect of forskolin was 3-fold greater in the growth cone fraction than in the pelleted homogenate. However, relative to basal levels in each of the two fractions, forskolin increased adenylate cyclase activity in the growth cone fraction by only approx. 5-fold compared to 15-fold in the pelleted homogenate. Dopamine (10(-4) M), vasoactive intestinal polypeptide (10(-6) M) and isoproterenol (10(-5) M) also augmented adenylate cyclase activity in the two fractions. In the growth cone fraction, dopamine and vasoactive intestinal polypeptide produced a stimulation over basal levels by approx. 20 pmol cyclic AMP/min/mg protein while isoproterenol produced a stimulation of approx. 10 pmol cAMP/min/mg protein. The incremental effects of these receptor agonists in the growth cone fraction are approx. 5-fold greater than in the pelleted homogenate. The dopamine-sensitive adenylate cyclase activity in the growth cone fraction could be blocked by the compound SCH23390, a selective D1 receptor antagonist. At saturating concentrations, all combinations of dopamine, vasoactive intestinal polypeptide and isoproterenol were found to be completely additive on adenylate cyclase activity in the growth cone fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii*

    PubMed Central

    Sugai, Yoshinori; Ueno, Yohei; Hayashi, Ken-ichiro; Oogami, Shingo; Toyomasu, Tomonobu; Matsumoto, Sadamu; Natsume, Masahiro; Nozaki, Hiroshi; Kawaide, Hiroshi

    2011-01-01

    Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-13C6]mevalonate, all carbons were labeled with 13C stable isotope (>99%). The fully 13C-labeled product was subjected to 13C-13C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and

  14. Insights into open/closed conformations of the catalytically active human guanylate kinase as investigated by small-angle X-ray scattering.

    PubMed

    Jain, Rohit; Khan, Nazimuddin; Menzel, Andreas; Rajkovic, Ivan; Konrad, Manfred; Techert, Simone

    2016-01-01

    Bio-catalysis is the outcome of a subtle interplay between internal motions in enzymes and chemical kinetics. Small-angle X-ray scattering (SAXS) investigation of an enzyme's internal motions during catalysis offers an integral view of the protein's structural plasticity, dynamics, and function, which is useful for understanding allosteric effects and developing novel medicines. Guanylate kinase (GMPK) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms. It is also required for the intracellular activation of numerous antiviral and anticancer purine nucleoside analog prodrugs. Catalytically active recombinant human GMPK (hGMPK) was purified for the first time and changes in the size and shape of open/closed hGMPK were tracked by SAXS. The binding of substrates (GMP + AMPPNP or Ap5G or GMP + ADP) resulted in the compaction of size and shape of hGMPK. The structural changes between open and completely closed hGMPK conformation were confirmed by observing differences in the hGMPK secondary structures with circular dichroism spectroscopy. PMID:26446352

  15. Two distinct alpha-interferon-dependent signal transduction pathways may contribute to activation of transcription of the guanylate-binding protein gene

    SciTech Connect

    Decker, T.; Lew, D.J.; Darnell, J.E. Jr. )

    1991-10-01

    The promoter of the gene encoding a cytoplasmic guanylate-binding (GBP) contains two overlapping elements: the interferon stimulation response element (ISRE), which mediates alpha interferon (IFN-{alpha})-dependent transcription, and the IFN-{gamma} activation site (GAS), which is required for INF-{gamma}-mediated stimulation. The ISRE binds a factor called ISGF-3 that is activated by IFN-{alpha} but not by IFN-{gamma}. The GAS binds a protein that is activated by IFN-{gamma}, which the authors have termed GAF. The authors now find that the GAS is also an IFN-{alpha}-responsive element in vivo and that IFN-{alpha} (in addition to activating ISGF-3) rapidly activates a GAS-binding factor, the IFN-{alpha} activation factor (AAF). The AAF has characteristics very similar to those of the previously described GAF. Through the use of inhibitors of protein synthesis and inhibitors of protein kinases, the activation conditions of AAF, GAF, and ISGF-3 could be distinguished. Therefore, not only do IFN-{alpha} and IFN-{gamma} stimulate transcription of GBP through different receptors linked to different signaling molecules, but occupation of the IFN-{alpha} receptor apparently leads to the rapid activation of two different DNA-binding proteins through the use of different intracellular pathways.

  16. Role of Dlg5/lp-dlg, a Membrane-Associated Guanylate Kinase Family Protein, in Epithelial-Mesenchymal Transition in LLc-PK1 Renal Epithelial Cells

    PubMed Central

    Sogabe, Takayuki; Kakuda, Kumiyo; Tomiyama, Lucia; Matsuno, Yohsuke; Ichikawa, Takafumi; Matsuo, Michinori; Ueda, Kazumitsu; Kioka, Noriyuki

    2012-01-01

    Discs large homolog 5 (Dlg5) is a member of the membrane-associated guanylate kinase adaptor family of proteins, some of which are involved in the regulation of epithelial-to-mesenchymal transition (EMT). Dlg5 has been described as a susceptibility gene for Crohn's disease; however, the physiological function of Dlg5 is unknown. We show here that transforming growth factor-β (TGF-β)-induced EMT suppresses Dlg5 expression in LLc-PK1 cells. Depletion of Dlg5 expression by knockdown promoted the expression of the mesenchymal marker proteins, fibronectin and α-smooth muscle actin, and suppressed the expression of E-cadherin. In addition, activation of JNK and p38, which are stimulated by TGF-β, was enhanced by Dlg5 depletion. Furthermore, inhibition of the TGF-β receptor suppressed the effects of Dlg5 depletion. These observations suggest that Dlg5 is involved in the regulation of TGF-βreceptor-dependent signals and EMT. PMID:22539977

  17. E. coli heat-stable enterotoxin and guanylyl cyclase C: new functions and unsuspected actions.

    PubMed Central

    Giannella, Ralph A.; Mann, Elizabeth A.

    2003-01-01

    Some E. coli cause diarrhea by elaborating heat-labile and heat-stable (ST) enterotoxins which stimulate intestinal secretion. E. coli ST's are small peptides which bind to intestinal luminal epithelial cell receptors. The ST receptor, one of a family of receptor-cyclases called guanylyl cyclase C (GC-C), is a membrane spanning protein containing an extracellular binding domain and intracellular protein kinase and catalytic domains. The intestine synthesizes and secretes homologous peptides, guanylin and uroguanylin. The kidney also synthesizes uroguanylin. ST, guanylin or uroguanylin binding to GC-C results in increased cGMP, phosphorylation of the CFTR Cl- channel and secretion. Proguanylin and prouroguanylin circulate in blood and bind to receptors in intestine, kidney, liver, brain etc. In the kidney, they stimulate the excretion of Na+ and K+. Study of GC-C "knock-out" mice reveal that GC-C is important to intestinal salt and water secretion, duodenal bicarbonate secretion, recovery from CCl4-induced liver injury, and to intestinal polyp formation in Min mice lacking GC-C. PMID:12813912

  18. Identification of an adenylyl cyclase inhibitor for treating neuropathic and inflammatory pain.

    PubMed

    Wang, Hansen; Xu, Hui; Wu, Long-Jun; Kim, Susan S; Chen, Tao; Koga, Kohei; Descalzi, Giannina; Gong, Bo; Vadakkan, Kunjumon I; Zhang, Xuehan; Kaang, Bong-Kiun; Zhuo, Min

    2011-01-12

    Neuropathic pain, often caused by nerve injury, is commonly observed among patients with different diseases. Because its basic mechanisms are poorly understood, effective medications are limited. Previous investigations of basic pain mechanisms and drug discovery efforts have focused mainly on early sensory neurons such as dorsal root ganglion and spinal dorsal horn neurons, and few synaptic-level studies or new drugs are designed to target the injury-related cortical plasticity that accompanies neuropathic pain. Our previous work has demonstrated that calcium-stimulated adenylyl cyclase 1 (AC1) is critical for nerve injury-induced synaptic changes in the anterior cingulate cortex. Through rational drug design and chemical screening, we have identified a lead candidate AC1 inhibitor, NB001, which is relatively selective for AC1 over other adenylate cyclase isoforms. Using a variety of behavioral tests and toxicity studies, we have found that NB001, when administered intraperitoneally or orally, has an analgesic effect in animal models of neuropathic pain, without any apparent side effects. Our study thus shows that AC1 could be a productive therapeutic target for neuropathic pain and describes a new agent for the possible treatment of neuropathic pain.

  19. Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase

    PubMed Central

    Abdo, Zaid; Byers, Sara Overstreet; Kriebel, Patrick; Rothrock, Michael J.

    2016-01-01

    Abstract Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition. PMID:27035032

  20. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    PubMed

    Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2015-12-01

    Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

  1. Adenylate cyclase activity in fish gills in relation to salt adaptation

    SciTech Connect

    Guibbolini, M.E.; Lahlou, B.

    1987-07-06

    The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of (..cap alpha..-/sup 32/P) - ATP into (..cap alpha..-/sup 32/P) - cAMP) was investigated in gill plasma membranes of rainbow trout (Salmo gairdneri) adapted to various salinities (deionized water, DW; fresh water, FW; 3/4 sea water, 3/4 SW; sea water, SW) and in sea water adapted- mullet (Mugil sp.). Basal activity declined by a factor of 2 in trout with increasing external salinity (pmoles cAMP/mg protein/10 min: 530 in DW, 440 in FW, 340 in 3/4 SW; 250 in SW) and was very low in SW adapted-mullet: 35. The Km for ATP was similar (0.5 mM) in both FW adapted- and SW adapted- trout in either the absence (basal activity) or in the presence of stimulating agents (isoproterenol; NaF) while the Vm varied. Analysis of stimulation ratios with respect to basal levels of the enzyme showed that hormones and pharmacological substances (isoproterenol, NaF) display a greater potency in high salt than in low salt adapted- fish gills. In contrast, salt adaptation did not have any effect on the regulation of adenylate cyclase by PGE/sub 1/. These results are interpreted in relation to the general process of osmoregulation. 27 references, 6 figures.

  2. Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade

    PubMed Central

    Mohan, Vijee; Pandey, Arun; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the “Beta type” promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening. PMID:27070417

  3. Diguanylate cyclase DgcP is involved in plant and human Pseudomonas spp. infections.

    PubMed

    Aragon, Isabel M; Pérez-Mendoza, Daniel; Moscoso, Joana A; Faure, Emmanuel; Guery, Benoit; Gallegos, María-Trinidad; Filloux, Alain; Ramos, Cayo

    2015-11-01

    The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosa PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosa PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections.

  4. Adenylyl cyclase 3 haploinsufficiency confers susceptibility to diet-induced obesity and insulin resistance in mice

    PubMed Central

    Tong, Tao; Shen, Ying; Lee, Han-Woong; Yu, Rina; Park, Taesun

    2016-01-01

    Adenylyl cyclase 3 (Adcy3), a member of the mammalian adenylyl cyclase family responsible for generating the second messenger cAMP, has long been known to play an essential role in olfactory signal transduction. Here, we demonstrated that Adcy3 heterozygous null mice displayed increased visceral adiposity in the absence of hyperphagia and developed abnormal metabolic features characterized by impaired insulin sensitivity, dyslipidemia, and increased plasma levels of proinflammatory cytokines on both chow and high-fat diet (HFD). Of note, HFD decreased the Adcy3 expression in white adipose tissue, liver, and muscle. We also report for the first time that Adcy3 haploinsufficiency resulted in reduced expression of genes involved in thermogenesis, fatty acid oxidation, and insulin signaling, with enhanced expression of genes related to adipogenesis in peripheral tissues of mice. In conclusion, these findings suggest that cAMP signals generated by Adcy3 in peripheral tissues may play a pivotal role in modulating obesity and insulin sensitivity. PMID:27678003

  5. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    PubMed

    Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2015-12-01

    Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC. PMID:26625288

  6. Bicarbonate-sensitive soluble and transmembrane adenylyl cyclases in peripheral chemoreceptors.

    PubMed

    Nunes, Ana R; Holmes, Andrew P S; Sample, Vedangi; Kumar, Prem; Cann, Martin J; Monteiro, Emília C; Zhang, Jin; Gauda, Estelle B

    2013-08-15

    Stimulation of the carotid body (CB) chemoreceptors by hypercapnia triggers a reflex ventilatory response via a cascade of cellular events, which includes generation of cAMP. However, it is not known if molecular CO2/HCO3(-) and/or H(+) mediate this effect and how these molecules contribute to cAMP production. We previously reported that the CB highly expresses HCO3(-)-sensitive soluble adenylyl cyclase (sAC). In the present study we systematically characterize the role of sAC in the CB, comparing the effect of isohydric hypercapnia (IH) in cAMP generation through activation of sAC or transmembrane-adenylyl cyclase (tmAC). Pharmacological deactivation of sAC and tmAC decreased the CB cAMP content in normocapnia and IH with no differences between these two conditions. Changes from normocapnia to IH did not effect the degree of PKA activation and the carotid sinus nerve discharge frequency. sAC and tmAC are functional in CB but intracellular elevations in CO2/HCO3(-) in IH conditions on their own are insufficient to further activate these enzymes, suggesting that the hypercapnic response is dependent on secondary acidosis.

  7. Inhibitory role of monovalent ions on rat brain cortex adenylyl cyclase activity.

    PubMed

    Nikolic, Ivana; Mitrovic, Marina; Zelen, Ivanka; Zaric, Milan; Kastratovic, Tatjana; Stanojevic, Marijana; Nenadovic, Milutin; Stojanovic, Tomislav

    2013-10-01

    Adenylyl cyclases, comprise of a large family of enzymes that catalyze synthesis of the cyclic AMP from ATP. The aim of our study was to determine the effect of monovalent ions on both basal, stimulated adenylate cyclase EC 4.6.1.1 (AC) activity and C unit of AC and on GTPase active G-protein in the synaptic membranes of rat brain cortex. The effect of ion concentration from 30 to 200 mM (1 mM MgCl2) showed dose-dependent and significant inhibition of the basal AC activity, stimulated and unstimulated C unit activity. Stimulation of AC with 5 μM GTPγS in the presence of 50-200 mM of tested salts showed inhibitory effect on the AC activity. From our results it could be postulated that the investigated monovalent ions exert inhibitory effect on the AC complex activity by affecting the intermolecular interaction of the activated α subunit of G/F protein and the C unit of AC complex an inhibitory influence of tested monovalent ions on these molecular interaction.

  8. Functional analysis of allene oxide cyclase, MpAOC, in the liverwort Marchantia polymorpha.

    PubMed

    Yamamoto, Yusuke; Ohshika, Jun; Takahashi, Tomohiro; Ishizaki, Kimitsune; Kohchi, Takayuki; Matusuura, Hideyuki; Takahashi, Kosaku

    2015-08-01

    12-Oxo-phytodienoic acid (OPDA) is an intermediate in jasmonic acid (JA) biosynthesis. OPDA exerts JA-dependent and JA-independent biological effects; therefore, it is considered a signaling molecule in flowering plants. OPDA is induced by bacterial infection and wounding and inhibits growth in the moss Physcomitrella patens. The functions of OPDA and allene oxide cyclase (AOC) in the liverwort Marchantia polymorpha were explored, which represents the most basal lineage of extant land plants. The analysis of OPDA showed that it is present in M. polymorpha and is increased by wounding. OPDA has been suggested to be involved in the response to environmental stresses. Moreover, OPDA showed growth inhibitory activity in M. polymorpha. Nonetheless JA in M. polymorpha was not found in this study. AOC synthesizes OPDA from an unstable allene oxide. A database search of the M. polymorpha genome identified only a putative gene encoding allene oxide cyclase (MpAOC). Recombinant MpAOC showed AOC activity similar to that in flowering plants. MpAOC was localized to chloroplasts, as in flowering plants. Expression of MpAOC was induced by wounding and OPDA treatment, and positive feedback regulation of OPDA was demonstrated in M. polymorpha. Overexpression of MpAOC increased the endogenous OPDA level and suppressed growth in M. polymorpha. These results indicate the role of OPDA as a signaling molecule regulating growth and the response to wounding in the liverwort M. polymorpha. PMID:25892411

  9. Adenylate cyclase 3: a new target for anti-obesity drug development.

    PubMed

    Wu, L; Shen, C; Seed Ahmed, M; Östenson, C-G; Gu, H F

    2016-09-01

    Obesity has become epidemic worldwide, and abdominal obesity has a negative impact on health. Current treatment options on obesity, however, still remain limited. It is then of importance to find a new target for anti-obesity drug development based upon recent molecular studies in obesity. Adenylate cyclase 3 (ADCY3) is the third member of adenylyl cyclase family and catalyses the synthesis of cAMP from ATP. Genetic studies with candidate gene and genome-wide association study approaches have demonstrated that ADCY3 genetic polymorphisms are associated with obesity in European and Chinese populations. Epigenetic studies have indicated that increased DNA methylation levels in the ADCY3 gene are involved in the pathogenesis of obesity. Furthermore, biological analyses with animal models have implicated that ADCY3 dysfunction resulted in increased body weight and fat mass, while reduction of body weight is partially explained by ADCY3 activation. In this review, we describe genomic and biological features of ADCY3, summarize genetic and epigenetic association studies of the ADCY3 gene with obesity and discuss dysfunction and activation of ADCY3. Based upon all data, we suggest that ADCY3 is a new target for anti-obesity drug development. Further investigation on the effectiveness of ADCY3 activator and its delivery approach to treat abdominal obesity has been taken into our consideration. PMID:27256589

  10. CO2/HCO3(-)- and calcium-regulated soluble adenylyl cyclase as a physiological ATP sensor.

    PubMed

    Zippin, Jonathan H; Chen, Yanqiu; Straub, Susanne G; Hess, Kenneth C; Diaz, Ana; Lee, Dana; Tso, Patrick; Holz, George G; Sharp, Geoffrey W G; Levin, Lonny R; Buck, Jochen

    2013-11-15

    The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo.

  11. Adenylate Cyclase Toxin (ACT) from Bordetella hinzii: Characterization and Differences from ACT of Bordetella pertussis

    PubMed Central

    Donato, Gina M.; Hsia, Hung-Lun J.; Green, Candace S.; Hewlett, Erik L.

    2005-01-01

    Bordetella hinzii is a commensal respiratory microorganism in poultry but is increasingly being recognized as an opportunistic pathogen in immunocompromised humans. Although associated with a variety of disease states, practically nothing is known about the mechanisms employed by this bacterium. In this study, we show by DNA sequencing and reverse transcription-PCR that both commensal and clinical strains of B. hinzii possess and transcriptionally express cyaA, the gene encoding adenylate cyclase toxin (ACT) in other pathogenic Bordetella species. By Western blotting, we also found that B. hinzii produces full-length ACT protein in quantities that are comparable to those made by B. pertussis. In contrast to B. pertussis ACT, however, ACT from B. hinzii is less extractable from whole bacteria, nonhemolytic, has a 50-fold reduction in adenylate cyclase activity, and is unable to elevate cyclic AMP levels in host macrophages (nontoxic). The decrease in enzymatic activity is attributable, at least in part, to a decreased binding affinity of B. hinzii ACT for calmodulin, the eukaryotic activator of B. pertussis ACT. In addition, we demonstrate that the lack of intoxication by B. hinzii ACT may be due to the absence of expression of cyaC, the gene encoding the accessory protein required for the acylation of B. pertussis ACT. These results demonstrate the expression of ACT by B. hinzii and represent the first characterization of a potential virulence factor of this organism. PMID:16267282

  12. Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade.

    PubMed

    Mohan, Vijee; Pandey, Arun; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the "Beta type" promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening.

  13. Diguanylate cyclase DgcP is involved in plant and human Pseudomonas spp. infections.

    PubMed

    Aragon, Isabel M; Pérez-Mendoza, Daniel; Moscoso, Joana A; Faure, Emmanuel; Guery, Benoit; Gallegos, María-Trinidad; Filloux, Alain; Ramos, Cayo

    2015-11-01

    The second messenger cyclic di-GMP (c-di-GMP) controls the transition between different lifestyles in bacterial pathogens. Here, we report the identification of DgcP (diguanylate cyclase conserved in Pseudomonads), whose activity in the olive tree pathogen Pseudomonas savastanoi pv. savastanoi is dependent on the integrity of its GGDEF domain. Furthermore, deletion of the dgcP gene revealed that DgcP negatively regulates motility and positively controls biofilm formation in both the olive tree pathogen P. savastanoi pv. savastanoi and the human opportunistic pathogen Pseudomonas aeruginosa. Overexpression of the dgcP gene in P. aeruginosa PAK led to increased exopolysaccharide production and upregulation of the type VI secretion system; in turn, it repressed the type III secretion system, which is a hallmark of chronic infections and persistence for P. aeruginosa. Deletion of the dgcP gene in P. savastanoi pv. savastanoi NCPPB 3335 and P. aeruginosa PAK reduced their virulence in olive plants and in a mouse acute lung injury model respectively. Our results show that diguanylate cyclase DgcP is a conserved Pseudomonas protein with a role in virulence, and confirm the existence of common c-di-GMP signalling pathways that are capable of regulating plant and human Pseudomonas spp. infections. PMID:25809128

  14. Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade.

    PubMed

    Mohan, Vijee; Pandey, Arun; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the "Beta type" promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening. PMID:27070417

  15. Characterization, Purification of Poncirin from Edible Citrus Ougan (Citrus reticulate cv. Suavissima) and Its Growth Inhibitory Effect on Human Gastric Cancer Cells SGC-7901

    PubMed Central

    Zhu, Xiaoyan; Luo, Fenglei; Zheng, Yixiong; Zhang, Jiukai; Huang, Jianzhen; Sun, Chongde; Li, Xian; Chen, Kunsong

    2013-01-01

    Poncirin is a bitter flavanone glycoside with various biological activities. Poncirin was isolated from four different tissues (flavedo, albedo, segment membrane, and juice sac) of Ougan fruit (Citrus reticulate cv. Suavissima). The highest content of poncirin was found in the albedo of Ougan fruit (1.37 mg/g DW). High speed counter-current chromatography (HSCCC) combined with D101 resin chromatography was utilized for the separation and purification of poncirin from the albedo of Ougan fruit. After this two-step purification, poncirin purity increased from 0.14% to 96.56%. The chemical structure of the purified poncirin was identified by both HPLC-PDA and LC-MS. Poncirin showed a significant in vitro inhibitory effect on the growth of the human gastric cancer cells, SGC-7901, in a dose-dependent manner. Thus, poncirin from Ougan fruit, may be beneficial for gastric cancer prevention. The purification method demonstrated here will be useful for further studies on the pharmacological mechanism of poncirin activity, as well as for guiding the consumption of Ougan fruit. PMID:23615464

  16. Novel mechanism of harmaline on inducing G2/M cell cycle arrest and apoptosis by up-regulating Fas/FasL in SGC-7901 cells.

    PubMed

    Wang, Yihai; Wang, Chunhua; Jiang, Chenguang; Zeng, Hong; He, Xiangjiu

    2015-01-01

    Harmaline (HAR), a natural occurrence β-carboline alkaloid, was isolated from the seeds of Peganum harmala and exhibited potent antitumor effect. In this study, the anti-gastric tumor effects of HAR were firstly investigated in vitro and in vivo. The results strongly showed that HAR could inhibit tumor cell proliferation and induce G2/M cell cycle arrest accompanied by an increase in apoptotic cell death in SGC-7901 cancer cells. HAR could up-regulate the expressions of cell cycle-related proteins of p-Cdc2, p21, p-p53, Cyclin B and down-regulate the expression of p-Cdc25C. In addition, HAR could up-regulate the expressions of Fas/FasL, activated Caspase-8 and Caspase-3. Moreover, blocking Fas/FasL signaling could markedly inhibit the apoptosis caused by HAR, suggesting that Fas/FasL mediated pathways were involved in HAR-induced apoptosis. Interestingly, HAR could also exert on antitumor activity with a dose of 15 mg/kg/day in vivo, which was also related with cell cycle arrest. These new findings provided a framework for further exploration of HAR which possess the potential antitumor activity by inducing cell cycle arrest and apoptosis. PMID:26678950

  17. Novel mechanism of harmaline on inducing G2/M cell cycle arrest and apoptosis by up-regulating Fas/FasL in SGC-7901 cells.

    PubMed

    Wang, Yihai; Wang, Chunhua; Jiang, Chenguang; Zeng, Hong; He, Xiangjiu

    2015-12-18

    Harmaline (HAR), a natural occurrence β-carboline alkaloid, was isolated from the seeds of Peganum harmala and exhibited potent antitumor effect. In this study, the anti-gastric tumor effects of HAR were firstly investigated in vitro and in vivo. The results strongly showed that HAR could inhibit tumor cell proliferation and induce G2/M cell cycle arrest accompanied by an increase in apoptotic cell death in SGC-7901 cancer cells. HAR could up-regulate the expressions of cell cycle-related proteins of p-Cdc2, p21, p-p53, Cyclin B and down-regulate the expression of p-Cdc25C. In addition, HAR could up-regulate the expressions of Fas/FasL, activated Caspase-8 and Caspase-3. Moreover, blocking Fas/FasL signaling could markedly inhibit the apoptosis caused by HAR, suggesting that Fas/FasL mediated pathways were involved in HAR-induced apoptosis. Interestingly, HAR could also exert on antitumor activity with a dose of 15 mg/kg/day in vivo, which was also related with cell cycle arrest. These new findings provided a framework for further exploration of HAR which possess the potential antitumor activity by inducing cell cycle arrest and apoptosis.

  18. Novel mechanism of harmaline on inducing G2/M cell cycle arrest and apoptosis by up-regulating Fas/FasL in SGC-7901 cells

    PubMed Central

    Wang, Yihai; Wang, Chunhua; Jiang, Chenguang; Zeng, Hong; He, Xiangjiu

    2015-01-01

    Harmaline (HAR), a natural occurrence β-carboline alkaloid, was isolated from the seeds of Peganum harmala and exhibited potent antitumor effect. In this study, the anti-gastric tumor effects of HAR were firstly investigated in vitro and in vivo. The results strongly showed that HAR could inhibit tumor cell proliferation and induce G2/M cell cycle arrest accompanied by an increase in apoptotic cell death in SGC-7901 cancer cells. HAR could up-regulate the expressions of cell cycle-related proteins of p-Cdc2, p21, p-p53, Cyclin B and down-regulate the expression of p-Cdc25C. In addition, HAR could up-regulate the expressions of Fas/FasL, activated Caspase-8 and Caspase-3. Moreover, blocking Fas/FasL signaling could markedly inhibit the apoptosis caused by HAR, suggesting that Fas/FasL mediated pathways were involved in HAR-induced apoptosis. Interestingly, HAR could also exert on antitumor activity with a dose of 15 mg/kg/day in vivo, which was also related with cell cycle arrest. These new findings provided a framework for further exploration of HAR which possess the potential antitumor activity by inducing cell cycle arrest and apoptosis. PMID:26678950

  19. Activation of locus coeruleus heme oxygenase-carbon monoxide pathway promoted an anxiolytic-like effect in rats.

    PubMed

    Carvalho-Costa, P G; Branco, L G S; Leite-Panissi, C R A

    2016-01-01

    The heme oxygenase-carbon monoxide pathway has been shown to play an important role in many physiological processes and is capable of altering nociception modulation in the nervous system by stimulating soluble guanylate cyclase (sGC). In the central nervous system, the locus coeruleus (LC) is known to be a region that expresses the heme oxygenase enzyme (HO), which catalyzes the metabolism of heme to carbon monoxide (CO). Additionally, several lines of evidence have suggested that the LC can be involved in the modulation of emotional states such as fear and anxiety. The purpose of this investigation was to evaluate the activation of the heme oxygenase-carbon monoxide pathway in the LC in the modulation of anxiety by using the elevated plus maze test (EPM) and light-dark box test (LDB) in rats. Experiments were performed on adult male Wistar rats weighing 250-300 g (n=182). The results showed that the intra-LC microinjection of heme-lysinate (600 nmol), a substrate for the enzyme HO, increased the number of entries into the open arms and the percentage of time spent in open arms in the elevated plus maze test, indicating a decrease in anxiety. Additionally, in the LDB test, intra-LC administration of heme-lysinate promoted an increase on time spent in the light compartment of the box. The intracerebroventricular microinjection of guanylate cyclase, an sGC inhibitor followed by the intra-LC microinjection of the heme-lysinate blocked the anxiolytic-like reaction on the EPM test and LDB test. It can therefore be concluded that CO in the LC produced by the HO pathway and acting via cGMP plays an anxiolytic-like role in the LC of rats.

  20. Activation of locus coeruleus heme oxygenase-carbon monoxide pathway promoted an anxiolytic-like effect in rats

    PubMed Central

    Carvalho-Costa, P.G.; Branco, L.G.S.; Leite-Panissi, C.R.A.

    2016-01-01

    The heme oxygenase-carbon monoxide pathway has been shown to play an important role in many physiological processes and is capable of altering nociception modulation in the nervous system by stimulating soluble guanylate cyclase (sGC). In the central nervous system, the locus coeruleus (LC) is known to be a region that expresses the heme oxygenase enzyme (HO), which catalyzes the metabolism of heme to carbon monoxide (CO). Additionally, several lines of evidence have suggested that the LC can be involved in the modulation of emotional states such as fear and anxiety. The purpose of this investigation was to evaluate the activation of the heme oxygenase-carbon monoxide pathway in the LC in the modulation of anxiety by using the elevated plus maze test (EPM) and light-dark box test (LDB) in rats. Experiments were performed on adult male Wistar rats weighing 250-300 g (n=182). The results showed that the intra-LC microinjection of heme-lysinate (600 nmol), a substrate for the enzyme HO, increased the number of entries into the open arms and the percentage of time spent in open arms in the elevated plus maze test, indicating a decrease in anxiety. Additionally, in the LDB test, intra-LC administration of heme-lysinate promoted an increase on time spent in the light compartment of the box. The intracerebroventricular microinjection of guanylate cyclase, an sGC inhibitor followed by the intra-LC microinjection of the heme-lysinate blocked the anxiolytic-like reaction on the EPM test and LDB test. It can therefore be concluded that CO in the LC produced by the HO pathway and acting via cGMP plays an anxiolytic-like role in the LC of rats. PMID:27074170

  1. The magnesium-protoporphyrin IX (oxidative) cyclase system. Studies on the mechanism and specificity of the reaction sequence.

    PubMed

    Walker, C J; Mansfield, K E; Rezzano, I N; Hanamoto, C M; Smith, K M; Castelfranco, P A

    1988-10-15

    Mg-protoporphyrin IX monomethyl ester cyclase activity was assayed in isolated developing cucumber (Cucumis sativus L. var. Beit Alpha) chloroplasts [Chereskin, Wong & Castelfranco (1982) Plant Physiol. 70, 987-993]. The presence of both 6- and 7-methyl esterase activities was detected, which permitted the use of diester porphyrins in a substrate-specificity study. It was found that: (1) the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester was inactive as a substrate for cyclization; (2) only one of the two enantiomers of 6-beta-hydroxy-Mg-protoporphyrin dimethyl ester had detectable activity as a substrate for the cyclase; (3) the 2-vinyl-4-ethyl-6-beta-oxopropionate derivatives of Mg-protoporphyrin mono- or di-methyl ester were approx. 4 times more active as substrates for cyclization than the corresponding divinyl forms; (4) at the level of Mg-protoporphyrin there was no difference in cyclase activity between the 4-vinyl and 4-ethyl substrates; (5) reduction of the side chain of Mg-protoporphyrin in the 2-position from a vinyl to an ethyl resulted in a partial loss of cyclase activity. This work suggests that the original scheme for cyclization proposed by Granick [(1950) Harvey Lect. 44, 220-245] should now be modified by the omission of the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester and the introduction of stereo-specificity at the level of the hydroxylated intermediate.

  2. Reduced basal and stimulated (isoprenaline, Gpp(NH)p, forskolin) adenylate cyclase activity in Alzheimer's disease correlated with histopathological changes.

    PubMed

    Ohm, T G; Bohl, J; Lemmer, B

    1991-02-01

    Cyclic adenosine monophosphate (cAMP) is an adenylate cyclase borne second messenger involved in basic metabolic events. The beta-adrenoceptor sensitive adenylate cyclase was studied in post-mortem hippocampi of controls and Alzheimer patients. Virtually identical subsets of each hippocampus homogenate were stimulated by 100 mumol isoprenaline, Gpp(NH)p and forskolin, respectively, in presence of an ATP-regenerating system. The determination of cAMP formed was carried out by means of a radioassay. The observed significant 50% reduction in basal as well as in stimulated adenylate cyclase activity in Alzheimer's disease is negatively correlated with semiquantitative evaluations of amyloid plaques (P less than 0.05) but not with neuritic plaques, neurofibrillary tangles or neuropil threads. This reduction in enzyme activity is obviously not due to simple cell loss alone. It is likely that the crucial point of the observed functional disturbance is at the level of the catalytic unit of the adenylate cyclase, since the same degree of reduction is maintained at all steps of the signal cascade. PMID:2054615

  3. Purification, characterization, and N-terminal amino acid sequence of the adenylyl cyclase-activating protease from bovine sperm.

    PubMed

    Adeniran, A J; Shoshani, I; Minuth, M; Awad, J A; Elce, J S; Johnson, R A

    1995-03-01

    We previously reported the extraction of a factor from bovine sperm that activated adenylyl cyclases of rat brain and human platelets, and identified it as a trypsin-like protease that was referred to as "ninhibin." This proteolytic activity was purified to near homogeneity from an alkaline extract of washed sperm particles by sequential chromatography on p-aminobenzamidine agarose and CM-Sephadex. Purification was greater than 100-fold with nearly 30% recovery of protease activity exhibiting a major band of approximately 40 kDa. An approximately 45-kDa form of the protease was also evident in crude extracts and was preferentially isolated when the enzyme was prepared in the presence of a mixture of protease inhibitors. The larger form of the protease was substantially less effective in stimulating adenylyl cyclase than was the smaller form; it is likely to be a zymogen form from which the smaller, more active form is derived. Purified forms of acrosin and ninhibin exhibited similar mobilities on PAGE, similar capacities for activating adenylyl cyclase, similar patterns of proteolytic fragmentation, and similar immunoblot patterns obtained with an antibody against purified bovine acrosin. More importantly, the N-terminal amino acid sequence of bovine ninhibin was found to be identical with that of bovine acrosin and caprine acrosin and more than 75% identical with porcine acrosin. The data support the conclusion that the adenylyl cyclase-activating protease previously referred to as ninhibin is, in fact, acrosin. PMID:7756444

  4. Multiplex PCR Assay Targeting a Diguanylate Cyclase-Encoding Gene, cgcA, To Differentiate Species within the Genus Cronobacter

    PubMed Central

    Carter, L.; Lindsey, L. A.; Grim, C. J.; Sathyamoorthy, V.; Jarvis, K. G.; Gopinath, G.; Lee, C.; Sadowski, J. A.; Trach, L.; Pava-Ripoll, M.; McCardell, B. A.; Tall, B. D.

    2013-01-01

    In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations. PMID:23144142

  5. The guanine cap of human guanylate-binding protein 1 is responsible for dimerization and self-activation of GTP hydrolysis.

    PubMed

    Wehner, Mark; Kunzelmann, Simone; Herrmann, Christian

    2012-01-01

    Human guanylate-binding protein 1 (hGBP1) belongs to the superfamily of large, dynamin-related GTPases. The expression of hGBP1 is induced by stimulation with interferons (mainly interferon-γ), and it plays a role in different cellular responses to inflammatory cytokines, e.g. pathogen defence, control of proliferation, and angiogenesis. Although other members of the dynamin superfamily show a diversity of cellular functions, they share a common GTPase mechanism that relies on nucleotide-controlled oligomerization and self-activation of the GTPase. Previous structural studies on hGBP1 have suggested a mechanism of GTPase and GDPase activity that, as a critical step, involves dimerization of the large GTP-binding domains. In this study, we show that the guanine cap of hGBP1 is the key structural element responsible for dimerization, and is thereby essential for self-activation of the GTPase activity. Studies of concentration-dependent GTP hydrolysis showed that mutations of residues in the guanine cap, in particular Arg240 and Arg244, resulted in higher dissociation constants of the dimer, whereas the maximum hydrolytic activity was largely unaffected. Additionally, we identified an intramolecular polar contact (Lys62-Asp255) whose mutation leads to a loss of self-activation capability and controlled oligomer formation. We suggest that this contact structurally couples the guanine cap to the switch regions of the GTPase, translating the structural changes that occur upon nucleotide binding to a change in oligomerization and self-activation. PMID:22059445

  6. Magnesium regulation of the beta-receptor-adenylate cyclase complex. II. Sc3+ as a Mg2 antagonist.

    PubMed

    Maguire, M E

    1982-09-01

    Sc3+ bears the same relationship to Mg2+ as La3+ to Ca2+, a similar ionic radius but increased charge. Therefore, the possibility was investigated that Sc3+ would be a Mg2+ antagonist at Mg2+ sites on the beta-adrenergic receptor-adenylate cyclase complex of the murine S49 lymphoma cell. Sc3+ is consistently much more potent than La3+ in inhibiting adenylate cyclase regardless of the mode of activation. IC50 values for Sc3+ of 10-30 microM were observed, whereas those for La3+ were about 300 microM. However, Sc3+ does not block the ability of Mg2+ to increase beta-receptor affinity for agonist nor alter agonist affinity by itself. Furthermore, Sc3+ is a weak inhibitor of the beta-receptor-mediated inhibition of Mg2+ influx. In cyc- S49 membranes, in which the catalytic subunit of cyclase cannot interact with the nucleotide-coupling protein(s), Sc3+ is as potent as in wild-type S49 membranes and again more potent than La3+. Substrate kinetics show that Sc3+, like Mg2+, modulates adenylate cyclase activity by affecting the Vmax without altering the Km for substrate. The data suggest that Sc3+ is a specific antagonist of Mg2+ at the Mg2+ site on the catalytic subunit and support the suggestion that there are two distinct sites for Mg2+ with different functions, one site on the coupling protein(s) and one on the catalytic subunit. It was also found that an apparent complex of Sc3+ and F-, ScF4-, is a potent inhibitor of adenylate cyclase, with an IC50 of 3 microM. PMID:6292689

  7. Calcium-Myristoyl Tug Is a New Mechanism for Intramolecular Tuning of Calcium Sensitivity and Target Enzyme Interaction for Guanylyl Cyclase-activating Protein 1

    PubMed Central

    Peshenko, Igor V.; Olshevskaya, Elena V.; Lim, Sunghyuk; Ames, James B.; Dizhoor, Alexander M.

    2012-01-01

    Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca2+ sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca2+ sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca2+ binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca2+-bound and Mg2+-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca2+ sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca2+ binding to EF-hand 4 and Ca2+ sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu80 and Val180) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or “tug”) between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca2+ sensitivity. PMID:22383530

  8. pH sensing via bicarbonate-regulated “soluble” adenylyl cyclase (sAC)

    PubMed Central

    Rahman, Nawreen; Buck, Jochen; Levin, Lonny R.

    2013-01-01

    Soluble adenylyl cyclase (sAC) is a source of the second messenger cyclic adenosine 3′, 5′ monophosphate (cAMP). sAC is directly regulated by bicarbonate (HCO−3) ions. In living cells, HCO−3 ions are in nearly instantaneous equilibrium with carbon dioxide (CO2) and pH due to the ubiquitous presence of carbonic anhydrases. Numerous biological processes are regulated by CO2, HCO−3, and/or pH, and in a number of these, sAC has been shown to function as a physiological CO2/HCO3/pH sensor. In this review, we detail the known pH sensing functions of sAC, and we discuss two highly-studied, pH-dependent pathways in which sAC might play a role. PMID:24324443

  9. An Approach to Mimicking the Sesquiterpene Cyclase Phase by Nickel-Promoted Diene/Alkyne Cooligomerization

    PubMed Central

    Holte, Dane; Götz, Daniel C. G.; Baran, Phil S.

    2012-01-01

    Artificially mimicking the cyclase phase of terpene biosynthesis inspires the invention of new methodologies, since working with carbogenic frameworks containing minimal functionality limits the chemist’s toolbox of synthetic strategies. For example, the construction of terpene skeletons from five-carbon building blocks would be an exciting pathway to mimic in the laboratory. Nature oligomerizes, cyclizes, and then oxidizes γ,γ-dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) to all of the known terpenes. Starting from isoprene, the goal of this work was to mimic Nature’s approach for rapidly building molecular complexity. In principle, the controlled oligomerization of isoprene would drastically simplify the synthesis of terpenes used in the medicine, perfumery, flavor, and materials industries. This article delineates our extensive efforts to cooligomerize isoprene or butadiene with alkynes in a controlled fashion by zero-valent nickel catalysis building off the classic studies by Günther Wilke and coworkers. PMID:22229741

  10. Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

    NASA Astrophysics Data System (ADS)

    Itoh, Takayuki; Tokunaga, Kinya; Matsuda, Yudai; Fujii, Isao; Abe, Ikuro; Ebizuka, Yutaka; Kushiro, Tetsuo

    2010-10-01

    Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

  11. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    SciTech Connect

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.; Seidler, F.J. )

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a {beta}-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of {beta}-adrenergic receptors: in fact, ({sup 125}I)pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.

  12. Endotoxic lipopolysaccharides stimulate steroidogenesis and adenylate cyclase in adrenal tumor cells.

    PubMed

    Wolff, J; Cook, G H

    1975-12-01

    Lipopolysaccharides (endotoxins) from Escherichia coli, Serratia marcesens and Salmonella typhosa stimulated steroid production in Y-1 adrenal tumor cells in culture with a latent period of 3-4 h. Lipid A, derived from Escherichia coli lipopolysaccharide, also stimulated steroidogenesis. Lipopolysaccharides and lipid A also stimulate adenylate cyclase activity and cause rounding of the cells. In contrast, lipopolysaccharides do not stimulate steroidogenesis in receptor-deficient adrenal tumor cells (OS-3) or Leydig tumor cells (I-10). This tends to rule out contamination by enterotoxin to which these lines respond. Although both hormone and lipopolysaccharide responses are lost in these lines, there was no interaction between these sites as judged by the failure of lipopolysaccharides to block, during their latency, the response to corticotropin in Y-1 cells. The possibility that the lipopolysaccharide effect is one on membrane conformation is discussed.

  13. Adenylate cyclase 5 is required for melanophore and male pattern development in the guppy (Poecilia reticulata).

    PubMed

    Kottler, Verena A; Künstner, Axel; Koch, Iris; Flötenmeyer, Matthias; Langenecker, Tobias; Hoffmann, Margarete; Sharma, Eshita; Weigel, Detlef; Dreyer, Christine

    2015-09-01

    Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects life span and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments.

  14. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

  15. Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases.

    PubMed

    Itoh, Takayuki; Tokunaga, Kinya; Matsuda, Yudai; Fujii, Isao; Abe, Ikuro; Ebizuka, Yutaka; Kushiro, Tetsuo

    2010-10-01

    Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities. PMID:20861902

  16. Squalene hopene cyclases are protonases for stereoselective Brønsted acid catalysis.

    PubMed

    Hammer, Stephan C; Marjanovic, Antonija; Dominicus, Jörg M; Nestl, Bettina M; Hauer, Bernhard

    2015-02-01

    For many important reactions catalyzed in chemical laboratories, the corresponding enzymes are missing, representing a restriction in biocatalysis. Although nature provides highly developed machineries appropriate to catalyze such reactions, their potential is often ignored. This also applies to Brønsted acid catalysis, a powerful method to promote a myriad of chemical transformations. Here, we report on the unique protonation machinery of a squalene hopene cyclase (SHC). Active site engineering of this highly evolvable enzyme yielded a platform for enzymatic Brønsted acid catalysis in water. This is illustrated by activation of different functional groups (alkenes, epoxides and carbonyls), enabling the highly stereoselective syntheses of various cyclohexanoids while uncoupling SHC from polycyclization chemistry. This work highlights the potential of systematic investigation on nature's catalytic machineries to generate unique catalysts.

  17. Characterization of the dopamine stimulated adenylate cyclase in the pedal ganglia of Mytilus edulis: interactions with etorphine, beta-endorphin, DALA, and methionine enkephalin.

    PubMed

    Stefano, G B; Catapane, E J; Kream, R M

    1981-03-01

    The dopamine-stimulated adenylate cyclase activity was studied both in vivo and in vitro in the central nervous system of the bivalve mollusc Mytilus edulis. Dopamine, epinine, and apomorphine stimulated the enzyme system. Fluphenazine, haloperidol, chlorpromaxine, and to a lesser extent BOL inhibited the dopamine-stimulated adenylate cyclase. Etorphine, beta-endorphine, DALA, and methionine enkephalin depressed cyclic AMP levels. This phenomena was naloxone reversible. In addition, the opioids inhibited the stimulation of adenylate cyclase by dopamine. This phenomena was also naloxone reversible. The study demonstrates an interaction among dopamine, the opioids, and cyclic AMP. PMID:6286125

  18. Topological mimicry and epitope duplication in the guanylyl cyclase C receptor.

    PubMed Central

    Nandi, A.; Suguna, K.; Surolia, A.; Visweswariah, S. S.

    1998-01-01

    Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence. PMID:9792105

  19. Activation of Soluble Adenylyl Cyclase Protects against Secretagogue Stimulated Zymogen Activation in Rat Pancreaic Acinar Cells

    PubMed Central

    Kolodecik, Thomas R.; Shugrue, Christine A.; Thrower, Edwin C.; Levin, Lonny R.; Buck, Jochen; Gorelick, Fred S.

    2012-01-01

    An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis. PMID:22844459

  20. Isolation and characterization of glutaminyl cyclases from Drosophila: evidence for enzyme forms with different subcellular localization.

    PubMed

    Schilling, Stephan; Lindner, Christiane; Koch, Birgit; Wermann, Michael; Rahfeld, Jens-Ulrich; von Bohlen, Alex; Rudolph, Thomas; Reuter, Gunter; Demuth, Hans-Ulrich

    2007-09-25

    Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes. PMID:17722885

  1. Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases

    PubMed Central

    Gao, Xiaohui; Dong, Xiao; Subramanian, Sundharraman; Matthews, Paige M.; Cooper, Caleb A.; Kearns, Daniel B.

    2014-01-01

    Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis. PMID:25085482

  2. Human serotonin1B receptor expression in Sf9 cells: phosphorylation, palmitoylation, and adenylyl cyclase inhibition.

    PubMed

    Ng, G Y; George, S R; Zastawny, R L; Caron, M; Bouvier, M; Dennis, M; O'Dowd, B F

    1993-11-01

    Analysis of the primary protein structure of the human serotonin1B (5-HT1B) receptor reveals consensus sites for phosphorylation and a putative site for palmitoylation. To investigate these posttranslational modifications, we have expressed a c-myc epitope-tagged 5-HT1B (m5-HT1B) receptor in Sf9 cells. This strategy enabled receptors to be detected by immunoblot analysis and purified by immunoprecipitation using a monoclonal antibody, 9E10, specific for the c-myc epitope. Agonist radioligand [3H]5-HT binding studies showed that the expressed 5-HT1B and m5-HT1B receptors displayed the characteristic pharmacological profile of the neuronal 5-HT1B receptor. The expressed receptors displayed both high- and low-affinity states for [3H]5-HT, suggesting that the receptors were coupled to endogenous G-proteins. Indeed, agonist binding to the high-affinity receptor state was regulated in the presence of GTP gamma S, Gpp(NH)p, and pertussis toxin. [32P]ADP-ribosylation experiments identified a major approximately 41-kDa ADP-ribosylated protein present in Sf9 membranes that comigrated with partially purified bovine brain Gi alpha/G(o) alpha subunits. Measurements of adenylyl cyclase activity in membranes from cells expressing m5-HT1B receptors showed that serotonergic agonists mediated the inhibition of adenylyl cyclase activity with a rank order of potency comparable to their affinity constants. Immunoblot analysis of membranes prepared from cells expressing m5-HT1B receptors and photoaffinity labeling of the immunoprecipitated material revealed photolabeled species at approximately 95 and at approximately 42 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Tachyphylaxis to PACAP-27 after inhibition of NO synthesis: a loss of adenylate cyclase activation

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The vasodilator effects of pituitary adenylate cyclase activating polypeptide (PACAP-27) are subject to tachyphylaxis in rats treated with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This study examined whether this tachyphylaxis is due to the loss of vasodilator potency of cAMP generated by activation of the G(s) protein-coupled PACAP receptors. Five successive treatments with PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats that were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME (50 micromol/kg iv)-treated rats produced vasodilator responses of similar magnitude to those in saline-treated rats, whereas four subsequent injections produced progressively and markedly smaller responses. The hemodynamic effects of the membrane-permeable cAMP analog 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 5-15 micromol/kg iv) were similar in L-NAME-treated rats and in L-NAME-treated rats that had received the five injections of PACAP-27. In addition, five injections of 8-CPT-cAMP (10 micromol/kg iv) produced pronounced vasodilator responses in saline- and L-NAME-treated rats that were not subject to the development of tachyphylaxis. These results suggest that a loss of biological potency of cAMP is not responsible for tachyphylaxis to PACAP-27 in L-NAME-treated rats. This tachyphylaxis may be due to the inability of the G(s) protein-coupled PACAP receptor to activate adenylate cyclase.

  4. Allosteric activation of Bordetella pertussis adenylyl cyclase by calmodulin: molecular dynamics and mutagenesis studies.

    PubMed

    Selwa, Edithe; Davi, Marilyne; Chenal, Alexandre; Sotomayor-Pérez, Ana-Cristina; Ladant, Daniel; Malliavin, Thérèse E

    2014-07-25

    Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

  5. Regulation of uterine adenylate cyclase by magnesium, manganese and calcium ions

    SciTech Connect

    Rayford, W.; Sanders, R.B.

    1987-05-01

    The regulation of rat uterine adenylate cyclase (AC) by Mg/sup 2 +/, Mn/sup 2 +/ and Ca/sup 2 +/ was examined during metestrus and proestrus of the estrous cycle and Days 1 and 4 of pseudopregnancy, before and after a mild trauma to the uterus. Mg/sup 2 +/ increased cyclase activity on all days measured. Maximal enzymic activity occurred with 5-10 mM Mg/sup 2 +/ during Day 4 following a mild traumatic stimulus to the uterus. The apparent Ka for Mg/sup 2 +/ was not significantly changed during these days. AC activity as a function of Mn/sup 2 +/ concentration was biphasic. It increased with increasing concentrations of Mn/sup 2 +/ and was maximal at 1.0-2.5 mM during Day 4 following uterine trauma. Higher concentrations of Mn/sup 2 +/ were inhibitory. The apparent Ka for Mn/sup 2 +/ was 0.36 +/- 0.05 mM and was not significantly altered during the days studied. Even though the Ka for Mn/sup 2 +/ was ten-fold lower than that for Mg/sup 2 +/, the Vmaxes shown with both ions were similar. Ca/sup 2 +/ is a potent inhibitor of uterine AC activity. When measured at its I.C./sub 50/, it lowered AC activity as Mg/sup 2 +/ concentrations were increased. Ca/sup 2 +/ did not have a significant effect on AC activated by Mn/sup 2 +/. The data showed that Mg/sup 2 +/, Mn/sup 2 +/ and Ca/sup 2 +/ might have important regulatory roles in the activation and inhibition of uterine AC in the rodent.

  6. Characterization of a novel sesquiterpene cyclase involved in (+)-caryolan-1-ol biosynthesis in Streptomyces griseus.

    PubMed

    Nakano, Chiaki; Horinouchi, Sueharu; Ohnishi, Yasuo

    2011-08-12

    Most terpenoids have been isolated from plants and fungi and only a few from bacteria. However, an increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. We characterized a sesquiterpene cyclase gene (SGR2079, named gcoA) found in Streptomyces griseus. When expressed in Streptomyces lividans, gcoA directed production of a sesquiterpene, isolated and determined to be (+)-caryolan-1-ol using spectroscopic analyses. (+)-Caryolan-1-ol was also detected in the crude cell lysate of wild-type S. griseus but not in a gcoA knockout mutant, indicating that GcoA is a genuine (+)-caryolan-1-ol synthase. Enzymatic properties were characterized using N-terminally histidine-tagged GcoA, produced in Escherichia coli. As expected, incubation of the recombinant GcoA protein with farnesyl diphosphate yielded (+)-caryolan-1-ol. However, a small amount of another sesquiterpene was also detected. This was identified as the bicyclic sesquiterpene hydrocarbon (+)-β-caryophyllene by comparison with an authentic sample using GC-MS. Incorporation of a deuterium atom into the C-9 methylene of (+)-caryolan-1-ol in an in vitro GcoA reaction in deuterium oxide indicated that (+)-caryolan-1-ol was synthesized by a proton attack on the C-8/C-9 double bond of (+)-β-caryophyllene. Several β-caryophyllene synthases have been identified from plants, but these cannot synthesize caryolan-1-ol. Although caryolan-1-ol has been isolated previously from several plants, the enzyme responsible for its biosynthesis has not been identified previously. GcoA is thus the first known caryolan-1-ol synthase. Isolation of caryolan-1-ol from microorganisms is unprecedented. PMID:21693706

  7. Novel metabotropic glutamate receptor negatively coupled to adenylyl cyclase in cultured rat cerebellar astrocytes.

    PubMed

    Kanumilli, Srinivasan; Toms, Nick J; Roberts, Peter J

    2004-04-01

    Several excitatory amino acid ligands were found potently to inhibit forskolin-stimulated cAMP accumulation in rat cultured cerebellar astrocytes: L-cysteine sulfinic acid (L-CSA) = L-aspartate > L-glutamate >/= the glutamate uptake inhibitor, L-PDC. This property did not reflect activation of conventional glutamate receptors, since the selective ionotropic glutamate receptor agonists NMDA, AMPA, and kainate, as well as several mGlu receptor agonists [(1S,3R)-ACPD, (S)-DHPG, DCG-IV, L-AP4, L-quisqualate, and L-CCG-I], were without activity. In addition, the mGlu receptor antagonists, L-AP3, (S)-4CPG, Eglu, LY341495, (RS)-CPPG, and (S)-MCPG failed to reverse 30 microM glutamate-mediated inhibitory responses. L-PDC-mediated inhibition was abolished by the addition of the enzyme glutamate-pyruvate transaminase. This finding suggests that the effect of L-PDC is indirect and that it is mediated through endogenously released L-glutamate. Interestingly, L-glutamate-mediated inhibitory responses were resistant to pertussis toxin, suggesting that G(i)/G(o) type G proteins were not involved. However, inhibition of protein kinase C (PKC, either via the selective PKC inhibitor GF109203X or chronic PMA treatment) augmented glutamate-mediated inhibitory responses. Although mGlu3 receptors (which are negatively coupled to adenylyl cyclase) are expressed in astrocyte populations, in our study Western blot analysis indicated that this receptor type was not expressed in cerebellar astrocytes. We therefore suggest that cerebellar astrocytes express a novel mGlu receptor, which is negatively coupled to adenylyl cyclase, and possesses an atypical pharmacological profile. PMID:14999808

  8. Cloning, sequencing, and expression of a 24-kDa Ca(2+)-binding protein activating photoreceptor guanylyl cyclase.

    PubMed

    Dizhoor, A M; Olshevskaya, E V; Henzel, W J; Wong, S C; Stults, J T; Ankoudinova, I; Hurley, J B

    1995-10-20

    Two vertebrate photoreceptor-specific membrane guanylyl cyclases, RetGC-1 and RetGC-2, are activated by a soluble 24-kDa retinal protein, p24, in a Ca(2+)-sensitive manner (Dizhoor, A.M., Lowe, D.G., Olshevskaya, E.V., Laura, R.P., and Hurley, J.B. (1994) Neuron 12, 1345-1352; Lowe, D.G., Dizhoor, A.M., Liu, K., Gu, O., Laura, R., Lu, L., and Hurley, J.B. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 5535-5539). The primary structure of bovine p24 has been derived from peptide sequencing and from its cDNA. p24 is a new EF-hand-type Ca(2+)-binding protein, related but not identical to another guanylyl cyclase-activating protein, GCAP (Palczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, H. Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S., Walsh, K.A., Gray-Keller, M.P., Detwiler, P.B., and Baehr, W. (1994) Neuron 13, 395-404) and other members of the recovering family of Ca(2+)-binding proteins. Antibodies against a truncated fusion protein and against a p24-specific synthetic peptide specifically recognize retinal p24 on immunoblot. Both antibodies inhibit activation of photoreceptor membrane guanylyl cyclase by purified p24. p24 is found only in retina, and it copurifies with outer segment membranes. Immunocytochemical analysis shows that it is present in rod photoreceptor cells. An immobilized antibody column was used to purify p24 from a heat-treated retinal extract. Purified p24 appears on SDS-polyacrylamide gel electrophoresis as a homogeneous protein not contaminated with GCAP, and it activates photoreceptor guanylyl cyclase in vitro at submicromolar concentrations. Ca2+ inhibits this activation with an EC50 near 200 nM and a Hill coefficient of 1.7. Recombinant p24 expressed in 293 cells effectively stimulates photoreceptor guanylyl cyclase. These findings demonstrate that p24, like GCAP, imparts Ca2+ sensitivity to photoreceptor membrane guanylyl cyclase. We propose that p24 be referred to as GCAP-2 and that GCAP be referred to as

  9. Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

    SciTech Connect

    Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.; Guillon, G.; Homburger, V.

    1987-06-25

    We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher mass (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.

  10. Cloning and bacterial expression of sesquiterpene cyclase, a key branch point enzyme for the synthesis of sesquiterpenoid phytoalexin capsidiol in UV-challenged leaves of Capsicum annuum.

    PubMed

    Back, K; He, S; Kim, K U; Shin, D H

    1998-09-01

    Sesquiterpene cyclase, a branch point enzyme in the general isoprenoid pathway for the synthesis of phytoalexin capsidiol, was induced in detached leaves of Capsicum annuum (pepper) by UV treatment. The inducibility of cyclase enzyme activities paralleled the absolute amount of cyclase protein(s) of pepper immunodetected by monoclonal antibodies raised against tobacco sesquiterpene cyclase. A cDNA library was constructed with poly(A)+ RNA isolated from 24 h UV-challenged leaves of pepper. A cDNA clone for sesquiterpene cyclase in pepper was isolated by using a tobacco 5-epi aristolochene synthase gene as a heterologous probe. The predicted protein encoded by this cDNA was comprised of 559 amino acids and had a relative molecular mass of 65,095. The primary structural information from the cDNA clone revealed that it shared 77%, 72% and 49% identity with 5-epi aristolochene, vetispiradiene, and cadinene synthase, respectively. The enzymatic product catalyzed by the cDNA clone in bacteria was identified as 5-epi aristolochene, as judged by argentation TLC. RNA blot hybridization demonstrated the induction of an mRNA consistent with the induction of cyclase enzyme activity in UV-treated pepper. PMID:9816674

  11. The Arabidopsis thaliana K(+)-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre.

    PubMed

    Al-Younis, Inas; Wong, Aloysius; Gehring, Chris

    2015-12-21

    Adenylate cyclases (ACs) catalyse the formation of the second messenger cyclic adenosine 3',5'-monophosphate (cAMP) from adenosine 5'-triphosphate (ATP). Although cAMP is increasingly recognised as an important signalling molecule in higher plants, ACs have remained somewhat elusive. Here we used a search motif derived from experimentally tested guanylyl cyclases (GCs), substituted the residues essential for substrate specificity and identified the Arabidopsis thaliana K(+)-uptake permease 7 (AtKUP7) as one of several candidate ACs. Firstly, we show that a recombinant N-terminal, cytosolic domain of AtKUP7(1-100) is able to complement the AC-deficient mutant cyaA in Escherichia coli and thus restoring the fermentation of lactose, and secondly, we demonstrate with both enzyme immunoassays and mass spectrometry that a recombinant AtKUP7(1-100) generates cAMP in vitro. PMID:26638082

  12. Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus.

    PubMed

    Sawai, Satoru; Uchiyama, Hiroshi; Mizuno, Syuhei; Aoki, Toshio; Akashi, Tomoyoshi; Ayabe, Shin-ichi; Takahashi, Takeyoshi

    2011-04-01

    Shionone is the major triterpenoid component of Aster tataricus possessing a unique all six-membered tetracyclic skeleton and 3-oxo-4-monomethyl structure. To clarify its biosynthetic process, an oxidosqualene cyclase cDNA was isolated from A. tataricus, and the function of the enzyme was determined in lanosterol synthase-deficient yeast. The cyclase yielded ca. 90% shionone and small amounts of β-amyrin, friedelin, dammara-20,24-dienol, and 4-epishionone and was designated as a shionone synthase (SHS). Transcripts of SHS were detected in A. tataricus organs, confirming its involvement in shionone biosynthesis. SHS was shown to have evolved in the Asteraceae from β-amyrin synthase lineages and acquired characteristic species- and product-specificities.

  13. Crystal structure of a squalene cyclase in complex with the potential anticholesteremic drug Ro48-8071.

    PubMed

    Lenhart, Alexander; Weihofen, Wilhelm A; Pleschke, Axel E W; Schulz, Georg E

    2002-05-01

    Squalene-hopene cyclase (SHC) catalyzes the conversion of squalene into pentacyclic compounds. It is the prokaryotic counterpart of the eukaryotic oxidosqualene cyclase (OSC) that catalyzes the steroid scaffold formation. Because of clear sequence homology, SHC can serve as a model for OSC, which is an attractive target for anticholesteremic drugs. We have established the crystal structure of SHC complexed with Ro48-8071, a potent inhibitor of OSC and therefore of cholesterol biosynthesis. Ro48-8071 is bound in the active-center cavity of SHC and extends into the channel that connects the cavity with the membrane. The binding site of Ro48-8071 is largely identical with the expected site of squalene; it differs from a previous model based on photoaffinity labeling. The knowledge of the inhibitor binding mode in SHC is likely to help develop more potent inhibitors for OSC.

  14. A Diguanylate Cyclase Acts as a Cell Division Inhibitor in a Two-Step Response to Reductive and Envelope Stresses

    PubMed Central

    Kim, Hyo Kyung

    2016-01-01

    ABSTRACT Cell division arrest is a universal checkpoint in response to environmental assaults that generate cellular stress. In bacteria, the cyclic di-GMP (c-di-GMP) signaling network is one of several signal transduction systems that regulate key processes in response to extra-/intracellular stimuli. Here, we find that the diguanylate cyclase YfiN acts as a bifunctional protein that produces c-di-GMP in response to reductive stress and then dynamically relocates to the division site to arrest cell division in response to envelope stress in Escherichia coli. YfiN localizes to the Z ring by interacting with early division proteins and stalls cell division by preventing the initiation of septal peptidoglycan synthesis. These studies reveal a new role for a diguanylate cyclase in responding to environmental change, as well as a novel mechanism for arresting cell division. PMID:27507823

  15. Isolation and functional characterization of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites

    PubMed Central

    2010-01-01

    Background Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061. Results A 908 bp region upstream to the initiation codon of the Lycopene β-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908 to -437 bp 5' to ATG led to

  16. Persistent Electrical Activity in Primary Nociceptors after Spinal Cord Injury Is Maintained by Scaffolded Adenylyl Cyclase and Protein Kinase A and Is Associated with Altered Adenylyl Cyclase Regulation

    PubMed Central

    Bavencoffe, Alexis; Li, Yong; Wu, Zizhen; Yang, Qing; Herrera, Juan; Kennedy, Eileen J.

    2016-01-01

    Little is known about intracellular signaling mechanisms that persistently excite neurons in pain pathways. Persistent spontaneous activity (SA) generated in the cell bodies of primary nociceptors within dorsal root ganglia (DRG) has been found to make major contributions to chronic pain in a rat model of spinal cord injury (SCI) (Bedi et al., 2010; Yang et al., 2014). The occurrence of SCI-induced SA in a large fraction of DRG neurons and the persistence of this SA long after dissociation of the neurons provide an opportunity to define intrinsic cell signaling mechanisms that chronically drive SA in pain pathways. The present study demonstrates that SCI-induced SA requires continuing activity of adenylyl cyclase (AC) and cAMP-dependent protein kinase (PKA), as well as a scaffolded complex containing AC5/6, A-kinase anchoring protein 150 (AKAP150), and PKA. SCI caused a small but significant increase in the expression of AKAP150 but not other AKAPs. DRG membranes isolated from SCI animals revealed a novel alteration in the regulation of AC. AC activity stimulated by Ca2+-calmodulin increased, while the inhibition of AC activity by Gαi showed an unexpected and dramatic decrease after SCI. Localized enhancement of the activity of AC within scaffolded complexes containing PKA is likely to contribute to chronic pathophysiological consequences of SCI, including pain, that are promoted by persistent hyperactivity in DRG neurons. SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major clinical problem with poorly understood mechanisms and inadequate treatments. Recent findings indicate that chronic pain in a rat SCI model depends upon hyperactivity in dorsal root ganglia (DRG) neurons. Although cAMP signaling is involved in many forms of neural plasticity, including hypersensitivity of nociceptors in the presence of inflammatory mediators, our finding that continuing cAMP-PKA signaling is required for persistent SA months after SCI and long after isolation of

  17. Protein-Protein Docking and Analysis Reveal That Two Homologous Bacterial Adenylyl Cyclase Toxins Interact with Calmodulin Differently*S⃞

    PubMed Central

    Guo, Qing; Jureller, Justin E.; Warren, Julia T.; Solomaha, Elena; Florián, Jan; Tang, Wei-Jen

    2008-01-01

    Calmodulin (CaM), a eukaryotic calcium sensor that regulates diverse biological activities, consists of N- and C-terminal globular domains (N-CaM and C-CaM, respectively). CaM serves as the activator of CyaA, a 188-kDa adenylyl cyclase toxin secreted by Bordetella pertussis, which is the etiologic agent for whooping cough. Upon insertion of the N-terminal adenylyl cyclase domain (ACD) of CyaA to its targeted eukaryotic cells, CaM binds to this domain tightly (∼200 pm affinity). This interaction activates the adenylyl cyclase activity of CyaA, leading to a rise in intracellular cAMP levels to disrupt normal cellular signaling. We recently solved the structure of CyaA-ACD in complex with C-CaM to elucidate the mechanism of catalytic activation. However, the structure of the interface between N-CaM and CyaA, the formation of which contributes a 400-fold increase of binding affinity between CyaA and CaM, remains elusive. Here, we used site-directed mutations and molecular dynamic simulations to generate several working models of CaM-bound CyaA-ACD. The validity of these models was evaluated by disulfide bond cross-linking, point mutations, and fluorescence resonance energy transfer experiments. Our study reveals that a β-hairpin region (amino acids 259–273) of CyaA-ACD likely makes contacts with the second calcium binding motif of the extended CaM. This mode of interaction differs from the interaction of N-CaM with anthrax edema factor, which binds N-CaM via its helical domain. Thus, two structurally conserved, bacterial adenylyl cyclase toxins have evolved to utilize distinct binding surfaces and modes of activation in their interaction with CaM, a highly conserved eukaryotic signaling protein. PMID:18583346

  18. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase.

    PubMed

    Ostrom, R S; Gregorian, C; Drenan, R M; Xiang, Y; Regan, J W; Insel, P A

    2001-11-01

    Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to beta(1)-adrenergic receptor (beta(1)AR)-selective activation 3.7-fold, to beta(2)AR-selective activation only 1.6-fold and to prostaglandin E(2) (PGE(2)) not at all. Therefore, the rank order of efficacy in coupling to AC6 is beta(1)AR > beta(2)AR > prostaglandin E(2) receptor (EP(2)R). beta(2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing betaARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, beta(1)AR, and beta(2)AR, but not EP(2)R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by betaAR subtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated with a betaAR agonist, beta(2)AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of betaARKct. Thus, agonist-induced translocation of beta(2)AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of beta(2)AR coupling to AC6 as compared with beta(1)AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors. PMID:11533056

  19. Extracellular Regulation of Sperm Transmembrane Adenylyl Cyclase by a Forward Motility Stimulating Protein

    PubMed Central

    Dey, Souvik; Roy, Debarun; Majumder, Gopal C.; Bhattacharyya, Debdas

    2014-01-01

    Forward motility stimulating factor (FMSF), a glycoprotein isolated from buffalo serum, binds to the surface of the mature sperm cells to promote their progressive motility. This article reports the mode of signal transduction of this extracellular factor in goat sperm. The mechanism was investigated by assaying intracellular second messenger level and forward motility in presence of different pharmacological modulators. Mg++-dependent Forskolin responsive form of transmembrane adenylyl cyclase (tmAC) of goat spermatozoa was probed for its involvement in FMSF action. Dideoxyadenosine, a selective inhibitor of tmACs, was used to identify the role of this enzyme in the scheme of FMSF-signaling. Involvement of the α-subunit of G-protein in this regard has been inspected using GTPγS. Participation of protein kinase A (PKA) and tyrosine kinase was checked using IP20 and genistein, respectively. FMSF promotes tmAC activity in a dose-dependent manner through receptor/G-protein activation to enhance intracellular cAMP and forward motility. Motility boosting effects of this glycoprotein are almost lost in presence of dideoxyadenosine. But, FMSF displayed substantial motility promoting activity when movement of spermatozoa was inhibited with KH7, the specific inhibitor of soluble adenylyl cyclase indicating tmAC to be the primary target of FMSF action. Involvement of cAMP in mediating FMSF action was confirmed by the application of dibutyryl cAMP. Observed motility regulatory effects with IP20 and genistein indicate contribution of PKA and tyrosine kinase in FMSF activity; enhanced phosphorylation of a tyrosine containing ≈50 kDa protein was detected in this regard. FMSF initiates a novel signaling cascade to stimulate tmAC activity that augments intracellular cAMP, which through downstream crosstalk of phosphokinases leads to enhanced forward motility in mature spermatozoa. Thus, this article for the first time describes conventional tmAC-dependent profound activation

  20. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase.

    PubMed

    Ostrom, R S; Gregorian, C; Drenan, R M; Xiang, Y; Regan, J W; Insel, P A

    2001-11-01

    Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to beta(1)-adrenergic receptor (beta(1)AR)-selective activation 3.7-fold, to beta(2)AR-selective activation only 1.6-fold and to prostaglandin E(2) (PGE(2)) not at all. Therefore, the rank order of efficacy in coupling to AC6 is beta(1)AR > beta(2)AR > prostaglandin E(2) receptor (EP(2)R). beta(2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing betaARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, beta(1)AR, and beta(2)AR, but not EP(2)R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by betaAR subtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated with a betaAR agonist, beta(2)AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of betaARKct. Thus, agonist-induced translocation of beta(2)AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of beta(2)AR coupling to AC6 as compared with beta(1)AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors.

  1. Isolation of adenylate cyclase-free, beta-adrenergic receptor from turkey erythrocyte membranes by affinity chromatography.

    PubMed Central

    Vauquelin, G; Geynet, P; Hanoune, J; Strosberg, A D

    1977-01-01

    The adenylate cyclase [ATP pyrophosphatelyase (cyclizing); EC 4.6.1.1] and beta-adrenergic receptor of plasma membranes of turkey erythrocytes were solubilized in an active form by treatment with either NaF or guanylylimidodiphosphate and digitonin. The solubilized enzyme was no longer stimulated by catecholamines, NaF, or guanine nucleotides. The digitonin extract was chromatographed on an alprenolol-agarose derivative. While the bulk of protein and all the adenylate cyclase activity passed unretarded through the column, the receptor was retained. It eluted free of enzyme activity with an alprenolol solution containing 1 M NaCl; the yield was 25-30%. The protein content of the alprenolol eluates was too low to be estimated by the Lowry technique and was assessed by a more sensitive fluorometric method. Under these conditions, the beta-adrenergic receptor was purified approximately 2000-fold in a single step with retention of all its pharmacological properties. These experiments establish that the beta-adrenergic receptor and the adenylate cyclase are independent entities which may be separated on a functional basis. PMID:198798

  2. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB. 20

    SciTech Connect

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT{sub 1C} receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing ({sup 3}H)serotonin, ({sup 3}H)lysergic acid diethylamide or ({sup 3}H)dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor.

  3. Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein.

    PubMed

    Vojtek, A B; Cooper, J A

    1993-07-01

    CAP, an adenylyl cyclase associated protein, is present in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both organisms, CAP is bifunctional: the N-terminal domain binds to adenylyl cyclase, thereby enabling adenylyl cyclase to respond appropriately to upstream regulatory signals, such as RAS in S. cerevisiae; the C-terminal domain is required for cellular morphogenesis. Here, we describe the isolation of a cDNA encoding a CAP homolog from a higher eukaryote. The mouse CAP cDNA contains an open reading frame capable of encoding a 474 amino acid protein. The protein encoded by the mouse CAP cDNA shows extensive homology to the yeast CAP proteins, particularly in the central poly-proline rich region and in the C-terminal domain. By northern analysis, the CAP message appears to be ubiquitous, but not uniform. By indirect immunofluorescence, ectopically expressed mouse CAP protein is found in the cytoplasm of fibroblasts and, in migrating cells, at the leading edge. Expression of the mouse CAP cDNA in S. cerevisiae complements defects associated with loss of the yeast CAP carboxy-terminal domain. Hence, the function of the CAP carboxy-terminal domain has been conserved from yeast to mouse.

  4. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1987-05-01

    The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mg protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.

  5. Hydroxamate based inhibitors of adenylyl cyclase. Part 1: the effect of acyclic linkers on P-site binding.

    PubMed

    Levy, Daniel; Marlowe, Charles; Kane-Maguire, Kim; Bao, Ming; Cherbavaz, Diana; Tomlinson, James; Sedlock, David; Scarborough, Robert

    2002-11-01

    The adenylyl cyclases (ACs) are a family of enzymes that are key elements of signal transduction by virtue of their ability to convert ATP to cAMP. The catalytic mechanism of this transformation proceeds through initial binding of ATP to the purine binding site (P-site) followed by metal mediated cyclization with loss of pyrophosphate. Crystallographic analysis of ACs with known inhibitors reveals the presence of two metals in the active site. Presently, nine isoforms of adenylyl cyclase are known and unique isoform combinations are expressed in a tissue specific manner. The development of isoform specific inhibitors of adenylyl cyclase may prove to be a useful strategy toward the design of novel therapeutic agents. In order to develop novel AC inhibitors, we have chosen a design approach utilizing molecules with the adenine ring system joined to a metal-coordinating hydroxamic acid via flexible acyclic linkers. The designed inhibitors were assayed against type V AC with the size and heteroatom content of the linkers varied to probe the interaction of the nucleotide and metal binding sites within the enzyme. PMID:12372507

  6. Type VI adenylyl cyclase negatively regulates GluN2B-mediated LTD and spatial reversal learning

    PubMed Central

    Chang, Ching-Pang; Lee, Cheng-Ta; Hou, Wen-Hsien; Lin, Meng-Syuan; Lai, Hsing-Lin; Chien, Chen-Li; Chang, Chen; Cheng, Pei-Lin; Lien, Cheng-Chang; Chern, Yijuang

    2016-01-01

    The calcium-sensitive type VI adenylyl cyclase (AC6) is a membrane-bound adenylyl cyclase (AC) that converts ATP to cAMP under stimulation. It is a calcium-inhibited AC and integrates negative inputs from Ca2+ and multiple other signals to regulate the intracellular cAMP level. In the present study, we demonstrate that AC6 functions upstream of CREB and negatively controls neuronal plasticity in the hippocampus. Genetic removal of AC6 leads to cyclase-independent and N-terminus of AC6 (AC6N)-dependent elevation of CREB expression, and enhances the expression of GluN2B-containing NMDA receptors in hippocampal neurons. Consequently, GluN2B-dependent calcium signaling and excitatory postsynaptic current, long-term depression, and spatial reversal learning are enhanced in the hippocampus of AC6−/− mice without altering the gross anatomy of the brain. Together, our results suggest that AC6 negatively regulates neuronal plasticity by modulating the levels of CREB and GluN2B in the hippocampus. PMID:26932446

  7. Intramolecular signaling in tandem-GAF domains from PDE5 and PDE10 studied with a cyanobacterial adenylyl cyclase reporter.

    PubMed

    Banjac, Ana; Zimmermann, Markus O; Boeckler, Frank M; Kurz, Ursula; Schultz, Anita; Schultz, Joachim E

    2012-03-01

    The dimeric mammalian phosphodiesterases (PDEs) are regulated by N-terminal domains. In PDE5, the GAF-A subdomain of a GAF-tandem (GAF-A and -B) binds the activator cGMP and in PDE10 GAF-B binds cAMP. GAF-tandem chimeras of PDE5 and 10 in which the 36 aa linker helix between GAF-A and -B was swapped lost allosteric regulation of a reporter adenylyl cyclase. In 16 consecutive constructs we substituted the PDE10 linker with that from PDE5. An initial stretch of 10 amino acids coded for isoform specificity. A C240Y substitution uncoupled cyclase activity from regulation, whereas C240F, L or G did not. The C240Y substitution increased basal activity to stimulated levels. Notably, over the next 12 substitutions basal cyclase activity decreased linearly. Further targeted substitutions were based on homology modeling using the PDE2 structure. No combination of substitutions within the initial 10 linker residues caused loss of regulation. The full 10 aa stretch was required. Modeling indicated a potential interaction of the linker with a loop from GAF-A. To interrupt H-bonding a glycine substitution of the loop segment was generated. Despite reduction of basal activity, loss of regulation was maintained. Possibly, the orientation of the linker helix is determined by formation of the dimer at the initial linker segment. Downstream deflections of the linker helix may have caused loss of regulation.

  8. A wheat allene oxide cyclase gene enhances salinity tolerance via jasmonate signaling.

    PubMed

    Zhao, Yang; Dong, Wei; Zhang, Naibo; Ai, Xinghui; Wang, Mengcheng; Huang, Zhigang; Xiao, Langtao; Xia, Guangmin

    2014-02-01

    One of the two branches of the α-linolenic acid metabolism pathway is catalyzed by 12-oxo-phytodienoic acid reductase I, and the other is involved in jasmonic acid (JA) synthesis. The former is known to be active in the response to salinity tolerance in wheat (Triticum aestivum), but the participation of the latter in this response has not been established as yet. Here, the salinity-responsive bread wheat gene TaAOC1, which encodes an allene oxide cyclase involved in the α-linolenic acid metabolism pathway, was constitutively expressed in both bread wheat and Arabidopsis (Arabidopsis thaliana). In both species, transgenic lines exhibited an enhanced level of tolerance to salinity. The transgenic plants accumulated a higher content of JA and developed shorter roots. Both the shortened roots and the salinity tolerance were abolished in a background lacking a functional AtMYC2, a key component of the JA and abscisic acid signaling pathway, but were still expressed in a background deficient with respect to abscisic acid synthesis. We provide the first evidence, to our knowledge, suggesting that JA is also involved in the plant salinity response and that the α-linolenic acid metabolism pathway has a regulatory role over this response. PMID:24326670

  9. A Chemoattractant-mediated Gi-coupled Pathway Activates Adenylyl Cyclase in Human Neutrophils

    PubMed Central

    Mahadeo, Dana C.; Janka-Junttila, Mirkka; Smoot, Rory L.; Roselova, Pavla

    2007-01-01

    Neutrophils and Dictyostelium use conserved signal transduction pathways to decipher chemoattractant gradients and migrate directionally. In both cell types, addition of chemoattractants stimulates the production of cAMP, which has been suggested to regulate chemotaxis. We set out to define the mechanism by which chemoattractants increase cAMP levels in human neutrophils. We show that chemoattractants elicit a rapid and transient activation of adenylyl cyclase (AC). This activation is sensitive to pertussis toxin treatment but independent of phosphoinositide-3 kinase activity and an intact cytoskeleton. Remarkably, and in sharp contrast to Gαs-mediated activation, chemoattractant-induced AC activation is lost in cell lysates. Of the nine, differentially regulated transmembrane AC isoforms in the human genome, we find that isoforms III, IV, VII, and IX are expressed in human neutrophils. We conclude that the signal transduction cascade used by chemoattractants to activate AC is conserved in Dictyostelium and human neutrophils and is markedly different from the canonical Gαs-meditated pathway. PMID:17135293

  10. Laboratory evolution of adenylyl cyclase independent learning in Drosophila and missing heritability.

    PubMed

    Cressy, M; Valente, D; Altick, A; Kockenmeister, E; Honegger, K; Qin, H; Mitra, P P; Dubnau, J

    2014-07-01

    Gene interactions are acknowledged to be a likely source of missing heritability in large-scale genetic studies of complex neurological phenotypes. However, involvement of rare variants, de novo mutations, genetic lesions that are not easily detected with commonly used methods and epigenetic factors also are possible explanations. We used a laboratory evolution study to investigate the modulatory effects of background genetic variation on the phenotypic effect size of a null mutation with known impact on olfactory learning. To accomplish this, we first established a population that contained variation at just 23 loci and used selection to evolve suppression of the learning defect seen with null mutations in the rutabaga adenylyl cyclase. We thus biased the system to favor relatively simplified outcomes by choosing a Mendelian trait and by restricting the genetic variation segregating in the population. This experimental design also assures that the causal effects are among the known 23 segregating loci. We observe a robust response to selection that requires the presence of the 23 variants. Analyses of the underlying genotypes showed that interactions between more than two loci are likely to be involved in explaining the selection response, with implications for the missing heritability problem.

  11. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis

    PubMed Central

    Zhou, Zhiwen; Tanaka, Kenji F.; Matsunaga, Shigeru; Iseki, Mineo; Watanabe, Masakatsu; Matsuki, Norio; Ikegaya, Yuji; Koyama, Ryuta

    2016-01-01

    Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways. PMID:26795422

  12. High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor.

    PubMed

    Pichlo, Magdalena; Bungert-Plümke, Stefanie; Weyand, Ingo; Seifert, Reinhard; Bönigk, Wolfgang; Strünker, Timo; Kashikar, Nachiket Dilip; Goodwin, Normann; Müller, Astrid; Pelzer, Patric; Van, Qui; Enderlein, Jörg; Klemm, Clementine; Krause, Eberhard; Trötschel, Christian; Poetsch, Ansgar; Kremmer, Elisabeth; Kaupp, U Benjamin; Körschen, Heinz G; Collienne, Ursel

    2014-08-18

    Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3',5'-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 10(5) GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces "molecule noise." Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons. PMID:25135936

  13. Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

    PubMed Central

    Tlili, Mounira; Rouatbi, Sonia; Sriha, Badreddine; Ben Rhouma, Khémais; Sakly, Mohsen; Vaudry, David; Wurtz, Olivier; Tebourbi, Olfa

    2015-01-01

    The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP), we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h) for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM) for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC) and cytokines (IL-1α and TNF-α) in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders. PMID:26199679

  14. Levels of Lycopene β-Cyclase 1 Modulate Carotenoid Gene Expression and Accumulation in Daucus carota

    PubMed Central

    Moreno, Juan Camilo; Pizarro, Lorena; Fuentes, Paulina; Handford, Michael; Cifuentes, Victor; Stange, Claudia

    2013-01-01

    Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and β-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and β-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots. PMID:23555569

  15. Type 3 adenylyl cyclase: a key enzyme mediating the cAMP signaling in neuronal cilia

    PubMed Central

    Qiu, Liyan; LeBel, Robert P; Storm, Daniel R; Chen, Xuanmao

    2016-01-01

    Cilia are rigid, centriole-derived, microtubule-based organelles present in a majority of vertebrate cells including neurons. They are considered the cellular “antennae” attuned for detecting a range of extracellular signals including photons, odorants, morphogens, hormones and mechanical forces. The ciliary microenvironment is distinct from most actin-based subcellular structures such as microvilli or synapses. In the nervous system, there is no evidence that neuronal cilia process any synaptic structure. Apparently, the structural features of neuronal cilia do not allow them to harbor any synaptic connections. Nevertheless, a large number of G protein-coupled receptors (GPCRs) including odorant receptors, rhodopsin, Smoothened, and type 6 serotonin receptor are found in cilia, suggesting that these tiny processes largely depend on metabotropic receptors and their tuned signals to impact neuronal functions. The type 3 adenylyl cyclase (AC3), widely known as a cilia marker, is highly and predominantly expressed in olfactory sensory cilia and primary cilia throughout the brain. We discovered that ablation of AC3 in mice leads to pleiotropic phenotypes including anosmia, failure to detect mechanical stimulation of airflow, cognitive deficit, obesity, and depression-like behaviors. Multiple lines of human genetic evidence also demonstrate that AC3 is associated with obesity, major depressive disorder (MDD), sarcoidosis, and infertility, underscoring its functional importance. Here we review recent progress on AC3, a key enzyme mediating the cAMP signaling in neuronal cilia. PMID:27785336

  16. Levels of lycopene β-cyclase 1 modulate carotenoid gene expression and accumulation in Daucus carota.

    PubMed

    Moreno, Juan Camilo; Pizarro, Lorena; Fuentes, Paulina; Handford, Michael; Cifuentes, Victor; Stange, Claudia

    2013-01-01

    Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and β-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and β-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots.

  17. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

    PubMed Central

    Hurley, Matthew M.; Maunze, Brian; Block, Megan E.; Frenkel, Mogen M.; Reilly, Michael J.; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A.; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive.

  18. Structural features of Escherichia coli heat-stable enterotoxin that activates membrane-associated guanylyl cyclase.

    PubMed

    Sato, T; Shimonishi, Y

    2004-03-01

    Heat-stable enterotoxin (ST), a small peptide of 18 or 19 amino acid residues produced by enterotoxigenic Escherichia coli, is the cause of acute diarrhea in infants and travelers in developing countries. ST triggers a biological response by binding to a membrane-associated guanylyl cyclase C (GC-C) which is located on intestinal epithelial cell membranes. This binding causes an increase in the concentration of cGMP as a second messenger in cells and activates protein kinase A and cystic fibrosis transmembrane conductance regulator. Here we describe the crystal structure of an ST at 0.89 A resolution. The molecule has a ring-shaped molecular architecture consisting of six peptide molecules with external and internal diameters of approximately 35 and 7 A, respectively and a thickness of approximately 11 A. The conserved residues at the central portion of ST are distributed on the outer surface of the ring-shaped peptide hexamer, suggesting that the hexamer may be implicated in the association with GC-C through these invariant residues. PMID:15049831

  19. A Cytochrome P450 Serves as an Unexpected Terpene Cyclase during Fungal Meroterpenoid Biosynthesis

    PubMed Central

    Chooi, Yit-Heng; Hong, Young J.; Cacho, Ralph A.; Tantillo, Dean J.; Tang, Yi

    2013-01-01

    Viridicatumtoxin (1) is a tetracycline-like fungal meroterpenoid with a unique, fused spirobicyclic ring system. Puzzlingly, no dedicated terpene cyclase is found in the gene cluster identified in Penicillium aethiopicum. The two cytochrome P450 enzymes VrtE and VrtK in the vrt gene cluster were shown to catalyze C5-hydroxylation and spirobicyclic ring formation, respectively. Feeding of acyclic previridicatumtoxin (2) to Saccharomyces cerevisiae expressing VrtK confirmed that VrtK is the sole enzyme required for cyclization of the geranyl moiety. Thus, VrtK is the first example of a P450 that can catalyze terpene cyclization, most likely via the initial oxidation of C17 to an allylic carbocation. Quantum chemical modeling revealed a possible new tertiary carbocation intermediate E that forms after the allylic carbocation formation. The intermediate E can readily undergo concerted 1,2-alkyl shift/1,3-hydride shift, either spontaneously or further aided by the active site configuration of VrtK, followed by C7 Friedel-Crafts alkylation to afford 1,. The most likely stereochemical course of the reaction was proposed based on the results of our computations. PMID:24161266

  20. Activity Regulation by Heteromerization of Arabidopsis Allene Oxide Cyclase Family Members

    PubMed Central

    Otto, Markus; Naumann, Christin; Brandt, Wolfgang; Wasternack, Claus; Hause, Bettina

    2016-01-01

    Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 + AOC1 and AOC4 + AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles. PMID:27135223

  1. Effects of forskolin on cerebral blood flow: implications for a role of adenylate cyclase

    SciTech Connect

    Wysham, D.G.; Brotherton, A.F.; Heistad, D.D.

    1986-11-01

    We have studied cerebral vascular effects of forskolin, a drug which stimulates adenylate cyclase and potentiates dilator effects of adenosine in other vascular beds. Our goals were to determine whether forskolin is a cerebral vasodilator and whether it potentiates cerebral vasodilator responses to adenosine. We measured cerebral blood flow with microspheres in anesthetized rabbits. Forskolin (10 micrograms/kg per min) increased blood flow (ml/min per 100 gm) from 39 +/- 5 (mean +/- S.E.) to 56 +/- 9 (p less than 0.05) in cerebrum, and increased flow to myocardium and kidney despite a decrease in mean arterial pressure. Forskolin did not alter cerebral oxygen consumption, which indicates that the increase in cerebral blood flow is a direct vasodilator effect and is not secondary to increased metabolism. We also examined effects of forskolin on the response to infusion of adenosine. Cerebral blood flow was measured during infusion of 1-5 microM/min adenosine into one internal carotid artery, under control conditions and during infusion of forskolin at 3 micrograms/kg per min i.v. Adenosine alone increased ipsilateral cerebral blood flow from 32 +/- 3 to 45 +/- 5 (p less than 0.05). Responses to adenosine were not augmented during infusion of forskolin. We conclude that forskolin is a direct cerebral vasodilator and forskolin does not potentiate cerebral vasodilator responses to adenosine.

  2. Characterisation of Two Oxidosqualene Cyclases Responsible for Triterpenoid Biosynthesis in Ilex asprella

    PubMed Central

    Zheng, Xiasheng; Luo, Xiuxiu; Ye, Guobing; Chen, Ye; Ji, Xiaoyu; Wen, Lingling; Xu, Yaping; Xu, Hui; Zhan, Ruoting; Chen, Weiwen

    2015-01-01

    Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expression in the yeast Saccharomyces cerevisiae. Analysis of the resulting products by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) showed that both genes encode a mixed amyrin synthase, producing α-amyrin and β-amyrin at different ratios. IaAS1, which mainly produces α-amyrin, is the second triterpene synthase so far identified in which the level of α-amyrin produced is ≥80% of total amyrin production. By contrast, IaAS2 mainly synthesises β-amyrin, with a yield of 95%. Gene expression patterns of these two amyrin synthases in roots and leaves of I. asprella were found to be consistent with the content patterns of total saponins. Finally, phylogenetic analysis and multiple sequence alignment of the two amyrin synthases against several known OSCs from other plants were conducted to further elucidate their evolutionary relationship. PMID:25664861

  3. Structure of the Class IV Adenylyl Cyclase Reveals a Novel Fold

    SciTech Connect

    Gallagher,D.; Smith, N.; Kim, S.; Heroux, A.; Robinson, H.; Reddy, P.

    2006-01-01

    The crystal structure of the class IV adenylyl cyclase (AC) from Yersinia pestis (Yp) is reported at 1.9 {angstrom} resolution. The class IV AC fold is distinct from the previously described folds for class II and class III ACs. The dimeric AC-IV folds into an antiparallel eight-stranded barrel whose connectivity has been seen in only three previous structures: yeast RNA triphosphatase and two proteins of unknown function from Pyrococcus furiosus and Vibrio parahaemolyticus. Eight highly conserved ionic residues E10, E12, K14, R63, K76, K111, D126, and E136 lie in the barrel core and form the likely binding sites for substrate and divalent cations. A phosphate ion is observed bound to R63, K76, K111, and R113 near the center of the conserved cluster. Unlike the AC-II and AC-III active sites that utilize two-Asp motifs for cation binding, the AC-IV active site is relatively enriched in glutamate and features an ExE motif as its most conserved element. Homologs of Y. pestis AC-IV, including human thiamine triphosphatase, span the three kingdoms of life and delineate an ancient family of phosphonucleotide processing enzymes.

  4. Contribution of Physical Interactions to Signaling Specificity between a Diguanylate Cyclase and Its Effector

    PubMed Central

    Dahlstrom, Kurt M.; Giglio, Krista M.; Collins, Alan J.; Sondermann, Holger

    2015-01-01

    ABSTRACT Cyclic diguanylate (c-di-GMP) is a bacterial second messenger that controls multiple cellular processes. c-di-GMP networks have up to dozens of diguanylate cyclases (DGCs) that synthesize c-di-GMP along with many c-di-GMP-responsive target proteins that can bind and respond to this signal. For such networks to have order, a mechanism(s) likely exists that allow DGCs to specifically signal their targets, and it has been suggested that physical interactions might provide such specificity. Our results show a DGC from Pseudomonas fluorescens physically interacting with its target protein at a conserved interface, and this interface can be predictive of DGC-target protein interactions. Furthermore, we demonstrate that physical interaction is necessary for the DGC to maximally signal its target. If such “local signaling” is a theme for even a fraction of the DGCs used by bacteria, it becomes possible to posit a model whereby physical interaction allows a DGC to directly signal its target protein, which in turn may help curtail undesired cross talk with other members of the network. PMID:26670387

  5. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

    PubMed Central

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. DOI: http://dx.doi.org/10.7554/eLife.10766.001 PMID:26650353

  6. Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis.

    PubMed

    Zhou, Zhiwen; Tanaka, Kenji F; Matsunaga, Shigeru; Iseki, Mineo; Watanabe, Masakatsu; Matsuki, Norio; Ikegaya, Yuji; Koyama, Ryuta

    2016-01-01

    Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways. PMID:26795422

  7. In vitro susceptibility of fungi to acyclic inhibitors of 2,3-oxidosqualene cyclases.

    PubMed

    Airaudi, D; Ceruti, M; Bianco, C; Filipello Marchisio, V

    1996-01-01

    In the present study we determine the antifungal properties of two acyclic inhibitors of 2,3-oxidosqualene cyclases: 22,23-epoxy-2-aza-2,3-dihydrosqualene (EAS) and azasqualene alcohol (ASA). Fungistatic and fungicidal activity towards dermatophytes and other fungi involved in cutaneous and systemic infections was tested (48 isolates from 10 species). The tests were carried out by inoculating 10 microliters of mycelial homogenate in 1 ml of Sabouraud glucose liquid medium containing serial dilutions of 100 to 0.25 micrograms ml-1 of the substance. For each isolate, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of both compounds were determined. EAS was more active (MIC range 1.5-25 micrograms ml-1) than ASA (MIC range 3-50 micrograms ml-1). At the highest concentration tested, EAS also showed fungicidal action towards some isolates of Trichophyton mentagrophytes, T. terrestre, Epidermophyton floccosum, Microsporum canis and Scopulariopsis brumptii. The most sensitive species was T. mentagrophytes, the most resistant T. rubrum. PMID:8786759

  8. Identification of Small Molecules That Antagonize Diguanylate Cyclase Enzymes To Inhibit Biofilm Formation

    PubMed Central

    Sambanthamoorthy, Karthik; Sloup, Rudolph E.; Parashar, Vijay; Smith, Joshua M.; Kim, Eric E.; Semmelhack, Martin F.; Neiditch, Matthew B.

    2012-01-01

    Bacterial biofilm formation is responsible for numerous chronic infections, causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. New strategies to treat biofilm-based infections are critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. As this signaling system is found only in bacteria, it is an attractive target for the development of new antibiofilm interventions. Here, we describe the results of a high-throughput screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP. We report seven small molecules that antagonize these enzymes and inhibit biofilm formation by Vibrio cholerae. Moreover, two of these compounds significantly reduce the total concentration of c-di-GMP in V. cholerae, one of which also inhibits biofilm formation by Pseudomonas aeruginosa in a continuous-flow system. These molecules represent the first compounds described that are able to inhibit DGC activity to prevent biofilm formation. PMID:22850508

  9. In silico prediction of tyrosinase and adenylyl cyclase inhibitors from natural compounds.

    PubMed

    Fong, Pedro; Tong, Henry H Y; Chao, Chi M

    2014-02-01

    Although many herbal medicines are effective in the treatment of hyperpigmentation, the potency of different constituents remains unknown. In this work, more than 20,000 herbal ingredients from 453 herbs were docked into the crystal structures of adenylyl cyclase and a human homology tyrosinase model using Surflex-Dock. These two enzymes are responsible for melanin production and inhibition of them may attain a skin-whitening effect superior to currently available agents. The essential drug properties for topical formulation of the herbal ingredients, including skin permeability, sensitization, irritation, corrosive and carcinogenic properties were predicted by Dermwin, Skin Sensitization Alerts (SSA), Skin Irritation Corrosion Rules Estimation Tool (SICRET) and Benigni/Bossa rulebase module of Toxtree. Moreover, similarity ensemble and pharmacophore mapping approaches were used to forecast other potential targets for these herbal compounds by the software, SEArch and PharmMapper. Overall, this study predicted seven compounds to have advanced drug-like properties over the well-known effective tyrosinase inhibitors, arbutin and kojic acid. These seven compounds have the highest potential for further in vitro and in vivo investigation with the aim of developing safe and high-efficacy skin-whitening agents.

  10. Heterogeneity of Bordetella bronchiseptica adenylate cyclase (cyaA) RTX domain.

    PubMed

    Wehmann, Enikő; Khayer, Bernadett; Magyar, Tibor

    2015-01-01

    Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A-D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR-RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica. PMID:25475014

  11. The adenylyl cyclase Rv2212 modifies the proteome and infectivity of Mycobacterium bovis BCG.

    PubMed

    Pedroza-Roldán, César; Aceves-Sánchez, Michel de Jesús; Zaveri, Anisha; Charles-Niño, Claudia; Elizondo-Quiroga, Darwin Eduardo; Hernández-Gutiérrez, Rodolfo; Allen, Kirk; Visweswariah, Sandhya S; Flores-Valdez, Mario Alberto

    2015-01-01

    All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.

  12. DgcA, a diguanylate cyclase from Xanthomonas oryzae pv. oryzae regulates bacterial pathogenicity on rice

    PubMed Central

    Su, Jianmei; Zou, Xia; Huang, Liangbo; Bai, Tenglong; Liu, Shu; Yuan, Meng; Chou, Shan-Ho; He, Ya-Wen; Wang, Haihong; He, Jin

    2016-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice blight disease as well as a serious phytopathogen worldwide. It is also one of the model organisms for studying bacteria-plant interactions. Current progress in bacterial signal transduction pathways has identified cyclic di-GMP as a major second messenger molecule in controlling Xanthomonas pathogenicity. However, it still remains largely unclear how c-di-GMP regulates the secretion of bacterial virulence factors in Xoo. In this study, we focused on the important roles played by DgcA (XOO3988), one of our previously identified diguanylate cyclases in Xoo, through further investigating the phenotypes of several dgcA-related mutants, namely, the dgcA-knockout mutant ΔdgcA, the dgcA overexpression strain OdgcA, the dgcA complemented strain CdgcA and the wild-type strain. The results showed that dgcA negatively affected virulence, EPS production, bacterial autoaggregation and motility, but positively triggered biofilm formation via modulating the intracellular c-di-GMP levels. RNA-seq data further identified 349 differentially expressed genes controlled by DgcA, providing a foundation for a more solid understanding of the signal transduction pathways in Xoo. Collectively, the present study highlights DgcA as a major regulator of Xoo virulence, and can serve as a potential target for preventing rice blight diseases. PMID:27193392

  13. High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor.

    PubMed

    Pichlo, Magdalena; Bungert-Plümke, Stefanie; Weyand, Ingo; Seifert, Reinhard; Bönigk, Wolfgang; Strünker, Timo; Kashikar, Nachiket Dilip; Goodwin, Normann; Müller, Astrid; Pelzer, Patric; Van, Qui; Enderlein, Jörg; Klemm, Clementine; Krause, Eberhard; Trötschel, Christian; Poetsch, Ansgar; Kremmer, Elisabeth; Kaupp, U Benjamin; Körschen, Heinz G; Collienne, Ursel

    2014-08-18

    Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3',5'-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 10(5) GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces "molecule noise." Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons.

  14. Identification of small molecules inhibiting diguanylate cyclases to control bacterial biofilm development.

    PubMed

    Sambanthamoorthy, Karthik; Luo, Chunyuan; Pattabiraman, Nagarajan; Feng, Xiarong; Koestler, Benjamin; Waters, Christopher M; Palys, Thomas J

    2014-01-01

    Biofilm formation by pathogenic bacteria is an important virulence factor in the development of numerous chronic infections, thereby causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. An urgent need for new strategies to treat biofilm-based infections is critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. The absence of this signalling system in higher eukaryotes makes it an attractive target for the development of new anti-biofilm agents. In this study, the results of an in silico pharmacophore-based screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP are described. Four small molecules, LP 3134, LP 3145, LP 4010 and LP 1062 that antagonize these enzymes and inhibit biofilm formation by Pseudomonas aeruginosa and Acinetobacter baumannii in a continuous-flow system are reported. All four molecules dispersed P. aeruginosa biofilms and inhibited biofilm development on urinary catheters. One molecule dispersed A. baumannii biofilms. Two molecules displayed no toxic effects on eukaryotic cells. These molecules represent the first compounds identified from an in silico screen that are able to inhibit DGC activity to prevent biofilm formation. PMID:24117391

  15. Characterisation of two oxidosqualene cyclases responsible for triterpenoid biosynthesis in Ilex asprella.

    PubMed

    Zheng, Xiasheng; Luo, Xiuxiu; Ye, Guobing; Chen, Ye; Ji, Xiaoyu; Wen, Lingling; Xu, Yaping; Xu, Hui; Zhan, Ruoting; Chen, Weiwen

    2015-02-05

    Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expression in the yeast Saccharomyces cerevisiae. Analysis of the resulting products by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) showed that both genes encode a mixed amyrin synthase, producing α-amyrin and β-amyrin at different ratios. IaAS1, which mainly produces α-amyrin, is the second triterpene synthase so far identified in which the level of α-amyrin produced is ≥80% of total amyrin production. By contrast, IaAS2 mainly synthesises β-amyrin, with a yield of 95%. Gene expression patterns of these two amyrin synthases in roots and leaves of I. asprella were found to be consistent with the content patterns of total saponins. Finally, phylogenetic analysis and multiple sequence alignment of the two amyrin synthases against several known OSCs from other plants were conducted to further elucidate their evolutionary relationship.

  16. Identification of small molecules inhibiting diguanylate cyclases to control bacterial biofilm development.

    PubMed

    Sambanthamoorthy, Karthik; Luo, Chunyuan; Pattabiraman, Nagarajan; Feng, Xiarong; Koestler, Benjamin; Waters, Christopher M; Palys, Thomas J

    2014-01-01

    Biofilm formation by pathogenic bacteria is an important virulence factor in the development of numerous chronic infections, thereby causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. An urgent need for new strategies to treat biofilm-based infections is critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. The absence of this signalling system in higher eukaryotes makes it an attractive target for the development of new anti-biofilm agents. In this study, the results of an in silico pharmacophore-based screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP are described. Four small molecules, LP 3134, LP 3145, LP 4010 and LP 1062 that antagonize these enzymes and inhibit biofilm formation by Pseudomonas aeruginosa and Acinetobacter baumannii in a continuous-flow system are reported. All four molecules dispersed P. aeruginosa biofilms and inhibited biofilm development on urinary catheters. One molecule dispersed A. baumannii biofilms. Two molecules displayed no toxic effects on eukaryotic cells. These molecules represent the first compounds identified from an in silico screen that are able to inhibit DGC activity to prevent biofilm formation.

  17. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3.

    PubMed

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. PMID:26650353

  18. Guanylyl cyclase C in colorectal cancer: susceptibility gene and potential therapeutic target.

    PubMed

    Lin, Jieru E; Li, Peng; Pitari, Giovanni M; Schulz, Stephanie; Waldman, Scott A

    2009-05-01

    Colorectal cancer is one of the leading causes of tumor-related morbidity and mortality worldwide. While mechanisms underlying this disease have been elucidated over the past two decades, these molecular insights have failed to translate into efficacious therapy. The oncogenomic view of cancer suggests that terminal transformation reflects the sequential corruption of signal transduction circuits regulating key homeostatic mechanisms, whose multiplicity underlies the therapeutic resistance of most tumors to interventions targeting individual pathways. Conversely, the paucity of mechanistic insights into proximal pathophysiological processes that initiate and amplify oncogenic circuits preceding accumulation of mutations and transformation impedes development of effective prevention and therapy. In that context, guanylyl cyclase C (GCC), the intestinal receptor for the paracrine hormones guanylin and uroguanylin, whose early loss characterizes colorectal transformation, has emerged as a component of lineage-specific homeostatic programs organizing spatiotemporal patterning along the crypt-surface axis. Dysregulation of GCC signaling, reflecting hormone loss, promotes tumorigenesis through reprogramming of replicative and bioenergetic circuits and genomic instability. Compensatory upregulation of GCC in response to hormone loss provides a unique translational opportunity for prevention and treatment of colorectal tumors by hormone-replacement therapy.

  19. Functional analysis of the sporulation-specific diadenylate cyclase CdaS in Bacillus thuringiensis

    PubMed Central

    Zheng, Cao; Ma, Yang; Wang, Xun; Xie, Yuqun; Ali, Maria K.; He, Jin

    2015-01-01

    Cyclic di-AMP (c-di-AMP) is a recently discovered bacterial secondary messenger molecule, which is associated with various physiological functions. In the genus Bacillus, the intracellular level and turnover of c-di-AMP are mainly regulated by three diadenylate cyclases (DACs), including DisA, CdaA and CdaS, and two c-di-AMP-specific phosphodiesterases (GdpP and PgpH). In this study, we demonstrated that CdaS protein from B. thuringiensis is a hexameric DAC protein that can convert ATP or ADP to c-di-AMP in vitro and the N-terminal YojJ domain is essential for the DAC activity. Based on the markerless gene knock-out method, we demonstrated that the transcription of cdaS was initiated by the sporulation-specific sigma factor σH and the deletion of cdaS significantly delayed sporulation and parasporal crystal formation. These findings contrast with similar experiments conducted using B. subtilis, wherein transcription of its cdaS was initiated by the sigma factor σG. Deletion of all the three DAC genes from a single strain was unsuccessful, suggesting that c-di-AMP is an indispensable molecule in B. thuringiensis. Phylogenetic analysis indicated increased diversity of CdaS in the B. cereus and B. subtilis Bacillus subgroups. In summary, this study identifies important aspects in the regulation of c-di-AMP in the genus Bacillus. PMID:26441857

  20. Multiple diguanylate cyclase-coordinated regulation of pyoverdine synthesis in Pseudomonas aeruginosa.

    PubMed

    Chen, Yicai; Yuan, Mingjun; Mohanty, Anee; Yam, Joey Kuok Hoong; Liu, Yang; Chua, Song Lin; Nielsen, Thomas E; Tolker-Nielsen, Tim; Givskov, Michael; Cao, Bin; Yang, Liang

    2015-06-01

    The nucleotide signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays an essential role in regulating microbial virulence and biofilm formation. C-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. One intrinsic feature of c-di-GMP signalling is the abundance of DGCs and PDEs encoded by many bacterial species. It is unclear whether the different DGCs or PDEs coordinately establish the c-di-GMP regulation or function independently of each other. Here, we provide evidence that multiple DGCs are involved in regulation of c-di-GMP on synthesis of the major iron siderophore pyoverdine in Pseudomonas aeruginosa. Constitutive expression of the WspG or YedQ DGC in P. aeruginosa is able to induce its pyoverdine synthesis. Induction of pyoverdine synthesis by high intracellular c-di-GMP depends on the synthesis of exopolysaccharides and another two DGCs, SiaD and SadC. SiaD was found to boost the c-di-GMP synthesis together with constitutively expressing YedQ. The exopolysaccharides and the SiaD DGC were found to modulate the expression of the RsmY/RsmZ ncRNAs. Induction of the RsmY/RsmZ ncRNAs might enhance the pyoverdine synthesis through SadC. Our study sheds light on a novel multiple DGC-coordinated c-di-GMP regulatory mechanism of bacteria. PMID:25683454

  1. Soluble adenylyl cyclase is not required for axon guidance to netrin-1.

    PubMed

    Moore, Simon W; Lai Wing Sun, Karen; Xie, Fang; Barker, Philip A; Conti, Marco; Kennedy, Timothy E

    2008-04-01

    During development, axons are directed to their targets by extracellular guidance cues. The axonal response to the guidance cue netrin-1 is profoundly influenced by the concentration of cAMP within the growth cone. In some cases, cAMP affects the sensitivity of the growth cone to netrin-1, whereas in others it changes the response to netrin-1 from attraction to repulsion. The effects of cAMP on netrin-1 action are well accepted, but the critical issue of whether cAMP production is activated by a netrin-1 induced signaling cascade remains uncertain. A previous report has suggested that axon guidance in response to netrin-1 requires cAMP production mediated by soluble adenyl cyclase (sAC). We have used genetic, molecular and biochemical strategies to assess this issue. Surprisingly, we found only extremely weak expression of sAC in embryonic neurons and determined that, under conditions where netrin-1 directs axonal pathfinding, exposure to netrin-1 does not alter cAMP levels. Furthermore, although netrin-1-deficient mice exhibit major axon guidance defects, we show that pathfinding is normal in sAC-null mice. Therefore, although cAMP can alter the response of axons to netrin-1, we conclude that netrin-1 does not alter cAMP levels in axons attracted by this cue, and that sAC is not required for axon attraction to netrin-1. PMID:18400890

  2. Adenylyl Cyclase Plays a Regulatory Role in Development, Stress Resistance and Secondary Metabolism in Fusarium fujikuroi

    PubMed Central

    García-Martínez, Jorge; Ádám, Attila L.; Avalos, Javier

    2012-01-01

    The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. Here we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments, reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, ΔacyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress (H2O2), but no change in heavy metal resistance or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group. PMID:22291883

  3. Progesterone prevents linkage of rabbit myometrial alpha 2-adrenergic receptors to inhibition of adenylate cyclase.

    PubMed

    Wu, Y Y; Riemer, R K; Goldfien, A; Roberts, J M

    1989-04-01

    The uterine response to adrenergic stimulation is determined by the hormonal milieu. This response is particularly well characterized in the rabbit. In this species, as in humans, the response of the uterus to sympathetic stimulation is alpha-adrenergically mediated contraction with elevated circulating estrogen. However, with progesterone predominance, similar stimulation inhibits uterine contractions, a response mediated by beta-adrenergic receptors acting through their second message, cyclic adenosine monophosphate. We studied the mechanisms by which sex steroids regulate myometrial adrenergic responses. In this study, we questioned whether part of the effect of sex steroids could be explained by an alteration of the coupling of the alpha 2-adrenergic receptor to the inhibition of adenylate cyclase. We found that in the progesterone-treated rabbit, although alpha 2-receptors are present, they are not linked to inhibition of cyclic adenosine monophosphate synthesis. The net synthesis of cyclic adenosine monophosphage in response to endogenous catecholamines is determined by their activation of beta-adrenergic receptors to increase and alpha 2-receptors to decrease cyclic adenosine monophosphate formation. Thus the uncoupling of alpha 2-receptors contributes to increased intracellular cyclic adenosine monophosphate in myometrium of progesterone-treated animals consistent with the reported predominance of beta-adrenergic contractile responses in this setting.

  4. Activity Regulation by Heteromerization of Arabidopsis Allene Oxide Cyclase Family Members.

    PubMed

    Otto, Markus; Naumann, Christin; Brandt, Wolfgang; Wasternack, Claus; Hause, Bettina

    2016-01-01

    Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 + AOC1 and AOC4 + AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles. PMID:27135223

  5. cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects.

    PubMed

    Beckert, Ulrike; Grundmann, Manuel; Wolter, Sabine; Schwede, Frank; Rehmann, Holger; Kaever, Volkhard; Kostenis, Evi; Seifert, Roland

    2014-09-01

    In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects. PMID:25108158

  6. Distribution of microbial terpenoid lipid cyclases in the global ocean metagenome.

    PubMed

    Pearson, Ann; Rusch, Douglas B

    2009-03-01

    The bacterial terpenoid lipids known as hopanoids are fundamental tools for interpreting ancient microbial communities. Their degradation products, the hopanes, are found in sedimentary rocks throughout the geologic record. These compounds are presumed to be analogous to the sterols of eukaryotes, yet although the eukaryotic requirement for sterols is universal, hopanoid biosynthetic capacity is not ubiquitous among marine bacteria. Among the 9.8 million shotgun reads from the Sorcerer II Global Ocean Sampling (GOS) expedition, 148 contain putative coding sequence for bacterial squalene-hopene cyclases (SHCs). SHCs encoded by alpha-Proteobacteria potentially related to Rhodospirillaceae dominate these hits, especially in the open ocean and in tropical regions. Planctomycetes and beta-Proteobacteria contribute more SHC-encoding sequences, and therefore presumably more hopanoid production, to coastal and temperate environments. Although sequences nominally related to alpha- and beta-Proteobacteria outnumber other taxa in marine and coastal environments, there is large phylogenetic distance between GOS sequences and known species. Assuming that the environments sampled here are broadly representative of a wide range of surface ocean climates, depositional settings and temporal periods, the data suggest a fundamental function for Proteobacteria in the development of the geologic record of hopanes.

  7. Heterogeneity of Bordetella bronchiseptica adenylate cyclase (cyaA) RTX domain.

    PubMed

    Wehmann, Enikő; Khayer, Bernadett; Magyar, Tibor

    2015-01-01

    Bordetella bronchiseptica is a widespread pathogen, with a broad host range, occasionally including humans. Diverse virulence factors (adhesins, toxins) allow its adaptation to its host, but this property of the adenylate cyclase (cyaA) toxin is not well understood. In this study, we analyzed the repeats-in-toxin domain of B. bronchiseptica cyaA with PCR, followed by restriction fragment length analysis. Of ninety-two B. bronchiseptica strains collected from different hosts and geographic regions, 72 (78.3 %) carried cyaA and four RFLP types (A-D) were established using NarI and SalI. However, in 20 strains, cyaA was replaced with a peptide transport protein operon. A phylogenetic tree based on partial nucleotide sequences of cyaA revealed that group 2 contains strains of specifically human origin, whereas subgroup 1a contains all but one of the strains from pigs. The human strains showed many PCR-RFLP and sequence variants, confirming the clonal population structure of B. bronchiseptica.

  8. Structure of RNA 3′-phosphate cyclase bound to substrate RNA

    PubMed Central

    Desai, Kevin K.; Bingman, Craig A.; Cheng, Chin L.; Phillips, George N.

    2014-01-01

    RNA 3′-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3′-phosphate to form a 2′,3′-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA–AMP and RNA(3′)pp(5′)A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3′-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3′-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3′-phosphate is poised for in-line attack on the P–N bond that links the phosphorous atom of AMP to Nε of His307. Thus, we provide the first insights into RNA 3′-phosphate termini recognition and the mechanism of 3′-phosphate activation by an Rtc enzyme. PMID:25161314

  9. KMUP-1 inhibits H441 lung epithelial cell growth, migration and proinflammation via increased NO/CGMP and inhibited RHO kinase/VEGF signaling pathways.

    PubMed

    Wu, B N; Chen, H Y; Liu, C P; Hsu, L Y; Chen, I J

    2011-01-01

    This study investigates whether KMUP-1 protects soluble guanylate cyclase (sGC) and inhibits vascular endothelial growth factor (VEGF) expression in lung epithelial cells in hypoxia, therapeutically targeting epithelial proinflammation. H441 cells were used as a representative epithelial cell line to examine the role of sGC and VEGF in hypoxia and the anti-proinflammatory activity of KMUP-1 in normoxia. Human H441 cells were grown in hypoxia for 24-72 h. KMUP-1 (1, 10, 100 microM) arrested cells at the G0/G1 phase of the cell cycle, reduced cell survival and migration, increased p21/p27, restored eNOS, increased soluble guanylate cyclase (sGC) and PKG and inhibited Rho kinase II (ROCK-II). KMUP-1 (0.001-0.1 microM) concentration dependently increased eNOS in normoxia and did not inhibit phosphodiesterase-5A (PDE-5A) in hypoxic cells. Hypoxia-induced factor-1alpha (HIF-1alpha) and VEGF were suppressed by KMUP-1 but not by L-NAME (100 microM). The PKG inhibitor Rp-8-CPT-cGMPS (10 microM) blunted the inhibition of ROCK-II by KMUP-1. KMUP-1 inhibited thromboxane A2-mimetic agonist U46619-induced PDE-5A, TNF-alpha (100 ng/ml)-induced iNOS, and ROCK-II and associated phospho-p38 MAPK, suggesting multiple anti-proinflammatory activities. In addition, increased p21/p27 by KMUP-1 at higher concentrations might contribute to an increased Bax/Bcl-2 and active caspase-3/procaspase-3 ratio, concomitantly causing apoptosis. KMUP-1 inhibited ROCK-II/VEGF in hypoxia, indicating its anti-neoplastic and anti-inflammatory properties. KMUP-1 inhibited TNF-alpha-induced iNOS and U46619-induced PDE-5A and phospho-p38 MAPK in normoxia, confirming its anti-proinflammatory action. KMUP-1 could be used as an anti-proinflammatory to reduce epithelial inflammation.

  10. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice

    PubMed Central

    Wang, Zhenshan; Zhou, Yanfen; Luo, Yingtao; Zhang, Jing; Zhai, Yunpeng; Yang, Dong; Zhang, Zhe; Li, Yongchao; Storm, Daniel R.; Ma, Runlin Z.

    2015-01-01

    Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/−) and wild-type (AC3+/+) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE. PMID:26633363

  11. Isolation and Functional Characterization of a Lycopene β-cyclase Gene Promoter from Citrus

    PubMed Central

    Lu, Suwen; Zhang, Yin; Zheng, Xiongjie; Zhu, Kaijie; Xu, Qiang; Deng, Xiuxin

    2016-01-01

    Lycopene β-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway. However, the transcriptional regulatory mechanisms of LCYb1 in citrus with abundant carotenoid accumulation are still unclear. To understand the molecular basis of CsLCYb1 expression, we isolated and functionally characterized the 5′ upstream sequences of CsLCYb1 from citrus. The full-length CsLCYb1 promoter and a series of its 5′ deletions were fused to the β-glucuronidase (GUS) reporter gene and transferred into different plants (tomato, Arabidopsis and citrus callus) to test the promoter activities. The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity, and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity. Furthermore, the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis, and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments. Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between -574 to -513 bp and conferring strong promoter activity. The copy numbers of the enhancer element differed among various citrus species, leading to the development of a derived simple sequence repeat marker to distinguish different species. In conclusion, this study elucidates the expression characteristics of the LCYb1 promoter from citrus and further identifies a novel enhancer element required for the promoter activity. The characterized promoter fragment would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements.

  12. Biochemical mechanisms of myocardial adenylate cyclase subsensitivity to isoproterenol in cardiac hypertrophy of spontaneously hypertensive rats

    SciTech Connect

    Cheon, J.W.

    1986-01-01

    The responsiveness of the myocardial adenylate cyclase (AC) system in generating cAMP was studied using isoproterenol (a beta-adrenergic receptor agonist), cholera toxin (a guanosinetriphosphatase inhibitor) and forskolin (a catalytic unit activator) in isolated myocytes of age-matched, 14-17 weeks old Wistar Kyoto normotensive rates (WKYs) and spontaneously hypertensive rats (SHRs). We found a reduction in isoproterenol-stimulated cAMP formation in myocytes of SHRs compared with WKYs. This reduction was not due to changes in isoproterenol-receptor interactions. Scatchard plot analysis of (/sup 3/H)CGP 12177 binding to beta-adrenergic receptors in isolated myocytes of WKYs and SHRs revealed to significant differences in the maximum number of binding sites or dissociation constant. There were no significant differences in Ki and IC/sub 50/ calculated from the competitive displacement of (/sup 3/H)CGP 12177 binding by (-) isoproterenol, suggesting no change in the affinity of the beta-adrenergic receptors for isoproterenol. We found no significant differences in forskolin-stimulated cAMP formation between the two groups. This suggest that the reduction in isoproterenol-stimulated cAMP formation observed in myocytes of SHRs is not due to changes in the ability of catalytic unit to convert ATP to cAMP. Interestingly, cholera toxin-stimulated cAMP formation was increased in myocytes of SHRs. One possible explanation for these observations may be increased guanosinetriphosphatase (GTPase) activation by isoproterenol in myocytes of SHRs. The activation of GTPase by isoproterenol in myocytes of SHRs. The activation of GTPase by isoproterenol was measured as the release of Pi from (..gamma..-/sup 32/P)GTP. There was an increase in isoproterenol-stimulated GTPase activity in myocytes of SHRs compared with WKYs.

  13. Isolation and Functional Characterization of a Lycopene β-cyclase Gene Promoter from Citrus.

    PubMed

    Lu, Suwen; Zhang, Yin; Zheng, Xiongjie; Zhu, Kaijie; Xu, Qiang; Deng, Xiuxin

    2016-01-01

    Lycopene β-cyclases are key enzymes located at the branch point of the carotenoid biosynthesis pathway. However, the transcriptional regulatory mechanisms of LCYb1 in citrus with abundant carotenoid accumulation are still unclear. To understand the molecular basis of CsLCYb1 expression, we isolated and functionally characterized the 5' upstream sequences of CsLCYb1 from citrus. The full-length CsLCYb1 promoter and a series of its 5' deletions were fused to the β-glucuronidase (GUS) reporter gene and transferred into different plants (tomato, Arabidopsis and citrus callus) to test the promoter activities. The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity, and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity. Furthermore, the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis, and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments. Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between -574 to -513 bp and conferring strong promoter activity. The copy numbers of the enhancer element differed among various citrus species, leading to the development of a derived simple sequence repeat marker to distinguish different species. In conclusion, this study elucidates the expression characteristics of the LCYb1 promoter from citrus and further identifies a novel enhancer element required for the promoter activity. The characterized promoter fragment would be an ideal candidate for genetic engineering and seeking of upstream trans-acting elements. PMID:27679644

  14. Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

    PubMed

    Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

    2013-11-15

    Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses.

  15. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

    PubMed Central

    Ohki, Mio; Sugiyama, Kanako; Kawai, Fumihiro; Tanaka, Hitomi; Nihei, Yuuki; Unzai, Satoru; Takebe, Masumi; Matsunaga, Shigeru; Adachi, Shin-ichi; Shibayama, Naoya; Zhou, Zhiwen; Koyama, Ryuta; Takahashi, Tetsuo; Tame, Jeremy R. H.; Iseki, Mineo; Park, Sam-Yong

    2016-01-01

    Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit. PMID:27247413

  16. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating.

    PubMed

    Hurley, Matthew M; Maunze, Brian; Block, Megan E; Frenkel, Mogen M; Reilly, Michael J; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive. PMID:27597817

  17. Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis

    PubMed Central

    Mann, Francis M.; Prisic, Sladjana; Hu, Huayou; Xu, Meimei; Coates, Robert M.; Peters, Reuben J.

    2009-01-01

    Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans. PMID:19574210

  18. Soluble Adenylyl Cyclase Is Required for Retinal Ganglion Cell and Photoreceptor Differentiation

    PubMed Central

    Shaw, Peter X.; Fang, Jiahua; Sang, Alan; Wang, Yan; Kapiloff, Michael S.; Goldberg, Jeffrey L.

    2016-01-01

    Purpose We have previously demonstrated that soluble adenylyl cyclase (sAC) is necessary for retinal ganglion cell (RGC) survival and axon growth. Here, we further investigate the role of sAC in neuronal differentiation during retinal development. Methods Chx10 or Math5 promoter-driven Cre-Lox recombination were used to conditionally delete sAC from early and intermediate retinal progenitor cells during retinal development. We examined cell type–specific markers expressed by retinal cells to estimate their relative numbers and characterize retinal laminar morphology by immunofluorescence in adult and newborn mice. Results Retinal ganglion cell and amacrine cell markers were significantly lower in the retinas of adult Math5cre/sACfl/fl and Chx10cre/sACfl/fl mice than in those of wild-type controls. The effect on RGC development was detectable as early as postnatal day 1 and deleting sAC in either Math5- or Chx10-expressing retinal progenitor cells also reduced nerve fiber layer thickness into adulthood. The thickness of the photoreceptor layer was slightly but statistically significantly decreased in both the newborn Chx10cre/sACfl/fl and Math5cre/sACfl/fl mice, but this reduction and abnormal morphology persisted in the adults in only the Chx10cre/sACfl/fl mice. Conclusions sAC plays an important role in the early retinal development of RGCs as well as in the development of amacrine cells and to a lesser degree photoreceptors. PMID:27679853

  19. The plant natriuretic peptide receptor is a guanylyl cyclase and enables cGMP-dependent signaling.

    PubMed

    Turek, Ilona; Gehring, Chris

    2016-06-01

    The functional homologues of vertebrate natriuretic peptides (NPs), the plant natriuretic peptides (PNPs), are a novel class of peptidic hormones that signal via guanosine 3',5'-cyclic monophosphate (cGMP) and systemically affect plant salt and water balance and responses to biotrophic plant pathogens. Although there is increasing understanding of the complex roles of PNPs in plant responses at the systems level, little is known about the underlying signaling mechanisms. Here we report isolation and identification of a novel Leucine-Rich Repeat (LRR) protein that directly interacts with A. thaliana PNP, AtPNP-A. In vitro binding studies revealed that the Arabidopsis AtPNP-A binds specifically to the LRR protein, termed AtPNP-R1, and the active region of AtPNP-A is sufficient for the interaction to occur. Importantly, the cytosolic part of the AtPNP-R1, much like in some vertebrate NP receptors, harbors a catalytic center diagnostic for guanylyl cyclases and the recombinant AtPNP-R1 is capable of catalyzing the conversion of guanosine triphosphate to cGMP. In addition, we show that AtPNP-A causes rapid increases of cGMP levels in wild type (WT) leaf tissue while this response is significantly reduced in the atpnp-r1 mutants. AtPNP-A also causes cGMP-dependent net water uptake into WT protoplasts, and hence volume increases, whereas responses of the protoplasts from the receptor mutant are impaired. Taken together, our results suggest that the identified LRR protein is an AtPNP-A receptor essential for the PNP-dependent regulation of ion and water homeostasis in plants and that PNP- and vertebrate NP-receptors and their signaling mechanisms share surprising similarities. PMID:26945740

  20. Hemodynamic actions of systemically injected pituitary adenylate cyclase activating polypeptide-27 in the rat

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The aims of this study were (1) to characterize the hemodynamic mechanisms underlying the hypotensive effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP-27 0.1-2.0 nmol/kg, i.v.) in pentobarbital-anesthetized rats, and (2) to determine the roles of the autonomic nervous system, adrenal catecholamines and endothelium-derived nitric oxide (NO) in the expression of PACAP-27-mediated effects on hemodynamic function. PACAP-27 produced dose-dependent decreases in mean arterial blood pressure and hindquarter and mesenteric vascular resistances in saline-treated rats. PACAP-27 also produced pronounced falls in mean arterial blood pressure in rats treated with the ganglion blocker, chlorisondamine (5 mg/kg, i.v.). The hypotensive and vasodilator actions of PACAP-27 were not attenuated by the beta-adrenoceptor antagonist, propranolol (1 mg/kg, i.v.), or the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME 50 micromol/kg, i.v.). PACAP-27 produced dose-dependent increases in heart rate whereas the hypotensive response produced by the nitrovasodilator, sodium nitroprusside (10 microg/kg, i.v.), was associated with a minimal tachycardia. The PACAP-27-induced tachycardia was unaffected by chlorisondamine, but was virtually abolished by propranolol. These results suggest that the vasodilator effects of PACAP-27 are due to actions in the microcirculation rather than to the release of adrenal catecholamines and that this vasodilation may not involve the release of endothelium-derived NO. These results also suggest that PACAP-27 produces tachycardia by directly releasing norepinephrine from cardiac sympathetic nerve terminals rather than by direct or baroreceptor reflex-mediated increases in sympathetic nerve activity.

  1. Effects of abstinence and family history for alcoholism on platelet adenylyl cyclase activity.

    PubMed

    Menninger, J A; Barón, A E; Tabakoff, B

    1998-12-01

    Platelet adenylyl cyclase (AC) activity was measured in 32 alcohol-dependent subjects and 27 control subjects who were categorized as either family history-positive (FHP) or family history-negative (FHN) for alcoholism. The interview and blood sample collections were performed shortly after cessation of heavy drinking in the alcoholic group, and repeat blood samples were obtained at the end of the first and second weeks of monitored abstinence. Control subjects received the same interview and provided blood samples at the time of the interview. When subjects were not segregated for FHP or FHN status, there were no statistically significant differences in basal, cesium fluoride (CsF)-, or forskolin-stimulated mean AC activities between the controls and the alcoholics, at study entry or with 1 or 2 weeks of abstinence. On the other hand, over the 2-week course of sobriety from heavy drinking, the CsF-stimulated AC activity of FHP alcohol-dependent subjects decreased significantly (p = 0.03). FHP alcohol-dependent subjects after 2 weeks of sobriety had significantly lower mean CsF-stimulated AC activity than FHN controls (p = 0.04), whereas the FHN alcoholic subjects' CsF-stimulated AC activity did not differ significantly from FHN controls at this point in time. When all subjects were pooled and then categorized as either FHP or FHN, there was a significant difference in mean CsF-stimulated AC activity (p = 0.02) between the FHP and FHN subject groups. Genetic factors and abstinence appear to have roles in determining low platelet AC activity in alcoholic and nonalcoholic subjects. CsF-stimulated platelet AC activity, in particular, appears to act as a trait marker for a genetic vulnerability to developing alcoholism, but recent heavy drinking in male alcoholics is a factor that can mask differences between FHP and FHN subjects.

  2. Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal.

    PubMed

    Dhar, Niha; Rana, Satiander; Razdan, Sumeer; Bhat, Wajid Waheed; Hussain, Aashiq; Dhar, Rekha S; Vaishnavi, Samantha; Hamid, Abid; Vishwakarma, Ram; Lattoo, Surrinder K

    2014-06-13

    Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, β-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS), and cycloartenol synthase (WsOSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool toward WsOSC/CS for increased withanolide production.

  3. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    D Gallagher; S Kim; H Robinson; P Reddy

    2011-12-31

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV) - two with substrate analogs and one with product. M