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Sample records for soluble guanylyl cyclases

  1. Characterization of two unusual guanylyl cyclases from Dictyostelium

    NARCIS (Netherlands)

    Roelofs, Jeroen; Haastert, Peter J.M. van

    2002-01-01

    Guanylyl cyclase A (GCA) and soluble guanylyl cyclase (sGC) encode GCs in Dictyostelium and have a topology similar to 12-transmembrane and soluble adenylyl cyclase, respectively. We demonstrate that all detectable GC activity is lost in a cell line in which both genes have been inactivated. Cell

  2. Hypoxic Vasospasm Mediated by cIMP: When Soluble Guanylyl Cyclase Turns Bad.

    Science.gov (United States)

    Gao, Yuansheng; Chen, Zhengju; Leung, Susan W S; Vanhoutte, Paul M

    2015-06-01

    In a number of isolated blood vessel types, hypoxia causes an acute contraction that is dependent on the presence of nitric oxide and activation of soluble guanylyl cyclase. It is more pronounced when the preparations are constricted and is therefore termed hypoxic augmentation of vasoconstriction. This hypoxic response is accompanied by increases in the intracellular level of inosine 5'-triphosphate and in the synthesis of inosine 3',5'-cyclic monophosphate (cIMP) by soluble guanylyl cyclase. The administration of exogenous cIMP or inosine 5'-triphosphate causes augmented vasoconstriction to hypoxia. Furthermore, the vasoconstriction evoked by hypoxia and cIMP is associated with increased activity of Rho kinase (ROCK), indicating that cIMP may mediate the hypoxic effect by sensitizing the myofilaments to Ca through ROCK. Hypoxia is implicated in exaggerated vasoconstriction in the pathogenesis of coronary artery disease, myocardial infarction, hypertension, and stroke. The newly found role of cIMP may help to identify unique therapeutic targets for certain cardiovascular disorders.

  3. The guanylyl cyclase family at Y2K.

    Science.gov (United States)

    Wedel, B; Garbers, D

    2001-01-01

    During the 1980s the purification, cloning, and expression of various forms of guanylyl cyclase (GC) revealed that they served as receptors for extracellular signals. Seven membrane forms, which presumably exist as homodimers, and four subunits of apparent heterodimers (commonly referred to as the soluble forms) are known, but in animals such as nematodes, much larger numbers of GCs are expressed. The number of transmembrane segments (none, one, or multiple) divide the GC family into three groups. Those with no or one transmembrane segment bind nitric oxide/carbon monoxide (NO/CO) or peptides. There are no known ligands for the multiple transmembrane segment class of GCs. Mutational and structural analyses support a model where catalysis requires a shared substrate binding site between the subunits, whether homomeric or heteromeric in nature. Because some cyclases or cyclase ligand genes lack specific GC inhibitors, disruption of either has been used to define the functions of individual cyclases, as well as to define human genetic disease counterparts.

  4. cCMP, cUMP, cTMP, cIMP and cXMP as possible second messengers: development of a hypothesis based on studies with soluble guanylyl cyclase α(1)β(1).

    Science.gov (United States)

    Beste, Kerstin Y; Seifert, Roland

    2013-02-01

    Adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate are second messengers that regulate multiple physiological functions. The existence of additional cyclic nucleotides in mammalian cells was postulated many years ago, but technical problems hampered development of the field. Using highly specific and sensitive mass spectrometry methods, soluble guanylyl cyclase has recently been shown to catalyze the formation of several cyclic nucleotides in vitro. This minireview discusses the broad substrate-specificity of soluble guanylyl cyclase and the possible second messenger roles of cyclic nucleotides other than adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate. We hope that this article stimulates productive and critical research in an area that has been neglected for many years.

  5. Biased activity of soluble guanylyl cyclase: the Janus face of thymoquinone.

    Science.gov (United States)

    Detremmerie, Charlotte; Vanhoutte, Paul M; Leung, Susan

    2017-07-01

    The natural compound thymoquinone, extracted from Nigella sativa (black cumin), is widely used in humans for its anti-oxidative properties. Thymoquinone is known for its acute endothelium-independent vasodilator effects in isolated rat aortae and pulmonary arteries, depending in part on activation of adenosine triphosphate-sensitive potassium channels and inhibition of voltage-dependent calcium channels. The compound also improves endothelial dysfunction in mesenteric arteries of ageing rodents and in aortae of rabbits treated with pyrogallol, by inhibiting oxidative stress. Serendipitously, thymoquinone was found to augment contractions in isolated arteries with endothelium of both rats and pigs. The endothelium-dependent augmentation it causes counterintuitively depends on biased activation of soluble guanylyl cyclase (sGC) producing inosine 3',5'-cyclic monophosphate (cyclic IMP) rather than guanosine 3',5'-cyclic monophosphate. This phenomenon shows a striking mechanistic similarity to the hypoxic augmentation previously observed in porcine coronary arteries. The cyclic IMP preferentially produced under thymoquinone exposure causes an increased contractility of arterial smooth muscle by interfering with calcium homeostasis. This brief review summarizes the vascular pharmacology of thymoquinone, focussing in particular on how the compound causes endothelium-dependent contractions by biasing the activity of sGC.

  6. Biased activity of soluble guanylyl cyclase: the Janus face of thymoquinone

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    Charlotte Detremmerie

    2017-07-01

    Full Text Available The natural compound thymoquinone, extracted from Nigella sativa (black cumin, is widely used in humans for its anti-oxidative properties. Thymoquinone is known for its acute endothelium-independent vasodilator effects in isolated rat aortae and pulmonary arteries, depending in part on activation of adenosine triphosphate-sensitive potassium channels and inhibition of voltage-dependent calcium channels. The compound also improves endothelial dysfunction in mesenteric arteries of ageing rodents and in aortae of rabbits treated with pyrogallol, by inhibiting oxidative stress. Serendipitously, thymoquinone was found to augment contractions in isolated arteries with endothelium of both rats and pigs. The endothelium-dependent augmentation it causes counterintuitively depends on biased activation of soluble guanylyl cyclase (sGC producing inosine 3ʹ,5ʹ-cyclic monophosphate (cyclic IMP rather than guanosine 3ʹ,5ʹ-cyclic monophosphate. This phenomenon shows a striking mechanistic similarity to the hypoxic augmentation previously observed in porcine coronary arteries. The cyclic IMP preferentially produced under thymoquinone exposure causes an increased contractility of arterial smooth muscle by interfering with calcium homeostasis. This brief review summarizes the vascular pharmacology of thymoquinone, focussing in particular on how the compound causes endothelium-dependent contractions by biasing the activity of sGC.

  7. Mapping Soluble Guanylyl Cyclase and Protein Disulfide Isomerase Regions of Interaction.

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    Erin J Heckler

    Full Text Available Soluble guanylyl cyclase (sGC is a heterodimeric nitric oxide (NO receptor that produces cyclic GMP. This signaling mechanism is a key component in the cardiovascular system. NO binds to heme in the β subunit and stimulates the catalytic conversion of GTP to cGMP several hundred fold. Several endogenous factors have been identified that modulate sGC function in vitro and in vivo. In previous work, we determined that protein disulfide isomerase (PDI interacts with sGC in a redox-dependent manner in vitro and that PDI inhibited NO-stimulated activity in cells. To our knowledge, this was the first report of a physical interaction between sGC and a thiol-redox protein. To characterize this interaction between sGC and PDI, we first identified peptide linkages between sGC and PDI, using a lysine cross-linking reagent and recently developed mass spectrometry analysis. Together with Flag-immunoprecipitation using sGC domain deletions, wild-type (WT and mutated PDI, regions of sGC involved in this interaction were identified. The observed data were further explored with computational modeling to gain insight into the interaction mechanism between sGC and oxidized PDI. Our results indicate that PDI interacts preferentially with the catalytic domain of sGC, thus providing a mechanism for PDI inhibition of sGC. A model in which PDI interacts with either the α or the β catalytic domain is proposed.

  8. The soluble guanylyl cyclase activator bay 58-2667 selectively limits cardiomyocyte hypertrophy.

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    Jennifer C Irvine

    Full Text Available Although evidence now suggests cGMP is a negative regulator of cardiac hypertrophy, the direct consequences of the soluble guanylyl cyclase (sGC activator BAY 58-2667 on cardiac remodeling, independent of changes in hemodynamic load, has not been investigated. In the present study, we tested the hypothesis that the NO(•-independent sGC activator BAY 58-2667 inhibits cardiomyocyte hypertrophy in vitro. Concomitant impact of BAY 58-2667 on cardiac fibroblast proliferation, and insights into potential mechanisms of action, were also sought. Results were compared to the sGC stimulator BAY 41-2272.Neonatal rat cardiomyocytes were incubated with endothelin-1 (ET(1, 60nmol/L in the presence and absence of BAY 41-2272 and BAY 58-2667 (0.01-0.3 µmol/L. Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. The impact of both sGC ligands on basal and stimulated cardiac fibroblast proliferation in vitro was also determined.We now demonstrate that BAY 58-2667 (0.01-0.3 µmol/L elicited concentration-dependent antihypertrophic actions, inhibiting ET(1-mediated increases in cardiomyocyte 2D area and de novo protein synthesis, as well as suppressing ET(1-induced cardiomyocyte superoxide generation. This was accompanied by potent increases in cardiomyocyte cGMP accumulation and activity of its downstream signal, vasodilator-stimulated phosphoprotein (VASP, without elevating cardiomyocyte cAMP. In contrast, submicromolar concentrations of BAY 58-2667 had no effect on basal or stimulated cardiac fibroblast proliferation. Indeed, only at concentrations ≥10 µmol/L was inhibition of cardiac fibrosis seen in vitro. The effects of BAY 58-2667 in both cell types were mimicked by BAY 41-2272.Our results demonstrate that BAY 58-2667 elicits protective, cardiomyocyte-selective effects in vitro. These actions are associated with sGC activation and are evident in the absence of confounding hemodynamic factors, at low (submicromolar

  9. The Arabidopsis thaliana proteome harbors undiscovered multi-domain molecules with functional guanylyl cyclase catalytic centers

    KAUST Repository

    Wong, Aloysius Tze; Gehring, Christoph A

    2013-01-01

    plants, guanylyl cyclases (GCs), enzymes that generate cGMP from guanosine-5'-triphosphate (GTP) have remained elusive until recently. GC search motifs constructed from the alignment of known GCs catalytic centers form vertebrates and lower eukaryotes

  10. Nitric oxide-soluble guanylyl cyclase-cyclic GMP signaling in the striatum: New targets for the treatment of Parkinson's disease?

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    Anthony R West

    2011-06-01

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

  11. Inferring biological functions of guanylyl cyclases with computational methods

    KAUST Repository

    Alquraishi, May Majed; Meier, Stuart Kurt

    2013-01-01

    A number of studies have shown that functionally related genes are often co-expressed and that computational based co-expression analysis can be used to accurately identify functional relationships between genes and by inference, their encoded proteins. Here we describe how a computational based co-expression analysis can be used to link the function of a specific gene of interest to a defined cellular response. Using a worked example we demonstrate how this methodology is used to link the function of the Arabidopsis Wall-Associated Kinase-Like 10 gene, which encodes a functional guanylyl cyclase, to host responses to pathogens. © Springer Science+Business Media New York 2013.

  12. Inferring biological functions of guanylyl cyclases with computational methods

    KAUST Repository

    Alquraishi, May Majed

    2013-09-03

    A number of studies have shown that functionally related genes are often co-expressed and that computational based co-expression analysis can be used to accurately identify functional relationships between genes and by inference, their encoded proteins. Here we describe how a computational based co-expression analysis can be used to link the function of a specific gene of interest to a defined cellular response. Using a worked example we demonstrate how this methodology is used to link the function of the Arabidopsis Wall-Associated Kinase-Like 10 gene, which encodes a functional guanylyl cyclase, to host responses to pathogens. © Springer Science+Business Media New York 2013.

  13. Degeneration of the olfactory guanylyl cyclase D gene during primate evolution.

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    Janet M Young

    2007-09-01

    Full Text Available The mammalian olfactory system consists of several subsystems that detect specific sets of chemical cues and underlie a variety of behavioral responses. Within the main olfactory epithelium at least three distinct types of chemosensory neurons can be defined by their expression of unique sets of signal transduction components. In rodents, one set of neurons expresses the olfactory-specific guanylyl cyclase (GC-D gene (Gucy2d, guanylyl cyclase 2d and other cell-type specific molecules. GC-D-positive neurons project their axons to a small group of atypical "necklace" glomeruli in the olfactory bulb, some of which are activated in response to suckling in neonatal rodents and to atmospheric CO2 in adult mice. Because GC-D is a pseudogene in humans, signaling through this system appears to have been lost at some point in primate evolution.Here we used a combination of bioinformatic analysis of trace-archive and genome-assembly data and sequencing of PCR-amplified genomic DNA to determine when during primate evolution the functional gene was lost. Our analysis reveals that GC-D is a pseudogene in a large number of primate species, including apes, Old World and New World monkeys and tarsier. In contrast, the gene appears intact and has evolved under purifying selection in mouse, rat, dog, lemur and bushbaby.These data suggest that signaling through GC-D-expressing cells was probably compromised more than 40 million years ago, prior to the divergence of New World monkeys from Old World monkeys and apes, and thus cannot be involved in chemosensation in most primates.

  14. Identification of residues in the heme domain of soluble guanylyl cyclase that are important for basal and stimulated catalytic activity.

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    Padmamalini Baskaran

    Full Text Available Nitric oxide signals through activation of soluble guanylyl cyclase (sGC, a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain to the effector domain (catalytic domain, in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105 of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.

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

    Science.gov (United States)

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

    2015-05-01

    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. 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. 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. 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. © 2014 The British Pharmacological Society.

  16. Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25.

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    Victor L Jensen

    2010-11-01

    Full Text Available In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-β and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis, but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia, implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT (required to build cilia is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.

  17. Role of Nitric Oxide, Nitric Oxide Synthase, Soluble Guanylyl Cyclase, and cGMP-Dependent Protein Kinase I in Mouse Stem Cell Cardiac Development

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    Valentina Spinelli

    2016-01-01

    Full Text Available Introduction and Aim. Nitric oxide (NO can trigger cardiac differentiation of embryonic stem cells (ESCs, indicating a cardiogenic function of the NO synthetizing enzyme(s (NOS. However, the involvement of the NO/NOS downstream effectors soluble guanylyl cyclase (sGC and cGMP activated protein kinase I (PKG-I is less defined. Therefore, we assess the involvement of the entire NO/NOS/sGC/PKG-I pathway during cardiac differentiation process. Methods. Mouse ESCs were differentiated toward cardiac lineages by hanging drop methodology for 21 days. NOS/sGC/PKG-I pathway was studied quantifying genes, proteins, enzymatic activities, and effects of inhibition during differentiation. Percentages of beating embryoid bodies (mEBs were evaluated as an index of cardiogenesis. Results and Discussion. Genes and protein expression of enzymes were increased during differentiation with distinctive kinetics and proteins possessed their enzymatic functions. Exogenous administered NO accelerated whereas the blockade of PKG-I strongly slowed cardiogenesis. sGC inhibition was effective only at early stages and NOS blockade ineffective. Of NOS/sGC/PKG-I pathway, PKG-I seems to play the prominent role in cardiac maturation. Conclusion. We concluded that exogenous administered NO and other pharmacological strategies able to increase the activity of PKG-I provide new tools to investigate and promote differentiation of cardiogenic precursors.

  18. Substrate specificity determinants of class III nucleotidyl cyclases.

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    Bharambe, Nikhil G; Barathy, Deivanayaga V; Syed, Wajeed; Visweswariah, Sandhya S; Colaςo, Melwin; Misquith, Sandra; Suguna, Kaza

    2016-10-01

    The two second messengers in signalling, cyclic AMP and cyclic GMP, are produced by adenylyl and guanylyl cyclases respectively. Recognition and discrimination of the substrates ATP and GTP by the nucleotidyl cyclases are vital in these reactions. Various apo-, substrate- or inhibitor-bound forms of adenylyl cyclase (AC) structures from transmembrane and soluble ACs have revealed the catalytic mechanism of ATP cyclization reaction. Previously reported structures of guanylyl cyclases represent ligand-free forms and inactive open states of the enzymes and thus do not provide information regarding the exact mode of substrate binding. The structures we present here of the cyclase homology domain of a class III AC from Mycobacterium avium (Ma1120) and its mutant in complex with ATP and GTP in the presence of calcium ion, provide the structural basis for substrate selection by the nucleotidyl cyclases at the atomic level. Precise nature of the enzyme-substrate interactions, novel modes of substrate binding and the ability of the binding pocket to accommodate diverse conformations of the substrates have been revealed by the present crystallographic analysis. This is the first report to provide structures of both the nucleotide substrates bound to a nucleotidyl cyclase. Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 5D15 (Ma1120 CHD +ATP.Ca 2+ ), 5D0E (Ma1120 CHD +GTP.Ca 2+ ), 5D0H (Ma1120 CHD (KDA→EGY)+ATP.Ca 2+ ), 5D0G (Ma1120 CHD (KDA→EGY)+GTP.Ca 2+ ). Adenylyl cyclase (EC number: 4.6.1.1). © 2016 Federation of European Biochemical Societies.

  19. Fluorescent fusion proteins of soluble guanylyl cyclase indicate proximity of the heme nitric oxide domain and catalytic domain.

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    Tobias Haase

    Full Text Available BACKGROUND: To examine the structural organisation of heterodimeric soluble guanylyl cyclase (sGC Förster resonance energy transfer (FRET was measured between fluorescent proteins fused to the amino- and carboxy-terminal ends of the sGC beta1 and alpha subunits. METHODOLOGY/PRINCIPAL FINDINGS: Cyan fluorescent protein (CFP was used as FRET donor and yellow fluorescent protein (YFP as FRET acceptor. After generation of recombinant baculovirus, fluorescent-tagged sGC subunits were co-expressed in Sf9 cells. Fluorescent variants of sGC were analyzed in vitro in cytosolic fractions by sensitized emission FRET. Co-expression of the amino-terminally tagged alpha subunits with the carboxy-terminally tagged beta1 subunit resulted in an enzyme complex that showed a FRET efficiency of 10% similar to fluorescent proteins separated by a helix of only 48 amino acids. Because these findings indicated that the amino-terminus of the alpha subunits is close to the carboxy-terminus of the beta1 subunit we constructed fusion proteins where both subunits are connected by a fluorescent protein. The resulting constructs were not only fluorescent, they also showed preserved enzyme activity and regulation by NO. CONCLUSIONS/SIGNIFICANCE: Based on the ability of an amino-terminal fragment of the beta1 subunit to inhibit activity of an heterodimer consisting only of the catalytic domains (alphacatbetacat, Winger and Marletta (Biochemistry 2005, 44:4083-90 have proposed a direct interaction of the amino-terminal region of beta1 with the catalytic domains. In support of such a concept of "trans" regulation of sGC activity by the H-NOX domains our results indicate that the domains within sGC are organized in a way that allows for direct interaction of the amino-terminal regulatory domains with the carboxy-terminal catalytic region. In addition, we constructed "fluorescent-conjoined" sGC's by fusion of the alpha amino-terminus to the beta1 carboxy-terminus leading to a

  20. Computational identification of candidate nucleotide cyclases in higher plants

    KAUST Repository

    Wong, Aloysius Tze; Gehring, Christoph A

    2013-01-01

    In higher plants guanylyl cyclases (GCs) and adenylyl cyclases (ACs) cannot be identified using BLAST homology searches based on annotated cyclic nucleotide cyclases (CNCs) of prokaryotes, lower eukaryotes, or animals. The reason is that CNCs

  1. Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

    KAUST Repository

    Qia, Zhi; Verma, Rajeev K.; Gehring, Christoph A; Yamaguchi, Yube; Zhao, Yichen; Ryan, Clarence A.; Berkowitz, Gerald A.

    2010-01-01

    receptor- like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2- dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33

  2. Gaseous ligand selectivity of the H-NOX sensor protein from Shewanella oneidensis and comparison to those of other bacterial H-NOXs and soluble guanylyl cyclase.

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    Wu, Gang; Liu, Wen; Berka, Vladimir; Tsai, Ah-Lim

    2017-09-01

    To delineate the commonalities and differences in gaseous ligand discrimination among the heme-based sensors with Heme Nitric oxide/OXygen binding protein (H-NOX) scaffold, the binding kinetic parameters for gaseous ligands NO, CO, and O 2 , including K D , k on , and k off , of Shewanella oneidensis H-NOX (So H-NOX) were characterized in detail in this study and compared to those of previously characterized H-NOXs from Clostridium botulinum (Cb H-NOX), Nostoc sp. (Ns H-NOX), Thermoanaerobacter tengcongensis (Tt H-NOX), Vibrio cholera (Vc H-NOX), and human soluble guanylyl cyclase (sGC), an H-NOX analogue. The K D (NO) and K D (CO) of each bacterial H-NOX or sGC follow the "sliding scale rule"; the affinities of the bacterial H-NOXs for NO and CO vary in a small range but stronger than those of sGC by at least two orders of magnitude. On the other hand, each bacterial H-NOX exhibits different characters in the stability of its 6c NO complex, reactivity with secondary NO, stability of oxyferrous heme and autoxidation to ferric heme. A facile access channel for gaseous ligands is also identified, implying that ligand access has only minimal effect on gaseous ligand selectivity of H-NOXs or sGC. This comparative study of the binding parameters of the bacterial H-NOXs and sGC provides a basis to guide future new structural and functional studies of each specific heme sensor with the H-NOX protein fold. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  3. Guanylyl cyclase C in colorectal cancer: susceptibility gene and potential therapeutic target.

    Science.gov (United States)

    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.

  4. High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

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    Pichlo, Magdalena; Bungert-Plümke, Stefanie; Weyand, Ingo; Seifert, Reinhard; Bönigk, Wolfgang; Strünker, Timo; Kashikar, Nachiket Dilip; Goodwin, Normann; Müller, Astrid; Körschen, Heinz G.; Collienne, Ursel; Pelzer, Patric; Van, Qui; Enderlein, Jörg; Klemm, Clementine; Krause, Eberhard; Trötschel, Christian; Poetsch, Ansgar; Kremmer, Elisabeth

    2014-01-01

    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 × 105 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

  5. Dysfunctional nitric oxide signalling increases risk of myocardial infarction.

    Science.gov (United States)

    Erdmann, Jeanette; Stark, Klaus; Esslinger, Ulrike B; Rumpf, Philipp Moritz; Koesling, Doris; de Wit, Cor; Kaiser, Frank J; Braunholz, Diana; Medack, Anja; Fischer, Marcus; Zimmermann, Martina E; Tennstedt, Stephanie; Graf, Elisabeth; Eck, Sebastian; Aherrahrou, Zouhair; Nahrstaedt, Janja; Willenborg, Christina; Bruse, Petra; Brænne, Ingrid; Nöthen, Markus M; Hofmann, Per; Braund, Peter S; Mergia, Evanthia; Reinhard, Wibke; Burgdorf, Christof; Schreiber, Stefan; Balmforth, Anthony J; Hall, Alistair S; Bertram, Lars; Steinhagen-Thiessen, Elisabeth; Li, Shu-Chen; März, Winfried; Reilly, Muredach; Kathiresan, Sekar; McPherson, Ruth; Walter, Ulrich; Ott, Jurg; Samani, Nilesh J; Strom, Tim M; Meitinger, Thomas; Hengstenberg, Christian; Schunkert, Heribert

    2013-12-19

    Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as β1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.

  6. Deducing the origin of soluble adenylyl cyclase, a gene lost in multiple lineages

    NARCIS (Netherlands)

    Roelofs, Jeroen; Haastert, Peter J.M. van

    2002-01-01

    The family of eukaryotic adenylyl cyclases consists of a very large group of 12 transmembrane adenylyl cyclases and a very small group of soluble adenylyl cyclase (sAC). Orthologs of human sAC are present in rat Diclyostelium and bacteria but absent from the completely sequenced genomes of

  7. Clathrin-dependent internalization, signaling, and metabolic processing of guanylyl cyclase/natriuretic peptide receptor-A.

    Science.gov (United States)

    Somanna, Naveen K; Mani, Indra; Tripathi, Satyabha; Pandey, Kailash N

    2018-04-01

    Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using 125 I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand-receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.

  8. Loss of guanylyl cyclase C (GCC signaling leads to dysfunctional intestinal barrier.

    Directory of Open Access Journals (Sweden)

    Xiaonan Han

    2011-01-01

    Full Text Available Guanylyl Cyclase C (GCC signaling via uroguanylin (UGN and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function.Paracellular permeability of intestinal segments was assessed in wild type (WT and GCC deficient (GCC-/- mice with and without lipopolysaccharide (LPS challenge, as well as in UGN deficient (UGN-/- mice. IFNγ and myosin light chain kinase (MLCK levels were determined by real time PCR. Expression of tight junction proteins (TJPs, phosphorylation of myosin II regulatory light chain (MLC, and STAT1 activation were examined in intestinal epithelial cells (IECs and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi. We found that intestinal permeability was increased in GCC-/- and UGN-/- mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC-/- mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC-/- and UGN-/- mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability.GCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury.

  9. The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

    Science.gov (United States)

    Schleede, Justin; Blair, Seth S

    2015-10-01

    The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog). However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

  10. In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

    KAUST Repository

    Raji, Misjudeen; Gehring, Christoph A

    2017-01-01

    Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

  11. In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

    KAUST Repository

    Raji, Misjudeen

    2017-05-31

    Cyclic nucleotides such as 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

  12. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    Science.gov (United States)

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Corruption of homeostatic mechanisms in the guanylyl cyclase C signaling pathway underlying colorectal tumorigenesis.

    Science.gov (United States)

    Li, Peng; Waldman, Scott A

    2010-08-01

    Colon cancer, the second leading cause of cancer-related mortality worldwide, originates from the malignant transformation of intestinal epithelial cells. The intestinal epithelium undergoes a highly organized process of rapid regeneration along the crypt-villus axis, characterized by proliferation, migration, differentiation and apoptosis, whose coordination is essential to maintaining the mucosal barrier. Disruption of these homeostatic processes predisposes cells to mutations in tumor suppressors or oncogenes, whose dysfunction provides transformed cells an evolutionary growth advantage. While sequences of genetic mutations at different stages along the neoplastic continuum have been established, little is known of the events initiating tumorigenesis prior to adenomatous polyposis coli (APC) mutations. Here, we examine a role for the corruption of homeostasis induced by silencing novel tumor suppressors, including the intestine-specific transcription factor CDX2 and its gene target guanylyl cyclase C (GCC), as early events predisposing cells to mutations in APC and other sequential genes that initiate colorectal cancer. CDX2 and GCC maintain homeostatic regeneration in the intestine by restricting cell proliferation, promoting cell maturation and adhesion, regulating cell migration and defending the intestinal barrier and genomic integrity. Elimination of CDX2 or GCC promotes intestinal tumor initiation and growth in aged mice, mice carrying APC mutations or mice exposed to carcinogens. The roles of CDX2 and GCC in suppressing intestinal tumorigenesis, universal disruption in their signaling through silencing of hormones driving GCC, and the uniform overexpression of GCC by tumors underscore the potential value of oral replacement with GCC ligands as targeted prevention and therapy for colorectal cancer.

  14. 17 beta-estradiol modifies nitric oxide-sensitive guanylyl cyclase expression and down-regulates its activity in rat anterior pituitary gland.

    Science.gov (United States)

    Cabilla, Jimena P; Díaz, María del Carmen; Machiavelli, Leticia I; Poliandri, Ariel H; Quinteros, Fernanda A; Lasaga, Mercedes; Duvilanski, Beatriz H

    2006-09-01

    Previous studies showed that 17 beta-estradiol (17 beta-E2) regulates the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP pathway in many tissues. Evidence from our laboratory indicates that 17 beta-E2 disrupts the inhibitory effect of NO on prolactin release, decreasing sGC activity and affecting the cGMP pathway in anterior pituitary gland of adult ovariectomized and estrogenized rats. To ascertain the mechanisms by which 17 beta-E2 affects sGC activity, we investigated the in vivo and in vitro effects of 17 beta-E2 on sGC protein and mRNA expression in anterior pituitary gland from immature female rats. In the present work, we showed that 17 beta-E2 acute treatment exerted opposite effects on the two sGC subunits, increasing alpha1 and decreasing beta1 subunit protein and mRNA expression. This action on sGC protein expression was maximal 6-9 h after 17 beta-E2 administration. 17beta-E2 also caused the same effect on mRNA expression at earlier times. Concomitantly, 17 beta-E2 dramatically decreased sGC activity 6 and 9 h after injection. These effects were specific of 17 beta-E2, because they were not observed with the administration of other steroids such as progesterone and 17 alpha-estradiol. This inhibitory action of 17beta-E2 on sGC also required the activation of estrogen receptor (ER), because treatment with the pure ER antagonist ICI 182,780 completely blocked 17 beta-E2 action. 17 beta-E2 acute treatment caused the same effects on pituitary cells in culture. These results suggest that 17 beta-E2 exerts an acute inhibitory effect on sGC in anterior pituitary gland by down-regulating sGC beta 1 subunit and sGC activity in a specific, ER-dependent manner.

  15. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

    Science.gov (United States)

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-09-08

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

  16. The Gyc76C Receptor Guanylyl Cyclase and the Foraging cGMP-Dependent Kinase Regulate Extracellular Matrix Organization and BMP Signaling in the Developing Wing of Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Justin Schleede

    2015-10-01

    Full Text Available The developing crossveins of the wing of Drosophila melanogaster are specified by long-range BMP signaling and are especially sensitive to loss of extracellular modulators of BMP signaling such as the Chordin homolog Short gastrulation (Sog. However, the role of the extracellular matrix in BMP signaling and Sog activity in the crossveins has been poorly explored. Using a genetic mosaic screen for mutations that disrupt BMP signaling and posterior crossvein development, we identify Gyc76C, a member of the receptor guanylyl cyclase family that includes mammalian natriuretic peptide receptors. We show that Gyc76C and the soluble cGMP-dependent kinase Foraging, likely linked by cGMP, are necessary for normal refinement and maintenance of long-range BMP signaling in the posterior crossvein. This does not occur through cell-autonomous crosstalk between cGMP and BMP signal transduction, but likely through altered extracellular activity of Sog. We identify a novel pathway leading from Gyc76C to the organization of the wing extracellular matrix by matrix metalloproteinases, and show that both the extracellular matrix and BMP signaling effects are largely mediated by changes in the activity of matrix metalloproteinases. We discuss parallels and differences between this pathway and other examples of cGMP activity in both Drosophila melanogaster and mammalian cells and tissues.

  17. Computational identification of candidate nucleotide cyclases in higher plants

    KAUST Repository

    Wong, Aloysius Tze

    2013-09-03

    In higher plants guanylyl cyclases (GCs) and adenylyl cyclases (ACs) cannot be identified using BLAST homology searches based on annotated cyclic nucleotide cyclases (CNCs) of prokaryotes, lower eukaryotes, or animals. The reason is that CNCs are often part of complex multifunctional proteins with different domain organizations and biological functions that are not conserved in higher plants. For this reason, we have developed CNC search strategies based on functionally conserved amino acids in the catalytic center of annotated and/or experimentally confirmed CNCs. Here we detail this method which has led to the identification of >25 novel candidate CNCs in Arabidopsis thaliana, several of which have been experimentally confirmed in vitro and in vivo. We foresee that the application of this method can be used to identify many more members of the growing family of CNCs in higher plants. © Springer Science+Business Media New York 2013.

  18. The plant natriuretic peptide receptor is a guanylyl cyclase and enables cGMP-dependent signaling

    KAUST Repository

    Turek, Ilona

    2016-03-05

    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. © 2016 Springer Science+Business Media Dordrecht

  19. Nitric oxide-sensitive guanylyl cyclase is differentially regulated by nuclear and non-nuclear estrogen pathways in anterior pituitary gland.

    Directory of Open Access Journals (Sweden)

    Jimena P Cabilla

    Full Text Available 17β-estradiol (E2 regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC, is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM and E2-dendrimer conjugate (EDC, 1 nM were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h, 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway.

  20. Nitric oxide-sensitive guanylyl cyclase is differentially regulated by nuclear and non-nuclear estrogen pathways in anterior pituitary gland.

    Science.gov (United States)

    Cabilla, Jimena P; Nudler, Silvana I; Ronchetti, Sonia A; Quinteros, Fernanda A; Lasaga, Mercedes; Duvilanski, Beatriz H

    2011-01-01

    17β-estradiol (E2) regulates hormonal release as well as proliferation and cell death in the pituitary. The main nitric oxide receptor, nitric oxide sensitive- or soluble guanylyl cyclase (sGC), is a heterodimer composed of two subunits, α and β, that catalyses cGMP formation. α1β1 is the most abundant and widely expressed heterodimer, showing the greater activity. Previously we have shown that E2 decreased sGC activity but exerts opposite effects on sGC subunits increasing α1 and decreasing β1 mRNA and protein levels. In the present work we investigate the mechanisms by which E2 differentially regulates sGC subunits' expression on rat anterior pituitary gland. Experiments were performed on primary cultures of anterior pituitary cells from adult female Wistar rats at random stages of estrous cycle. After 6 h of E2 treatment, α1 mRNA and protein expression is increased while β1 levels are down-regulated. E2 effects on sGC expression are partially dependent on de novo transcription while de novo translation is fully required. E2 treatment decreased HuR mRNA stabilization factor and increased AUF1 p37 mRNA destabilization factor. E2-elicited β1 mRNA decrease correlates with a mRNA destabilization environment in the anterior pituitary gland. On the other hand, after 6 h of treatment, E2-BSA (1 nM) and E2-dendrimer conjugate (EDC, 1 nM) were unable to modify α1 or β1 mRNA levels, showing that nuclear receptor is involved in E2 actions. However, at earlier times (3 h), 1 nM EDC causes a transient decrease of α1 in a PI3k-dependent fashion. Our results show for the first time that E2 is able to exert opposite actions in the anterior pituitary gland, depending on the activation of classical or non-classical pathways. Thus, E2 can also modify sGC expression through membrane-initiated signals bringing to light a new point of regulation in NO/sGC pathway. © 2011 Cabilla et al.

  1. Metabolic Communication between Astrocytes and Neurons via Bicarbonate-Responsive Soluble Adenylyl Cyclase

    OpenAIRE

    Choi, Hyun B.; Gordon, Grant R.J.; Zhou, Ning; Tai, Chao; Rungta, Ravi L.; Martinez, Jennifer; Milner, Teresa A.; Ryu, Jae K.; McLarnon, James G.; Tresguerres, Martin; Levin, Lonny R.; Buck, Jochen; MacVicar, Brian A.

    2012-01-01

    Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO3−) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response t...

  2. 3',5'-cIMP as Potential Second Messenger in the Vascular Wall.

    Science.gov (United States)

    Leung, Susan W S; Gao, Yuansheng; Vanhoutte, Paul M

    2017-01-01

    Traditionally, only the 3',5'-cyclic monophosphates of adenosine and guanosine (produced by adenylyl cyclase and guanylyl cyclase, respectively) are regarded as true "second messengers" in the vascular wall, despite the presence of other cyclic nucleotides in different tissues. Among these noncanonical cyclic nucleotides, inosine 3',5'-cyclic monophosphate (cIMP) is synthesized by soluble guanylyl cyclase in porcine coronary arteries in response to hypoxia, when the enzyme is activated by endothelium-derived nitric oxide. Its production is associated with augmentation of vascular contraction mediated by stimulation of Rho kinase. Based on these findings, cIMP appears to meet most, if not all, of the criteria required for it to be accepted as a "second messenger," at least in the vascular wall.

  3. Role of the bicarbonate-responsive soluble adenylyl cyclase in pH sensing and metabolic regulation

    NARCIS (Netherlands)

    Chang, Jung-Chin; Oude-Elferink, Ronald P. J.

    2014-01-01

    The evolutionarily conserved soluble adenylyl cyclase (sAC, adcy10) was recently identified as a unique source of cAMP in the cytoplasm and the nucleus. Its activity is regulated by bicarbonate and fine tuned by calcium. As such, and in conjunction with carbonic an hydrase ( CA), sAC constitutes an

  4. The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

    KAUST Repository

    Meier, Stuart; Ruzvidzo, Oziniel; Morse, Monique; Donaldson, Lara; Kwezi, Lusisizwe; Gehring, Christoph A

    2010-01-01

    Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

  5. The arabidopsis wall associated kinase-like 10 gene encodes a functional guanylyl cyclase and is co-expressed with pathogen defense related genes

    KAUST Repository

    Meier, Stuart

    2010-01-26

    Background: Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3?,5?-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants. Principal Findings: We have identified an Arabidopsis receptor type wall associated kinase-like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10431-700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently coexpressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors. Conclusions: We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP. © 2010 Meier et al.

  6. The Arabidopsis thaliana proteome harbors undiscovered multi-domain molecules with functional guanylyl cyclase catalytic centers

    KAUST Repository

    Wong, Aloysius Tze

    2013-07-08

    Background: Second messengers link external cues to complex physiological responses. One such messenger, 3\\',5\\'-cyclic guanosine monophosphate (cGMP), has been shown to play a key role in many physiological responses in plants. However, in higher plants, guanylyl cyclases (GCs), enzymes that generate cGMP from guanosine-5\\'-triphosphate (GTP) have remained elusive until recently. GC search motifs constructed from the alignment of known GCs catalytic centers form vertebrates and lower eukaryotes have led to the identification of a number of plant GCs that have been characterized in vitro and in vivo.Presentation of the hypothesis.Recently characterized GCs in Arabidopsis thaliana contributed to the development of search parameters that can identify novel candidate GCs in plants. We hypothesize that there are still a substantial number (> 40) of multi-domain molecules with potentially functional GC catalytic centers in plants that remain to be discovered and characterized. Testing the hypothesis. The hypothesis can be tested, firstly, by computational methods constructing 3D models of selected GC candidates using available crystal structures as templates. Homology modeling must include substrate docking that can provide support for the structural feasibility of the GC catalytic centers in those candidates. Secondly, recombinant peptides containing the GC domain need to be tested in in vitro GC assays such as the enzyme-linked immune-sorbent assay (ELISA) and/or in mass spectrometry based cGMP assays. In addition, quantification of in vivo cGMP transients with fluorescent cGMP-reporter assays in wild-type or selected mutants will help to elucidate the biological role of novel GCs.Implications of the hypothesis.If it turns out that plants do harbor a large number of functional GC domains as part of multi-domain enzymes, then major new insights will be gained into the complex signal transduction pathways that link cGMP to fundamental processes such as ion transport

  7. Impact of the NO-Sensitive Guanylyl Cyclase 1 and 2 on Renal Blood Flow and Systemic Blood Pressure in Mice.

    Science.gov (United States)

    Mergia, Evanthia; Thieme, Manuel; Hoch, Henning; Daniil, Georgios; Hering, Lydia; Yakoub, Mina; Scherbaum, Christina Rebecca; Rump, Lars Christian; Koesling, Doris; Stegbauer, Johannes

    2018-03-23

    Nitric oxide (NO) modulates renal blood flow (RBF) and kidney function and is involved in blood pressure (BP) regulation predominantly via stimulation of the NO-sensitive guanylyl cyclase (NO-GC), existing in two isoforms, NO-GC1 and NO-GC2. Here, we used isoform-specific knockout (KO) mice and investigated their contribution to renal hemodynamics under normotensive and angiotensin II-induced hypertensive conditions. Stimulation of the NO-GCs by S -nitrosoglutathione (GSNO) reduced BP in normotensive and hypertensive wildtype (WT) and NO-GC2-KO mice more efficiently than in NO-GC1-KO. NO-induced increase of RBF in normotensive mice did not differ between the genotypes, but the respective increase under hypertensive conditions was impaired in NO-GC1-KO. Similarly, inhibition of endogenous NO increased BP and reduced RBF to a lesser extent in NO-GC1-KO than in NO-GC2-KO. These findings indicate NO-GC1 as a target of NO to normalize RBF in hypertension. As these effects were not completely abolished in NO-GC1-KO and renal cyclic guanosine monophosphate (cGMP) levels were decreased in both NO-GC1-KO and NO-GC2-KO, the results suggest an additional contribution of NO-GC2. Hence, NO-GC1 plays a predominant role in the regulation of BP and RBF, especially in hypertension. However, renal NO-GC2 appears to compensate the loss of NO-GC1, and is able to regulate renal hemodynamics under physiological conditions.

  8. Endothelium-independent and endothelium-dependent ...

    African Journals Online (AJOL)

    This endothelium-independent relaxant effect was also sensitive to combination of 1H-[1,2,4]-oxadiazole-[4,3-á]-quinoxalin- 1-one (ODQ, 10 ìM, soluble guanylyl cyclase inhibitor) and N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H89, 100 nM, Protein Kinase A inhibitor). Taken together ...

  9. Effect of Vericiguat, a Soluble Guanylate Cyclase Stimulator, on Natriuretic Peptide Levels in Patients With Worsening Chronic Heart Failure and Reduced Ejection Fraction

    DEFF Research Database (Denmark)

    Gheorghiade, Mihai; Greene, Stephen J; Butler, Javed

    2015-01-01

    IMPORTANCE: Worsening chronic heart failure (HF) is a major public health problem. OBJECTIVE: To determine the optimal dose and tolerability of vericiguat, a soluble guanylate cyclase stimulator, in patients with worsening chronic HF and reduced left ventricular ejection fraction (LVEF). DESIGN, ...

  10. Soluble adenylyl cyclase is an acid-base sensor in epithelial base-secreting cells.

    Science.gov (United States)

    Roa, Jinae N; Tresguerres, Martin

    2016-08-01

    Blood acid-base regulation by specialized epithelia, such as gills and kidney, requires the ability to sense blood acid-base status. Here, we developed primary cultures of ray (Urolophus halleri) gill cells to study mechanisms for acid-base sensing without the interference of whole animal hormonal regulation. Ray gills have abundant base-secreting cells, identified by their noticeable expression of vacuolar-type H(+)-ATPase (VHA), and also express the evolutionarily conserved acid-base sensor soluble adenylyl cyclase (sAC). Exposure of cultured cells to extracellular alkalosis (pH 8.0, 40 mM HCO3 (-)) triggered VHA translocation to the cell membrane, similar to previous reports in live animals experiencing blood alkalosis. VHA translocation was dependent on sAC, as it was blocked by the sAC-specific inhibitor KH7. Ray gill base-secreting cells also express transmembrane adenylyl cyclases (tmACs); however, tmAC inhibition by 2',5'-dideoxyadenosine did not prevent alkalosis-dependent VHA translocation, and tmAC activation by forskolin reduced the abundance of VHA at the cell membrane. This study demonstrates that sAC is a necessary and sufficient sensor of extracellular alkalosis in ray gill base-secreting cells. In addition, this study indicates that different sources of cAMP differentially modulate cell biology. Copyright © 2016 the American Physiological Society.

  11. The Arabidopsis thalianaK+-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre

    KAUST Repository

    Al-Younis, Inas

    2015-11-27

    Adenylate Cyclases (ACs) catalyze the formation of the second messenger cyclic adenosine 3′, 5′-monophosphate (cAMP) from adenosine 5’-triphosphate (ATP). Although cAMP is increasingly recognized as an important signaling 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 AtKUP71-100 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 AtKUP71-100 generates cAMP in vitro.

  12. (1)H, (13)C, (15)N backbone and side-chain resonance assignment of Nostoc sp. C139A variant of the heme-nitric oxide/oxygen binding (H-NOX) domain.

    Science.gov (United States)

    Alexandropoulos, Ioannis I; Argyriou, Aikaterini I; Marousis, Kostas D; Topouzis, Stavros; Papapetropoulos, Andreas; Spyroulias, Georgios A

    2016-10-01

    The H-NOX (Heme-nitric oxide/oxygen binding) domain is conserved across eukaryotes and bacteria. In human soluble guanylyl cyclase (sGC) the H-NOX domain functions as a sensor for the gaseous signaling agent nitric oxide (NO). sGC contains the heme-binding H-NOX domain at its N-terminus, which regulates the catalytic site contained within the C-terminal end of the enzyme catalyzing the conversion of GTP (guanosine 5'-triphosphate) to GMP (guanylyl monophosphate). Here, we present the backbone and side-chain assignments of the (1)H, (13)C and (15)N resonances of the 183-residue H-NOX domain from Nostoc sp. through solution NMR.

  13. Metabolic communication between astrocytes and neurons via bicarbonate-responsive soluble adenylyl cyclase.

    Science.gov (United States)

    Choi, Hyun B; Gordon, Grant R J; Zhou, Ning; Tai, Chao; Rungta, Ravi L; Martinez, Jennifer; Milner, Teresa A; Ryu, Jae K; McLarnon, James G; Tresguerres, Martin; Levin, Lonny R; Buck, Jochen; MacVicar, Brian A

    2012-09-20

    Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO₃⁻) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO₃⁻ entry via the electrogenic NaHCO₃ cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K⁺](ext) and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    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

  15. Molecular characterization of a novel intracellular ADP-ribosyl cyclase.

    Directory of Open Access Journals (Sweden)

    Dev Churamani

    2007-08-01

    Full Text Available ADP-ribosyl cyclases are remarkable enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent intracellular calcium mobilizing messengers, cyclic ADP-ribose and NAADP. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Moreover, those that have are located predominately at the outer cell surface and thus away from their cytosolic substrates.Here we report the molecular cloning of a novel expanded family of ADP-ribosyl cyclases from the sea urchin, an extensively used model organism for the study of inositol trisphosphate-independent calcium mobilization. We provide evidence that one of the isoforms (SpARC1 is a soluble protein that is targeted exclusively to the endoplasmic reticulum lumen when heterologously expressed. Catalytic activity of the recombinant protein was readily demonstrable in crude cell homogenates, even under conditions where luminal continuity was maintained.Our data reveal a new intracellular location for ADP-ribosyl cyclases and suggest that production of calcium mobilizing messengers may be compartmentalized.

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

    Energy Technology Data Exchange (ETDEWEB)

    Misono, K. S.; Philo, J. S.; Arakawa, T.; Ogata, C. M.; Qiu, Y.; Ogawa, H.; Young, H. S. (Biosciences Division); (Univ. of Nevada); (Alliance Protein Labs.)

    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.

  17. Consistent expression of guanylyl cyclase-C in primary and metastatic gastrointestinal cancers.

    Directory of Open Access Journals (Sweden)

    Hadi Danaee

    Full Text Available The transmembrane receptor guanylate cyclase-C (GCC has been found to be expressed in colorectal cancers. However, limited data are available on GCC protein expression in non-colorectal gastrointestinal tumors and few studies have reported whether GCC protein expression was consistently preserved in synchronous primary and metastatic cancer tissues.GCC protein status was assessed by immunohistochemistry in tumor specimens from individuals (n = 627 with gastrointestinal tumors, including esophageal (n = 130, gastric (n = 276, pancreatic (n = 136, and colorectal (n = 85 primary and metastatic tumors. Tissue specimens consisted of tissue microarrays containing esophageal, gastric, pancreatic tumors, and whole-slide tissue sections from colorectal cancer patients with matching primary and metastatic tumors.Among the evaluated esophageal, gastric, and pancreatic tumors, the frequency of GCC positivity at the protein level ranged from 59% to 68%. GCC was consistently expressed in primary and matched/synchronous metastatic lesions of colorectal cancer tissues derived from the same patients.This observational study demonstrated the protein expression of GCC across various gastrointestinal malignancies. In all cancer histotypes, GCC protein localization was observed predominantly in the cytoplasm compared to the membrane region of tumor cells. Consistent immunohistochemistry detection of GCC protein expression in primary colorectal cancers and in their matched liver metastases suggests that the expression of GCC is maintained throughout the process of tumor progression and formation of metastatic disease.

  18. Ca 2+ signaling by plant Arabidopsis thaliana Pep peptides depends on AtPepR1, a receptor with guanylyl cyclase activity, and cGMP-activated Ca 2+ channels

    KAUST Repository

    Qia, Zhi

    2010-11-18

    A family of peptide signaling molecules (AtPeps) and their plasma membrane receptor AtPepR1 are known to act in pathogendefense signaling cascades in plants. Little is currently known about the molecular mechanisms that link these signaling peptides and their receptor, a leucine-rich repeat receptor-like kinase, to downstream pathogen-defense responses. We identify some cellular activities of these molecules that provide the context for a model for their action in signaling cascades. AtPeps activate plasma membrane inwardly conducting Ca 2+ permeable channels in mesophyll cells, resulting in cytosolic Ca 2+ elevation. This activity is dependent on their receptor as well as a cyclic nucleotide-gated channel (CNGC2). We also show that the leucine-rich repeat receptor- like kinase receptor AtPepR1 has guanylyl cyclase activity, generating cGMP from GTP, and that cGMP can activate CNGC2- dependent cytosolic Ca 2+ elevation. AtPep-dependent expression of pathogen-defense genes (PDF1.2, MPK3, and WRKY33) is mediated by the Ca 2+ signaling pathway associated with AtPep peptides and their receptor. The work presented here indicates that extracellular AtPeps, which can act as danger-associated molecular patterns, signal by interaction with their receptor, AtPepR1, a plasma membrane protein that can generate cGMP. Downstream from AtPep and AtPepR1 in a signaling cascade, the cGMP-activated channel CNGC2 is involved in AtPep- and AtPepR1-dependent inward Ca 2+ conductance and resulting cytosolic Ca 2+ elevation. The signaling cascade initiated by AtPeps leads to expression of pathogen- defense genes in a Ca 2+-dependent manner.

  19. Spatial resolution of cAMP signaling by soluble adenylyl cyclase

    Science.gov (United States)

    Caldieri, Giusi

    2016-01-01

    G protein–coupled receptor signaling starts at the plasma membrane and continues at endosomal stations. In this issue, Inda et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201512075) show that different forms of adenylyl cyclase are activated at the plasma membrane versus endosomes, providing a rationale for the spatial encoding of cAMP signaling. PMID:27402955

  20. N-hydroxylamine is not an intermediate in the conversion of L-arginine to an activator of soluble guanylate cyclase in neuroblastoma N1E-115 cells.

    Science.gov (United States)

    Pou, S; Pou, W S; Rosen, G M; el-Fakahany, E E

    1991-01-01

    This study evaluates the role of N-hydroxylamine (NH2OH) in activating soluble guanylate cyclase in the mouse neuroblastoma clone N1E-115. It has been proposed that NH2OH is a putative intermediate in the biochemical pathway for the generation of nitric oxide (NO)/endothelium-derived relaxing factor (EDRF) from L-arginine. NH2OH caused a time- and concentration-dependent increase in cyclic GMP formation in intact cells. This response was not dependent on Ca2+. In cytosol preparations the activation of guanylate cyclase by L-arginine was dose-dependent and required Ca2+ and NADPH. In contrast, NH2OH itself did not activate cytosolic guanylate cyclase but it inhibited the basal activity of this enzyme in a concentration-dependent manner. The formation of cyclic GMP in the cytosolic fractions in response to NH2OH required the addition of catalase and H2O2. On the other hand, catalase and/or H2O2 lead to a decrease in L-arginine-induced cyclic GMP formation. Furthermore, NH2OH inhibited L-arginine- and sodium nitroprusside-induced cyclic GMP formation in the cytosol. The inhibition of L-arginine-induced cyclic GMP formation in the cytosol by NH2OH was not reversed by the addition of superoxide dismutase. These data strongly suggest that NH2OH is not a putative intermediate in the metabolism of L-arginine to an activator of guanylate cyclase. PMID:1671745

  1. Role of the bicarbonate-responsive soluble adenylyl cyclase in pH sensing and metabolic regulation

    Directory of Open Access Journals (Sweden)

    Jung-Chin eChang

    2014-02-01

    Full Text Available The evolutionarily conserved soluble adenylyl cyclase (sAC, adcy10 was recently identified as a unique source of cAMP in the cytoplasm and the nucleus. Its activity is regulated by bicarbonate and fine-tuned by calcium. As such, and in conjunction with carbonic anhydrase (CA, sAC constitutes an HCO3-/CO¬2/pH sensor. In both alpha-intercalated cells of the collecting duct and the clear cells of the epididymis, sAC is expressed at significant level and involved in pH homeostasis via apical recruitment of vacuolar H+-ATPase (VHA in a PKA-dependent manner. In addition to maintenance of pH homeostasis, sAC is also involved in metabolic regulation such as coupling of Krebs cycle to oxidative phosphorylation via bicarbonate/CO2 sensing. Additionally, sAC also regulates CFTR channel and plays an important role in regulation of barrier function and apoptosis. These observations suggest that sAC, via bicarbonate-sensing, plays an important role in maintaining homeostatic status of cells against fluctuations in their microenvironment.

  2. Adenylyl cyclases in the digestive system.

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Heterodimerization with the β1 subunit directs the α2 subunit of nitric oxide-sensitive guanylyl cyclase to calcium-insensitive cell-cell contacts in HEK293 cells: Interaction with Lin7a.

    Science.gov (United States)

    Hochheiser, Julia; Haase, Tobias; Busker, Mareike; Sömmer, Anne; Kreienkamp, Hans-Jürgen; Behrends, Sönke

    2016-12-15

    Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of an α and a β subunit. Two different α subunits (α 1 and α 2 ) give rise to two heterodimeric enzymes α 1 /β 1 and α 2 /β 1 . Both coexist in a wide range of tissues including blood vessels and the lung, but expression of the α 2 /β 1 form is generally much lower and approaches levels similar to the α 1 /β 1 form in the brain only. In the present paper, we show that the α 2 /β 1 form interacts with Lin7a in mouse brain synaptosomes based on co-precipitation analysis. In HEK293 cells, we found that the overexpressed α 2 /β 1 form, but not the α 1 /β 1 form is directed to calcium-insensitive cell-cell contacts. The isolated PDZ binding motif of an amino-terminally truncated α 2 subunit was sufficient for cell-cell contact localization. For the full length α 2 subunit with the PDZ binding motif this was only the case in the heterodimer configuration with the β 1 subunit, but not as isolated α 2 subunit. We conclude that the PDZ binding motif of the α 2 subunit is only accessible in the heterodimer conformation of the mature nitric oxide-sensitive enzyme. Interaction with Lin7a, a small scaffold protein important for synaptic function and cell polarity, can direct this complex to nectin based cell-cell contacts via MPP3 in HEK293 cells. We conclude that heterodimerization is a prerequisite for further protein-protein interactions that direct the α 2 /β 1 form to strategic sites of the cell membrane with adjacent neighbouring cells. Drugs increasing the nitric oxide-sensitivity of this specific form may be particularly effective. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

    Science.gov (United States)

    Lee, S; Parent, C A; Insall, R; Firtel, R A

    1999-09-01

    We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras

  5. Identification and Characterization of Novel Plant Adenylate Cyclases – The Arabidopsis Thaliana Potassium Uptake Permeases

    KAUST Repository

    Al-Younis, Inas M.

    2018-05-01

    Adenylyl Cyclases (ACs) catalyze the formation of the key universal second messenger adenosine 3’, 5’-cyclic monophosphate (cAMP) from adenosine 5’- triphosphate. Cyclic AMP participates in several signal transduction pathways and is present in bacteria and higher and lower eukaryotes including higher plants. Previous studies in plants have shown a role for cAMP in signal transduction during e.g. the cell cycle, elongation of the pollen tube and stimulation of protein kinase activity. More recently cAMP has been shown to play a role in stress responses. Interestingly, cAMP has also been shown to regulate ion transport in plant cells. Here we used a similar strategy that led to the discovery of the first guanylyl cyclase in plants that was based on the alignment of conserved and functionally assigned amino acids in the catalytic centre of annotated nucleotide cyclases from lower and higher eukaryotes, to identify a novel candidate ACs in Arabidopsis (Arabidopsis thaliana K+ Uptake 5 and 7). ATKUP5 and 7 are homologous to K+ uptake permeases (KUPs) from bacteria and high-affinity K+ transporters (HAKs) from fungi. The AC activity was investigated by recombinantly expressing the ATKUP5 and 7 AC domain in vitro and by complementation of an E. coli AC mutant (cyaA). Furthermore, ATKUP5 was tested for its ability to functionally complement a yeast mutant deficient in Trk1 and Trk2 high affinity potassium uptake transporters. Site-mutagenesis in the AC domain was used to test the effect of both functions in each other. Furthermore, ATKUP5 was characterized electrophysiologically in HEK-293 cells to characterize the nature of this transporter. The localization of the ATKUP5 in Arabidopsis was examined using a Green Fluorescent Protein (GFP) fusion with the ATKUP5 to determine whether ATKUP5 is expressed at the plasma or tonoplast membrane. Arabiodpsis thaliana of the wild type, overexpressing ATKUP5 and atkup5 mutant lines were used to examine phenotypic differences.

  6. Regulation of anterior chamber drainage by bicarbonate-sensitive soluble adenylyl cyclase in the ciliary body.

    Science.gov (United States)

    Lee, Yong S; Tresguerres, Martin; Hess, Kenneth; Marmorstein, Lihua Y; Levin, Lonny R; Buck, Jochen; Marmorstein, Alan D

    2011-12-02

    Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (C(t)). Modulation of "inflow" and "outflow" pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in C(t) with no effect on inflow. In mice deficient in sAC IOP is elevated, and C(t) is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or C(t) in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate C(t) and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure.

  7. Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

    Science.gov (United States)

    Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

    2018-06-01

    Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Vasodilator effects and putative guanylyl cyclase stimulation by 2-nitro-1-phenylethanone and 2-nitro-2-phenyl-propane-1,3-diol on rat aorta.

    Science.gov (United States)

    Vasconcelos, Thiago Brasileiro de; Ribeiro-Filho, Helder Veras; Lahlou, Saad; Pereira, José Geraldo de Carvalho; Oliveira, Paulo Sérgio Lopes de; Magalhães, Pedro Jorge Caldas

    2018-07-05

    Compounds containing a nitro group may reveal vasodilator properties. Several nitro compounds have a NO 2 group in a short aliphatic chain connected to an aromatic group. In this study, we evaluated in rat aorta the effects of two nitro compounds, with emphasis on a putative recruitment of the soluble guanylate cyclase (sGC) pathway to induce vasodilation. Isolated aortic rings were obtained from male Wistar rats to compare the effects induced by 2-nitro-1-phenylethanone (NPeth) or 2-nitro-2-phenyl-propane-1,3-diol (NPprop). In aortic preparations contracted with phenylephrine or KCl, NPeth and NPprop induced vasorelaxant effects that did not depend on the integrity of vascular endothelium. NPeth had a lesser vasorelaxant efficacy than NPprop and only the NPprop effects were inhibited by pretreatment with the sGC inhibitors, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or methylene blue. In an ODQ-preventable manner, NPprop inhibited the contractile component of the phenylephrine-induced response mediated by intracellular Ca 2+ release or by extracellular Ca 2+ recruitment through receptor- or voltage-operated Ca 2+ channels. In contrast, NPprop was inert against the transient contraction induced by caffeine in Ca 2+ -free medium. In an ODQ-dependent manner, NPprop inhibited the contraction induced by the protein kinase C activator phorbol 12,13-dibutyrate or by the tyrosine phosphatase inhibitor sodium orthovanadate. In silico docking analysis of a sGC homologous protein revealed preferential site for NPprop. In conclusion, the nitro compounds NPeth and NPprop induced vasorelaxation in rat aortic rings. Aliphatic chain substituents selectively interfered in the ability of these compounds to induce vasorelaxant effects, and only NPprop relaxed aortic rings via a sGC pathway. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Regulation of brain adenylate cyclase by calmodulin

    International Nuclear Information System (INIS)

    Harrison, J.K.

    1988-01-01

    This thesis examined the interaction between the Ca 2+ -binding protein, calmodulin (CaM), and the cAMP synthesizing enzyme, adenylate cyclase. The regulation of guanyl nucleotide-dependent adenylate cyclase by CaM was examined in a particulate fraction from bovine striatum. CaM stimulated basal adenylate cyclase activity and enhanced the stimulation of the enzyme by GTP and dopamine (DA). The potentiation of GTP- and DA-stimulated adenylate cyclase activities by CaM was more sensitive to the concentration of CaM than was the stimulation of basal activity. A photoreactive CaM derivative was developed in order to probe the interactions between CaM and the adenylate cyclase components of bovine brain. Iodo-[ 125 I]-CaM-diazopyruvamide ( 125 I-CAM-DAP) behaved like native CaM with respect to Ca 2+ -enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and Ca 2+ -dependent stimulation of adenylate cyclase. 125 I-CaM-DAP cross-linked to CaM-binding proteins in a Ca 2+ -dependent, concentration-dependent, and CaM-specific manner. Photolysis of 125 I-CaM-DAP and forskolin-agarose purified CaM-sensitive adenylate cyclase produced an adduct with a molecular weight of 140,000

  10. Soluble guanylate cyclase stimulation prevents fibrotic tissue remodeling and improves survival in salt-sensitive Dahl rats.

    Directory of Open Access Journals (Sweden)

    Sandra Geschka

    Full Text Available 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.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.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.

  11. The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacle Balanus (=Amphibalanus) amphitrite

    KAUST Repository

    Zhang, Y.

    2012-08-01

    The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling.

  12. The regulatory role of the NO/cGMP signal transduction cascade during larval attachment and metamorphosis of the barnacle Balanus (=Amphibalanus) amphitrite

    KAUST Repository

    Zhang, Y.; He, L.-S.; Zhang, G.; Xu, Y.; Lee, O.-O.; Matsumura, K.; Qian, P.-Y.

    2012-01-01

    The barnacle Balanus amphitrite is among the most dominant fouling species on intertidal rocky shores in tropical and subtropical areas and is thus a target organism in antifouling research. After being released from adults, the swimming nauplius undertakes six molting cycles and then transforms into a cyprid. Using paired antennules, a competent cyprid actively explores and selects a suitable substratum for attachment and metamorphosis (collectively known as settlement). This selection process involves the reception of exogenous signals and subsequent endogenous signal transduction. To investigate the involvement of nitric oxide (NO) and cyclic GMP (cGMP) during larval settlement of B. amphitrite, we examined the effects of an NO donor and an NO scavenger, two nitric oxide synthase (NOS) inhibitors and a soluble guanylyl cyclase (sGC) inhibitor on settling cyprids. We found that the NO donor sodium nitroprusside (SNP) inhibited larval settlement in a dose-dependent manner. In contrast, both the NO scavenger carboxy-PTIO and the NOS inhibitors aminoguanidine hemisulfate (AGH) and S-methylisothiourea sulfate (SMIS) significantly accelerated larval settlement. Suppression of the downstream guanylyl cyclase (GC) activity using a GC-selective inhibitor ODQ could also significantly accelerate larval settlement. Interestingly, the settlement inhibition effects of SNP could be attenuated by ODQ at all concentrations tested. In the developmental expression profiling of NOS and sGC, the lowest expression of both genes was detected in the cyprid stage, a crucial stage for the larval decision to attach and metamorphose. In summary, we concluded that NO regulates larval settlement via mediating downstream cGMP signaling.

  13. Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

    Science.gov (United States)

    Vilches-Flores, Alonso; Tovar, Armando R; Marin-Hernandez, Alvaro; Rojas-Ochoa, Alberto; Fernandez-Mejia, Cristina

    2010-07-01

    Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression. (c) 2010 Elsevier Inc. All rights reserved.

  14. Effect of thuringiensin on adenylate cyclase in rat cerebral cortex

    International Nuclear Information System (INIS)

    Tsai, S.-F.; Yang Chi; Wang, S.-C.; Wang, J.-S.; Hwang, J.-S.; Ho, S.-P.

    2004-01-01

    The purpose of this work is to evaluate the effect of thuringiensin on the adenylate cyclase activity in rat cerebral cortex. The cyclic adenosine 3'5'-monophosphate (cAMP) levels were shown to be dose-dependently elevated 17-450% or 54-377% by thuringiensin at concentrations of 10 μM-100 mM or 0.5-4 mM, due to the activation of basal adenylate cyclase activity of rat cerebral cortical membrane preparation. Thuringiensin also activated basal activity of a commercial adenylate cyclase from Escherichia coli. However, the forskolin-stimulated adenylate cyclase activity in rat cerebral cortex was inhibited by thuringiensin at concentrations of 1-100 μM, thus cAMP production decreased. Furthermore, thuringiensin or adenylate cyclase inhibitor (MDL-12330A) reduced the forskolin (10 μM)-stimulated adenylate cyclase activity at concentrations of 10 μM, 49% or 43% inhibition, respectively. In conclusion, this study demonstrated that thuringiensin could activate basal adenylate cyclase activity and increase cAMP concentrations in rat cerebral cortex or in a commercial adenylate cyclase. Comparing the dose-dependent effects of thuringiensin on the basal and forskolin-stimulated adenylate cyclase activity, thuringiensin can be regarded as a weak activator of adenylate cyclase or an inhibitor of forskolin-stimulated adenylate cyclase

  15. Salt-induced Na+/K+-ATPase-α/β expression involves soluble adenylyl cyclase in endothelial cells.

    Science.gov (United States)

    Mewes, Mirja; Nedele, Johanna; Schelleckes, Katrin; Bondareva, Olga; Lenders, Malte; Kusche-Vihrog, Kristina; Schnittler, Hans-Joachim; Brand, Stefan-Martin; Schmitz, Boris; Brand, Eva

    2017-10-01

    High dietary salt intake may lead to vascular stiffness, which predicts cardiovascular diseases such as heart failure, and myocardial and cerebral infarctions as well as renal impairment. The vascular endothelium is a primary target for deleterious salt effects leading to dysfunction and endothelial stiffness. We hypothesize that the Ca 2+ - and bicarbonate-activated soluble adenylyl cyclase (sAC) contributes to Na + /K + -ATPase expression regulation in vascular endothelial cells and is an important regulator of endothelial stiffness. In vitro stimulation of vascular endothelial cells with high sodium (150 mM Na + )-induced Na + /K + -ATPase-α and Na + /K + -ATPase-β protein expression determined by western blot. Promoter analyses revealed increased cAMP response element (CRE)-mediated Na + /K + -ATPase-α transcriptional activity under high sodium concentrations. Inhibition of sAC by the specific inhibitor KH7 or siRNA reduced the sodium effects. Flame photometry revealed increased intracellular sodium concentrations in response to high sodium stimulations, which were paralleled by elevated ATP levels. Using atomic force microscopy, a nano-technique that measures cellular stiffness and deformability, we detected significant endothelial stiffening under increased sodium concentrations, which was prevented by inhibition of sAC using KH7 and Na + /K + -ATPase using ouabain. Furthermore, analysis of primary aortic endothelial cells in an in vitro aging model revealed an impaired Na + /K + -ATPase-α sodium response and elevated intracellular sodium levels with cellular aging. We conclude that sAC mediates sodium-induced Na + /K + -ATPase expression in vascular endothelium and is an important regulator of endothelial stiffness. The reactivity of Na + /K + -ATPase-α expression regulation in response to high sodium seems to be impaired in aging endothelial cells and might be a component of endothelial dysfunction.

  16. Chronic intratracheal application of the soluble guanylyl cyclase stimulator BAY 41-8543 ameliorates experimental pulmonary hypertension.

    Science.gov (United States)

    Amirjanians, Matthieu; Egemnazarov, Bakytbek; Sydykov, Akylbek; Kojonazarov, Baktybek; Brandes, Ralf; Luitel, Himal; Pradhan, Kabita; Stasch, Johannes-Peter; Redlich, Gorden; Weissmann, Norbert; Grimminger, Friedrich; Seeger, Werner; Ghofrani, Hossein; Schermuly, Ralph

    2017-05-02

    Dysfunction of the NO/sGC/cGMP signaling pathway has been implicated in the pathogenesis of pulmonary hypertension (PH). Therefore, agents stimulating cGMP synthesis via sGC are important therapeutic options for treatment of PH patients. An unwanted effect of this novel class of drugs is their systemic hypotensive effect. We tested the hypothesis that aerosolized intra-tracheal delivery of the sGC stimulator BAY41-8543 could diminish its systemic vasodilating effect.Pharmacodynamics and -kinetics of BAY41-8543 after single intra-tracheal delivery was tested in healthy rats. Four weeks after a single injection of monocrotaline (MCT, 60 mg/kg s.c.), rats were randomized to a two-week treatment with either placebo, BAY 41-8543 (10 mg/kg per os (PO)) or intra-tracheal (IT) instillation (3 mg/kg or 1 mg/kg).Circulating concentrations of the drug 10 mg/kg PO and 3 mg/kg IT were comparable. BAY 41-8543 was detected in the lung tissue and broncho-alveolar fluid after IT delivery at higher concentrations than after PO administration. Systemic arterial pressure transiently decreased after oral BAY 41-8543 and was unaffected by intratracheal instillation of the drug. PO 10 mg/kg and IT 3 mg/kg regimens partially reversed pulmonary hypertension and improved heart function in MCT-injected rats. Minor efficacy was noted in rats treated IT with 1 mg/kg. The degree of pulmonary vascular remodeling was largely reversed in all treatment groups.Intratracheal administration of BAY 41-8543 reverses PAH and vascular structural remodeling in MCT-treated rats. Local lung delivery is not associated with systemic blood pressure lowering and represents thus a further development of PH treatment with sGC stimulators.

  17. Calmodulin-regulated adenylyl cyclases and neuromodulation.

    Science.gov (United States)

    Xia, Z; Storm, D R

    1997-06-01

    Coincidence detection and crosstalk between signal transduction systems play very important regulatory roles in the nervous system, particularly in the regulation of transcription. Coupling of the Ca2+ and cAMP regulatory systems by calmodulin-regulated adenylyl cyclases is hypothesized to be important for some forms of synaptic plasticity, neuroendocrine function, and olfactory detection. Recent studies of a mutant mouse deficient in type I calmodulin-sensitive adenylyl cyclase have provided the first evidence that adenylyl cyclases are important for synaptic plasticity, as well as for learning and memory in vertebrates.

  18. Defying the stereotype: non-canonical roles of the peptide hormones guanylin and uroguanylin

    Directory of Open Access Journals (Sweden)

    Nirmalya eBasu

    2011-06-01

    Full Text Available The peptide hormones uroguanylin and guanylin have been traditionally thought to be mediators of fluid-ion homeostasis in the vertebrate intestine. They serve as ligands for receptor guanylyl cyclase C (GC-C, and both receptor and ligands are expressed predominantly in the intestine. Ligand binding to GC-C results in increased cGMP production in the cell which governs downstream signaling. In the last decade, a significant amount of research has unraveled novel functions for this class of peptide hormones, in addition to their action as intestinal secretagogues. An additional receptor for uroguanylin, receptor guanylyl cyclase D, has also been identified. Thus, unconventional roles of these peptides in regulating renal filtration, olfaction, reproduction and cell proliferation have begun to be elucidated in detail. These varied effects suggest that these peptide hormones act in an autocrine, paracrine as well as endocrine manner to regulate diverse cellular processes.

  19. Stimulation of cyclic GMP efflux in human melanocytes by hypergravity generated by centrifugal acceleration

    NARCIS (Netherlands)

    Ivanova, Krassimira; Zadeh, Nahid Hamidi; Block, Ingrid; Das, Pranab K.; Gerzer, Rupert

    2004-01-01

    Gravity alteration (micro- and hypergravity) is known to influence cell functions. As guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in human melanocyte functions and different guanylyl cyclase isoforms are responsible for cGMP synthesis in human non-metastatic and metastatic

  20. Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease.

    Science.gov (United States)

    Cortese-Krott, Miriam M; Mergia, Evanthia; Kramer, Christian M; Lückstädt, Wiebke; Yang, Jiangning; Wolff, Georg; Panknin, Christina; Bracht, Thilo; Sitek, Barbara; Pernow, John; Stasch, Johannes-Peter; Feelisch, Martin; Koesling, Doris; Kelm, Malte

    2018-04-01

    Endothelial dysfunction is associated with decreased NO bioavailability and impaired activation of the NO receptor soluble guanylate cyclase (sGC) in the vasculature and in platelets. Red blood cells (RBCs) are known to produce NO under hypoxic and normoxic conditions; however evidence of expression and/or activity of sGC and downstream signaling pathway including phopshodiesterase (PDE)-5 and protein kinase G (PKG) in RBCs is still controversial. In the present study, we aimed to investigate whether RBCs carry a functional sGC signaling pathway and to address whether this pathway is compromised in coronary artery disease (CAD). Using two independent chromatographic procedures, we here demonstrate that human and murine RBCs carry a catalytically active α 1 β 1 -sGC (isoform 1), which converts 32 P-GTP into 32 P-cGMP, as well as PDE5 and PKG. Specific sGC stimulation by NO+BAY 41-2272 increases intracellular cGMP-levels up to 1000-fold with concomitant activation of the canonical PKG/VASP-signaling pathway. This response to NO is blunted in α1-sGC knockout (KO) RBCs, but fully preserved in α2-sGC KO. In patients with stable CAD and endothelial dysfunction red cell eNOS expression is decreased as compared to aged-matched controls; by contrast, red cell sGC expression/activity and responsiveness to NO are fully preserved, although sGC oxidation is increased in both groups. Collectively, our data demonstrate that an intact sGC/PDE5/PKG-dependent signaling pathway exists in RBCs, which remains fully responsive to NO and sGC stimulators/activators in patients with endothelial dysfunction. Targeting this pathway may be helpful in diseases with NO deficiency in the microcirculation like sickle cell anemia, pulmonary hypertension, and heart failure. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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. Copyright © 2016 the American Physiological Society.

  2. Ruthenium Complexes as NO Donors for Vascular Relaxation Induction

    Directory of Open Access Journals (Sweden)

    Renata Galvão de Lima

    2014-07-01

    Full Text Available Nitric oxide (NO donors are substances that can release NO. Vascular relaxation induction is among the several functions of NO, and the administration of NO donors is a pharmacological alternative to treat hypertension. This review will focus on the physicochemical description of ruthenium-derived NO donor complexes that release NO via reduction and light stimulation. In particular, we will discuss the complexes synthesized by our research group over the last ten years, and we will focus on the vasodilation and arterial pressure control elicited by these complexes. Soluble guanylyl cyclase (sGC and potassium channels are the main targets of the NO species released from the inorganic compounds. We will consider the importance of the chemical structure of the ruthenium complexes and their vascular effects.

  3. Investigations for a multi-marker RT-PCR to improve sensitivity of disseminated tumor cell detection.

    NARCIS (Netherlands)

    Vlems, F.A.; Diepstra, J.H.S.; Cornelissen, I.M.; Ligtenberg, M.J.L.; Wobbes, Th.; Punt, C.J.A.; Krieken, J.H.J.M. van; Ruers, T.J.M.; Muijen, G.N.P. van

    2003-01-01

    BACKGROUND: In order to develop a multi-marker RT-PCR, which as such may be more sensitive than a single marker assay for the detection of disseminated tumor cells, we evaluated six RT-PCR markers: cytokeratin 20 (CK20), carcinoembryonic antigen (CEA), guanylyl cyclase C (GCC), epidermal growth

  4. Investigations for a multi-marker RT-PCR to improve sensitivity of disseminated tumor cell detection

    NARCIS (Netherlands)

    Vlems, F. A.; Diepstra, J. H. S.; Cornelissen, I. M. H. A.; Ligtenberg, M. J. L.; Wobbes, Th; Punt, C. J. A.; van Krieken, J. H. J. M.; Ruers, T. J. M.; van Muijen, G. N. P.

    2003-01-01

    In order to develop a multi-marker RT-PCR, which as such may be more sensitive than a single marker assay for the detection of disseminated tumor cells, we evaluated six RT-PCR markers: cytokeratin 20 (CK20), carcinoembryonic antigen (CEA), guanylyl cyclase C (GCC), epidermal growth factor receptor

  5. Identification of a fourth family of lycopene cyclases in photosynthetic bacteria.

    Science.gov (United States)

    Maresca, Julia A; Graham, Joel E; Wu, Martin; Eisen, Jonathan A; Bryant, Donald A

    2007-07-10

    A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.

  6. Compartmentalization of NO signaling cascade in skeletal muscles

    International Nuclear Information System (INIS)

    Buchwalow, Igor B.; Minin, Evgeny A.; Samoilova, Vera E.; Boecker, Werner; Wellner, Maren; Schmitz, Wilhelm; Neumann, Joachim; Punkt, Karla

    2005-01-01

    Skeletal muscle functions regulated by NO are now firmly established. However, the literature on the compartmentalization of NO signaling in myocytes is highly controversial. To address this issue, we examined localization of enzymes engaged in L-arginine-NO-cGMP signaling in the rat quadriceps muscle. Employing immunocytochemical labeling complemented with tyramide signal amplification and electron microscopy, we found NO synthase expressed not only in the sarcolemma, but also along contractile fibers, in the sarcoplasmic reticulum and mitochondria. The expression pattern of NO synthase in myocytes showed striking parallels with the enzymes engaged in L-arginine-NO-cGMP signaling (arginase, phosphodiesterase, and soluble guanylyl cyclase). Our findings are indicative of an autocrine fashion of NO signaling in skeletal muscles at both cellular and subcellular levels, and challenge the notion that the NO generation is restricted to the sarcolemma

  7. Interaction of glutamate- and adenosine-induced decrease of acetylcholine quantal release at frog neuromuscular junction

    Czech Academy of Sciences Publication Activity Database

    Adámek, S.; Shakirzyanova, V.; Malomouzh, A. I.; Naumenko, N. V.; Vyskočil, František

    2010-01-01

    Roč. 59, č. 5 (2010), s. 803-810 ISSN 0862-8408 R&D Projects: GA AV ČR(CZ) IAA500110905; GA ČR GA202/09/0806 Institutional research plan: CEZ:AV0Z50110509 Keywords : Endplate potentials * Guanylyl cyclase Subject RIV: ED - Physiology Impact factor: 1.646, year: 2010

  8. Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease

    Directory of Open Access Journals (Sweden)

    Miriam M. Cortese-Krott

    2018-04-01

    Full Text Available Endothelial dysfunction is associated with decreased NO bioavailability and impaired activation of the NO receptor soluble guanylate cyclase (sGC in the vasculature and in platelets. Red blood cells (RBCs are known to produce NO under hypoxic and normoxic conditions; however evidence of expression and/or activity of sGC and downstream signaling pathway including phopshodiesterase (PDE-5 and protein kinase G (PKG in RBCs is still controversial. In the present study, we aimed to investigate whether RBCs carry a functional sGC signaling pathway and to address whether this pathway is compromised in coronary artery disease (CAD. Using two independent chromatographic procedures, we here demonstrate that human and murine RBCs carry a catalytically active α1β1-sGC (isoform 1, which converts 32P-GTP into 32P-cGMP, as well as PDE5 and PKG. Specific sGC stimulation by NO+BAY 41-2272 increases intracellular cGMP-levels up to 1000-fold with concomitant activation of the canonical PKG/VASP-signaling pathway. This response to NO is blunted in α1-sGC knockout (KO RBCs, but fully preserved in α2-sGC KO. In patients with stable CAD and endothelial dysfunction red cell eNOS expression is decreased as compared to aged-matched controls; by contrast, red cell sGC expression/activity and responsiveness to NO are fully preserved, although sGC oxidation is increased in both groups. Collectively, our data demonstrate that an intact sGC/PDE5/PKG-dependent signaling pathway exists in RBCs, which remains fully responsive to NO and sGC stimulators/activators in patients with endothelial dysfunction. Targeting this pathway may be helpful in diseases with NO deficiency in the microcirculation like sickle cell anemia, pulmonary hypertension, and heart failure. Keywords: cGMP, Nitric oxide, Protein kinase G, Signaling, Non -canonical functions of RBCs

  9. Inhaled nitric oxide pretreatment but not posttreatment attenuates ischemia-reperfusion-induced pulmonary microvascular leak.

    Science.gov (United States)

    Chetham, P M; Sefton, W D; Bridges, J P; Stevens, T; McMurtry, I F

    1997-04-01

    Ischemia-reperfusion (I/R) pulmonary edema probably reflects a leukocyte-dependent, oxidant-mediated mechanism. Nitric oxide (NO) attenuates leukocyte-endothelial cell interactions and I/R-induced microvascular leak. Cyclic adenosine monophosphate (cAMP) agonists reverse and prevent I/R-induced microvascular leak, but reversal by inhaled NO (INO) has not been tested. In addition, the role of soluble guanylyl cyclase (sGC) activation in the NO protection effect is unknown. Rat lungs perfused with salt solution were grouped as either I/R, I/R with INO (10 or 50 ppm) on reperfusion, or time control. Capillary filtration coefficients (Kfc) were estimated 25 min before ischemia (baseline) and after 30 and 75 min of reperfusion. Perfusate cell counts and lung homogenate myeloperoxidase activity were determined in selected groups. Additional groups were treated with either INO (50 ppm) or isoproterenol (ISO-10 microM) after 30 min of reperfusion. Guanylyl cyclase was inhibited with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ-15 microM), and Kfc was estimated at baseline and after 30 min of reperfusion. (1) Inhaled NO attenuated I/R-induced increases in Kfc. (2) Cell counts were similar at baseline. After 75 min of reperfusion, lung neutrophil retention (myeloperoxidase activity) and decreased perfusate neutrophil counts were similar in all groups. (3) In contrast to ISO, INO did not reverse microvascular leak. (4) 8-bromoguanosine 3',5'-cyclic monophosphate (8-br-cGMP) prevented I/R-induced microvascular leak in ODQ-treated lungs, but INO was no longer effective. Inhaled NO attenuates I/R-induced pulmonary microvascular leak, which requires sGC activation and may involve a mechanism independent of inhibition of leukocyte-endothelial cell interactions. In addition, INO is ineffective in reversing I/R-induced microvascular leak.

  10. Soluble adenylyl cyclase in vascular endothelium: gene expression control of epithelial sodium channel-α, Na+/K+-ATPase-α/β, and mineralocorticoid receptor.

    Science.gov (United States)

    Schmitz, Boris; Nedele, Johanna; Guske, Katrin; Maase, Martina; Lenders, Malte; Schelleckes, Michael; Kusche-Vihrog, Kristina; Brand, Stefan-Martin; Brand, Eva

    2014-04-01

    The Ca(2+)- and bicarbonate-activated soluble adenylyl cyclase (sAC) has been identified recently as an important mediator of aldosterone signaling in the kidney. Nuclear sAC has been reported to stimulate cAMP response element-binding protein 1 phosphorylation via protein kinase A, suggesting an alternative cAMP pathway in the nucleus. In this study, we analyzed the sAC as a potential modulator of endothelial stiffness in the vascular endothelium. We determined the contribution of sAC to cAMP response element-mediated transcriptional activation in vascular endothelial cells and kidney collecting duct cells. Inhibition of sAC by the specific inhibitor KH7 significantly reduced cAMP response element-mediated promoter activity and affected cAMP response element-binding protein 1 phosphorylation. Furthermore, KH7 and anti-sAC small interfering RNA significantly decreased mRNA and protein levels of epithelial sodium channel-α and Na(+)/K(+)-ATPase-α. Using atomic force microscopy, a nano-technique that measures stiffness and deformability of living cells, we detected significant endothelial cell softening after sAC inhibition. Our results suggest that the sAC is a regulator of gene expression involved in aldosterone signaling and an important regulator of endothelial stiffness. Additional studies are warranted to investigate the protective action of sAC inhibitors in humans for potential clinical use.

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

    Directory of Open Access Journals (Sweden)

    Melissa H Costell

    2012-07-01

    Full Text Available 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 NO-sensitive sGC stimulation. The cardiovascular effects of the NO-insensitive sGC activator GSK2181236A (a non-depressor dose and a higher dose which lowered mean arterial pressure [MAP] by 5-10mmHg and equi-efficacious doses of the NO-sensitive sGC stimulator BAY 60-4552 were assessed in Sprague Dawley rats during coronary artery ischemia/reperfusion (I/R and spontaneously hypertensive stroke prone rats (SHR-SP on a high salt/fat diet (HSFD. In I/R, neither compound reduced infarct size. In SHR-SP, HSFD increased MAP, urine output, microalbuminuria and mortality, caused left ventricular hypertrophy and impaired endothelium-dependent vasorelaxation. The low dose of BAY 60-4552 but not GSK2181236A decreased urine output and mortality. Conversely, the low dose of GSK2181236A attenuated cardiac hypertrophy. The high doses of both compounds similarly attenuated cardiac hypertrophy and mortality. In addition, the high dose of BAY 60-4552 reduced urine output, microalbuminuria and MAP. Neither compound improved endothelium-dependent vasorelaxation. In SHR-SP aorta, the vasodilatory responses to the NO-dependent compounds carbachol and sodium nitroprusside were attenuated by HSFD. In contrast, the vasodilatory responses to GSK2181236A and BAY 60-4552 were unaltered by HSFD, indicating that reduced NO-bioavailability and not changes in the sGC oxidative state is responsible for the vascular dysfunction. In summary, GSK2181236A and BAY 60-4552 provide partial benefit against hypertension-induced end organ damage. The differential beneficial effects observed between these compounds could reflect tissue-specific changes in the s

  12. Monospecific antibody against Bordetella pertussis Adenylate Cyclase protects from Pertussis

    Directory of Open Access Journals (Sweden)

    Yasmeen Faiz Kazi

    2012-06-01

    Full Text Available Objectives: Acellular pertussis vaccines has been largely accepted world-wide however, there are reports about limitedantibody response against these vaccines suggesting that multiple antigens should be included in acellular vaccinesto attain full protection. The aim of present study was to evaluate the role of Bordetella pertussis adenylate cyclase as aprotective antigen.Materials and methods: Highly mono-specific antibody against adenylate cyclase (AC was raised in rabbits usingnitrocellulose bound adenylate cyclase and the specificity was assessed by immuoblotting. B.pertussis 18-323, wasincubated with the mono-specific serum and without serum as a control. Mice were challenged intra-nasally and pathophysiolgicalresponses were recorded.Results: The production of B.pertussis adenylate cyclase monospecific antibody that successfully recognized on immunoblotand gave protection against fatality (p< 0.01 and lung consolidation (p <0.01. Mouse weight gain showedsignificant difference (p< 0.05.Conclusion: These preliminary results highlight the role of the B.pertussis adenylate cyclase as a potential pertussisvaccine candidate. B.pertussis AC exhibited significant protection against pertussis in murine model. J Microbiol InfectDis 2012; 2(2: 36-43Key words: Pertussis; monospecific; antibody; passive-protection

  13. Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors

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    Thomas Pleli

    2018-03-01

    Full Text Available Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA and Epac, and an efflux of cAMP, the function of which is still unclear. Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2 inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors. In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

  14. [Adenylate cyclase from rabbit heart: substrate binding site].

    Science.gov (United States)

    Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

    1981-08-01

    The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

  15. Overexpression of functional human oxidosqualene cyclase in Escherichia coli

    DEFF Research Database (Denmark)

    Kürten, Charlotte; Uhlén, Mathias; Syrén, Per-Olof

    2015-01-01

    The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the te......The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation...... of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7mg protein/g cells (hOSC in Pichia pastoris) after 48h...... of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9mg/g bacterial cells...

  16. pH sensing via bicarbonate-regulated ‘soluble’ adenylyl cyclase (sAC

    Directory of Open Access Journals (Sweden)

    Nawreen eRahman

    2013-11-01

    Full Text Available Soluble adenylyl cyclase (sAC is a source of the second messenger cyclic adenosine 3',5' monophosphate (cAMP. sAC is directly regulated by bicarbonate (HCO3- ions. In living cells, HCO3- 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, HCO3-, 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.

  17. Developmental changes of beta-adrenergic receptor-linked adenylate cyclase of rat liver

    International Nuclear Information System (INIS)

    Katz, M.S.; Boland, S.R.; Schmidt, S.J.

    1985-01-01

    beta-Adrenergic agonist-sensitive adenylate cyclase activity and binding of the beta-adrenergic antagonist(-)-[ 125 I]iodopindolol were studied in rat liver during development of male Fischer 344 rats ages 6-60 days. In liver homogenates maximum adenylate cyclase response to beta-adrenergic agonist (10(-5) M isoproterenol or epinephrine) decreased by 73% (P less than 0.01) between 6 and 60 days, with most of the decrease (56%; P less than 0.01) occurring by 20 days. beta-adrenergic receptor density (Bmax) showed a corresponding decrease of 66% (P less than 0.01) by 20 days without subsequent change. Binding characteristics of stereospecificity, pharmacological specificity, saturability with time, and reversibility were unchanged with age. GTP-, fluoride-, forskolin-, and Mn2+-stimulated adenylate cyclase activities also decreased during development, suggesting a decrease of activity of the catalytic component and/or guanine nucleotide regulatory component of adenylate cyclase. These results indicate that the developmental decrease of beta-adrenergic agonist-sensitive adenylate cyclase activity may result from decreased numbers of beta-adrenergic receptors. Developmental alterations of nonreceptor components of the enzyme may also contribute to changes of catecholamine-sensitive adenylate cyclase

  18. Ocean acidification stimulates alkali signal pathway: A bicarbonate sensing soluble adenylyl cyclase from oyster Crassostrea gigas mediates physiological changes induced by CO2 exposure.

    Science.gov (United States)

    Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Wang, Hao; Jiang, Shuai; Chen, Hao; Wang, Lingling; Song, Linsheng

    2016-12-01

    Ocean acidification (OA) has been demonstrated to have severe effects on marine organisms, especially marine calcifiers. However, the impacts of OA on the physiology of marine calcifiers and the underlying mechanisms remain unclear. Soluble adenylyl cyclase (sAC) is an acid-base sensor in response to [HCO 3 - ] and an intracellular source of cyclic AMP (cAMP). In the present study, an ortholog of sAC was identified from pacific oyster Crassostrea gigas (designated as CgsAC) and the catalytic region of CgsAC was cloned and expressed. Similar to the native CgsAC from gill tissues, the recombinant CgsAC protein (rCgsAC) exhibited [HCO 3 - ] mediated cAMP-forming activity, which could be inhibited by a small molecule KH7. After 16days of CO 2 exposure (pH=7.50), the mRNA transcripts of CgsAC increased in muscle, mantle, hepatopancreas, gill, male gonad and haemocytes, and two truncated CgsAC forms of 45kD and 20kD were produced. Cytosolic CgsAC could be translocated from the cytoplasm and nuclei to the membrane in response to CO 2 exposure. Besides, CO 2 exposure could increase the production of cAMP and intracellular pH of haemocytes, which was regulated by CgsAC (pocean acidification on marine calcifiers. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Reduced ability of C-type natriuretic peptide (CNP) to activate natriuretic peptide receptor B (NPR-B) causes dwarfism in lbab−/− mice

    Science.gov (United States)

    Yoder, Andrea R.; Kruse, Andrew C.; Earhart, Cathleen A.; Ohlendorf, Douglas H.; Potter, Lincoln R.

    2015-01-01

    C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality (lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that thirty to greater than one hundred-fold more CNPlbab was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNPlbab to activate NPR-B was explained, at least in part, by decreased binding since ten-fold more CNPlbab than wild-type CNP was required to compete with [125I][Tyr0]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab−/− mice. PMID:18554750

  20. Food restriction modulates β-adrenergic-sensitive adenylate cyclase in rat liver during aging

    International Nuclear Information System (INIS)

    Katz, M.S.

    1988-01-01

    Adenylate cyclase activities were studied in rat liver during postmaturational aging of male Fischer 344 rats fed ad libitum or restricted to 60% of the ad libitum intake. Catecholamine-stimulated adenylate cyclase activity increased by 200-300% between 6 and 24-27 mo of age in ad libitum-fed rats, whereas in food-restricted rats catecholamine response increased by only 58-84% between 6 and 30 mo. In ad libitum-fed rats, glucagon-stimulated enzyme activity also increased by 40% between 6 and 12 mo and in restricted rats a similar age-related increase was delayed until 18 mo. β-Adrenergic receptor density increased by 50% between 6 and 24 mo in livers from ad libitum-fed but not food-restricted rats and showed a highly significant correlation with maximal isoproterenol-stimulated adenylate cyclase activity over the postmaturational life span. Age-related increases in unstimulated (basal) adenylate cyclase activity and nonreceptor-mediated enzyme activation were retarded by food restriction. The results demonstrate that food restriction diminishes a marked age-related increase in β-adrenergic-sensitive adenylate cyclase activity of rat liver. Alterations of adrenergic-responsive adenylate cyclase with age and the modulatory effects of food restriction appear to be mediated by changes in both receptor and nonreceptor components of adenylate cyclase

  1. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    Unknown

    **Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, National Institute of Health, .... This construct expresses the N-terminal 330 amino acids ... C-terminal 112 amino acids of GCC as fusion with GST.

  2. Vascular nitric oxide: Beyond eNOS

    Directory of Open Access Journals (Sweden)

    Yingzi Zhao

    2015-10-01

    Full Text Available As the first discovered gaseous signaling molecule, nitric oxide (NO affects a number of cellular processes, including those involving vascular cells. This brief review summarizes the contribution of NO to the regulation of vascular tone and its sources in the blood vessel wall. NO regulates the degree of contraction of vascular smooth muscle cells mainly by stimulating soluble guanylyl cyclase (sGC to produce cyclic guanosine monophosphate (cGMP, although cGMP-independent signaling [S-nitrosylation of target proteins, activation of sarco/endoplasmic reticulum calcium ATPase (SERCA or production of cyclic inosine monophosphate (cIMP] also can be involved. In the blood vessel wall, NO is produced mainly from l-arginine by the enzyme endothelial nitric oxide synthase (eNOS but it can also be released non-enzymatically from S-nitrosothiols or from nitrate/nitrite. Dysfunction in the production and/or the bioavailability of NO characterizes endothelial dysfunction, which is associated with cardiovascular diseases such as hypertension and atherosclerosis.

  3. Effect of age and posture on human lymphocyte adenylate cyclase activity.

    Science.gov (United States)

    Mader, S L; Robbins, A S; Rubenstein, L Z; Tuck, M L; Scarpace, P J

    1988-03-01

    1. A number of age-related changes have been reported in the catecholamine-adrenoceptor-adenylate cyclase system. Most of the data available on these alterations come from resting subjects; the response to acute stress may provide additional insights into the age effect on these responses. 2. We measured supine and 10 min upright plasma noradrenaline and lymphocyte adenylate cyclase activity in ten healthy elderly subjects (age 66-80 years) and seven healthy young subjects (age 27-34 years). 3. Isoprenaline stimulation of lymphocyte adenylate cyclase activity was not significantly different between supine and upright positions or between elderly and young subjects. There was a marked increase in forskolin-stimulated adenylate cyclase activity in the upright posture in both elderly and young subjects. The increment over supine levels was 70% in the elderly (P less than 0.025) and 73% in the young (P less than 0.05). This enhanced forskolin activity was not seen in two young subjects who became syncopal. 4. These data suggest that enhanced forskolin-stimulated adenylate cyclase activity occurs after 10 min of upright posture in both elderly and young subjects, and may be relevant to immediate blood pressure regulation. We were unable to demonstrate any age-related differences in these acute adrenergic responses.

  4. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

    International Nuclear Information System (INIS)

    Masure, H.R.; Donovan, M.G.; Storm, D.R.

    1991-01-01

    An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca 2+ to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca 2+ and this interaction may be important for its invasion into animal cells

  5. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

    Energy Technology Data Exchange (ETDEWEB)

    Masure, H.R.; Donovan, M.G.; Storm, D.R.

    1991-01-01

    An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.

  6. Characterization of the functional domains of the natriuretic peptide receptor/guanylate cyclase by radiation inactivation

    International Nuclear Information System (INIS)

    Tremblay, J.; Huot, C.; Koch, C.; Potier, M.

    1991-01-01

    Radiation inactivation has been used to evaluate the molecular size of domains responsible for atrial natriuretic peptide (ANP)-binding and cyclase functions of the ANP receptor/guanylate cyclase. Two types of inactivation curves were observed for cyclase function in both adrenal cortex and aortic smooth muscle cells: (1) biphasic with enhanced guanylate cyclase activity after exposure to low radiation doses and (2) linear after preincubation of membrane proteins with 0.5 microM ANP or solubilization with Triton X-100. The existence of an inhibitory component was the simplest model that best explained the types of radiation curves obtained. Activation of guanylate cyclase by ANP or Triton X-100 could occur via the dissociation of this inhibitory component from the catalytic domain. On the other hand, the loss of ANP-binding activity was linear with increasing radiation exposures under basal, ANP treatment, and Triton X-100 solubilization conditions. Radiation inactivation sizes of about 30 kDa for cyclase function, 20 kDa for ANP-binding function, and 90 kDa for inhibitory function were calculated. These studies suggest that the ANP receptor/guanylate cyclase behaves as a multidomain protein. The results obtained by radiation inactivation of the various biological functions of this receptor are compatible with the hypothesis of an intramolecular inhibitory domain repressing the guanylate cyclase catalytic domain within its membrane environment

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

    Science.gov (United States)

    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

  8. The role of transcriptional regulation in maintaining the availability of mycobacterial adenylate cyclases

    Directory of Open Access Journals (Sweden)

    Sarah J. Casey

    2014-03-01

    Full Text Available Mycobacterium species have a complex cAMP regulatory network indicated by the high number of adenylate cyclases annotated in their genomes. However the need for a high level of redundancy in adenylate cyclase genes remains unknown. We have used semiquantitiative RT-PCR to examine the expression of eight Mycobacterium smegmatis cyclases with orthologs in the human pathogen Mycobacterium tuberculosis, where cAMP has recently been shown to be important for virulence. All eight cyclases were transcribed in all environments tested, and only four demonstrated environmental-mediated changes in transcription. M. smegmatis genes MSMEG_0545 and MSMEG_4279 were upregulated during starvation conditions while MSMEG_0545 and MSMEG_4924 were downregulated in H2O2 and MSMEG_3780 was downregulated in low pH and starvation. Promoter fusion constructs containing M. tuberculosis H37Rv promoters showed consistent regulation compared to their M. smegmatis orthologs. Overall our findings indicate that while low levels of transcriptional regulation occur, regulation at the mRNA level does not play a major role in controlling cellular cyclase availability in a given environment.

  9. Differential Contribution of the Guanylyl Cyclase-Cyclic GMP-Protein Kinase G Pathway to the Proliferation of Neural Stem Cells Stimulated by Nitric Oxide

    Directory of Open Access Journals (Sweden)

    Bruno P. Carreira

    2012-02-01

    Full Text Available Nitric oxide (NO is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP and the cGMP-dependent kinase (PKG are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ were used. We observed that long-term exposure to the NO donor (24 h, NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner.

  10. LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

    Science.gov (United States)

    Clayton, R N; Shakespear, R A; Marshall, J C

    1978-06-01

    Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

  11. Pituitary adenylate cyclase activating polypeptide and migraine

    DEFF Research Database (Denmark)

    Zagami, Alessandro S; Edvinsson, Lars; Goadsby, Peter J

    2014-01-01

    Pituitary adenylate cyclase activating peptide (PACAP) is found in human trigeminocervical complex and can trigger migraine. PACAP levels were measured using a sensitive radioimmunoassay. Stimulation of the superior sagittal sinus (SSS) in cat elevated PACAP levels in cranial blood. Patients...

  12. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} 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.

  13. Effects of ionizing radiation and cysteamine (MEA) on activity of mouse spleen adenyl cyclase

    International Nuclear Information System (INIS)

    Soltysiak-Pawluczuk, D.; Bitny-Szlachto, S.

    1976-01-01

    In mice X-irradiated with doses of 200 R and 400 R, there was a substantial increase in spleen adenyl cyclase activity; there was similar activation by MEA. In mice given MEA before irradiation, an additive effect of radiation and the radioprotective drug was observed. On the other hand, a dose of 800 R given either alone or after pre-treatment with MEA failed to elicit any change in cyclase activity. The results indicate the importance of the adenyl cyclase system in the response of cells to irradiation and action of MEA. (author)

  14. Porcine CD38 exhibits prominent secondary NAD(+) cyclase activity.

    Science.gov (United States)

    Ting, Kai Yiu; Leung, Christina F P; Graeff, Richard M; Lee, Hon Cheung; Hao, Quan; Kotaka, Masayo

    2016-03-01

    Cyclic ADP-ribose (cADPR) mobilizes intracellular Ca(2+) stores and activates Ca(2+) influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD(+) by the multifunctional CD38/ADP-ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD(+) can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD(+) to form linear ADPR while Aplysia ADP-ribosyl cyclase prefers cyclizing NAD(+) to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD(+) cyclase activity producing cADPR. We also determined the X-ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD(+) reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively. © 2016 The Protein Society.

  15. Radiation inactivation of multimeric enzymes: application to subunit interactions of adenylate cyclase

    International Nuclear Information System (INIS)

    Verkman, A.S.; Skorecki, K.L.; Ausiello, D.A.

    1986-01-01

    Radiation inactivation has been applied extensively to determine the molecular weight of soluble enzyme and receptor systems from the slope of a linear ln (activity) vs. dose curve. Complex nonlinear inactivation curves are predicted for multimeric enzyme systems, composed of distinct subunits in equilibrium with multimeric complexes. For the system A1 + A2----A1A2, with an active A1A2 complex (associative model), the ln (activity) vs. dose curve is linear for high dissociation constant, K. If a monomer, A1, has all the enzyme activity (dissociative model), the ln (activity) vs. dose curve has an activation hump at low radiation dose if the inactive subunit, A2, has a higher molecular weight than A1 and has upward concavity when A2 is smaller than A1. In general, a radiation inactivation model for a multistep mechanism for enzyme activation fulfills the characteristics of an associative or dissociative model if the reaction step forming active enzyme is an associative or dissociative reaction. Target theory gives the molecular weight of the active enzyme subunit or complex from the limiting slope of the ln (activity) vs. dose curve at high radiation dose. If energy transfer occurs among subunits in the multimer, the ln (activity) vs. dose curve is linear for a single active component and is concave upward for two or more active components. The use of radiation inactivation as a method to determine enzyme size and multimeric subunit assembly is discussed with specific application to the hormone-sensitive adenylate cyclase system. It is shown that the complex inactivation curves presented in the accompanying paper can be used select the best mechanism out of a series of seven proposed mechanisms for the activation of adenylate cyclase by hormone

  16. Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

    OpenAIRE

    Maresca, Julia A.; Graham, Joel E.; Wu, Martin; Eisen, Jonathan A.; Bryant, Donald A.

    2007-01-01

    A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechoc...

  17. Age-associated alterations in hepatic β-adrenergic receptor/adenylate cyclase complex

    International Nuclear Information System (INIS)

    Graham, S.M.; Herring, P.A.; Arinze, I.J.

    1987-01-01

    The effect of age on catecholamine regulation of hepatic glycogenolysis and on hepatic adenylate cyclase was studied in male rats up to 24 mo of age. Epinephrine and norepinephrine stimulated glycogenolysis in isolated hepatocytes at all age groups studied. Isoproterenol, however, stimulated glycogenolysis only at 24 mo. In isolated liver membranes, usual activators of adenylate cyclase increased the activity of the enzyme considerably more in membranes from 24-mo-old rats than in membranes from either 3- or 22-mo-old rats. The Mn 2+ -dependent activity of the cyclase was increased by 2.9-fold in 3-mo-old animals and ∼ 5.7-fold in 24-mo-old rats, indicating a substantial age-dependent increase in the intrinsic activity of the catalytic unit. The density of the β-adrenergic receptor, as measured by the binding of [ 125 I]-iodocyanopindolol to plasma membranes, was 5-8 fmol/mg protein in rats aged 3-12 mo but increased to 19 fmol/mg protein in 24-mo-old rats. Computer-aided analysis of isoproterenol competition of the binding indicated a small age-dependent increase in the proportion of β-receptors in the high-affinity state. These observations suggest that β-receptor-mediated hepatic glycogenolysis in the aged rat is predicated upon increases in the density of β-receptors as well as increased intrinsic activity of the catalytic unit of adenylate cyclase

  18. Pituitary adenylate cyclase activating polypeptide reduces A-type K+ currents and caspase activity in cultured adult mouse olfactory neurons.

    Science.gov (United States)

    Han, P; Lucero, M T

    2005-01-01

    Pituitary adenylate cyclase activating polypeptide has been shown to reduce apoptosis in neonatal cerebellar and olfactory receptor neurons, however the underlying mechanisms have not been elucidated. In addition, the neuroprotective effects of pituitary adenylate cyclase activating polypeptide have not been examined in adult tissues. To study the effects of pituitary adenylate cyclase activating polypeptide on neurons in apoptosis, we measured caspase activation in adult olfactory receptor neurons in vitro. Interestingly, we found that the protective effects of pituitary adenylate cyclase activating polypeptide were related to the absence of a 4-aminopyridine (IC50=144 microM) sensitive rapidly inactivating potassium current often referred to as A-type current. In the presence of 40 nM pituitary adenylate cyclase activating polypeptide 38, both A-type current and activated caspases were significantly reduced. A-type current reduction by pituitary adenylate cyclase activating polypeptide was blocked by inhibiting the phospholipase C pathway, but not the adenylyl cyclase pathway. Our observation that 5 mM 4-aminopyridine mimicked the caspase inhibiting effects of pituitary adenylate cyclase activating polypeptide indicates that A-type current is involved in apoptosis. This work contributes to our growing understanding that potassium currents are involved with the activation of caspases to affect the balance between cell life and death.

  19. Cyclic Nucleotide Signalling in Kidney Fibrosis

    Directory of Open Access Journals (Sweden)

    Elisabeth Schinner

    2015-01-01

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

  20. Cloning and Functional Characterization of a Lycopene β-Cyclase from Macrophytic Red Alga Bangia fuscopurpurea

    Directory of Open Access Journals (Sweden)

    Tian-Jun Cao

    2017-04-01

    Full Text Available Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene into β- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, β-carotene (β,β-carotene is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene β-cyclase activity in these organisms. In this work, we isolated a gene (BfLCYB encoding a lycopene β-cyclase from Bangia fuscopurpurea, a red alga that is considered to be one of the primitive multicellular eukaryotic photosynthetic organisms and accumulates carotenoid constituents with both β- and ε-rings, including β-carotene, zeaxanthin, α-carotene (β,ε-carotene and lutein. Functional complementation in Escherichia coli demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (β,ψ-carotene and bicyclic β-carotene, and cyclization of the open end of monocyclic δ-carotene (ε,ψ-carotene to produce α-carotene. No ε-cyclization activity was identified for BfLCYB. Sequence comparison showed that BfLCYB shares conserved domains with other functionally characterized lycopene cyclases from different organisms and belongs to a group of ancient lycopene cyclases. Although B. fuscopurpurea also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.

  1. Agonist-induced desensitization of adenylyl cyclase in Y1 adrenocortical tumor cells

    International Nuclear Information System (INIS)

    Olson, M.F.; Tsao, J.; Pon, D.J.; Schimmer, B.P.

    1991-01-01

    Y1 adrenocortical tumor cells (Y1DS) and Y1 mutants resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were transfected with a gene encoding the mouse beta 2-adrenergic receptor (beta 2-AR). Transfectants expressed beta 2-ARs that were able to stimulate adenylyl cyclase activity and steroid biosynthesis. These transfectants were used to explore the basis for the DR mutation in Y1 cells. The authors demonstrate that beta-adrenergic agonists desensitize the adenylyl cyclase system in transfected Y1DS cells whereas transfected Y1DR cells are resistant to desensitization by beta-adrenergic agonists. The fate of the beta 2-ARs during desensitization was evaluated by photoaffinity labelling with [125I]iodocyanopindolol diazerine. Desensitization of Y1DS transfectants was accompanied by a modest loss in receptor density that was insufficient to account for the complete loss of responsiveness to beta-adrenergic agonists. The extent of receptor loss induced by beta-adrenergic agonists in Y1DR transfectants exceeded that in the Y1DS transfectants indicating that the mutation which protects Y1DR cells from agonist-induced desensitization is prior to receptor down-regulation in the desensitization pathway. From these results we infer that ACTH and isoproterenol desensitize adenylyl cyclase by a common pathway and that receptor loss is not a major component of the desensitization process in these cells

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

    Science.gov (United States)

    Yamanashi, Kaori; Minamizaki, Kei; Fujita, Yuichi

    2015-08-07

    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." Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Sensitive method for the assay of guanylate cyclase activity

    Energy Technology Data Exchange (ETDEWEB)

    Karczewski, P; Krause, E G [Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Herz- und Kreislauf-Regulationsforschung

    1978-07-01

    A method for the assay of guanylate cyclase is described utilizing ..cap alpha..-(/sup 32/P)-GTP as substrate for the enzyme reaction. 100-150 ..mu..g of enzyme protein is incubated in a 15.6 mM Tris-HCl buffer incubation mixture, pH 7.6. The reaction is stopped by the addition of EDTA. The (/sup 32/P)-cyclic GMP formed is separated by a two-step column chromatography on Dowex 50W-X4 ion-exchange resin and neutral alumina. The recovery for cyclic GMP was about 70%. The blank values ranged from 0.001-0.003 % of the added ..cap alpha..-(/sup 32/P)-GTP which had been purified by Dowex 50W-X4 column chromatography. This method was employed for the assay of guanylate cyclase activities in different tissues.

  4. Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme

    Czech Academy of Sciences Publication Activity Database

    Mašín, Jiří; Osička, Radim; Bumba, Ladislav; Šebo, Peter

    2015-01-01

    Roč. 73, č. 8 (2015) ISSN 2049-632X R&D Projects: GA ČR GAP302/12/0460; GA ČR GA15-09157S; GA ČR(CZ) GA15-11851S Institutional support: RVO:61388971 Keywords : adenylate cyclase toxin * membrane penetration * pore-formation Subject RIV: EE - Microbiology, Virology Impact factor: 2.483, year: 2015

  5. Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

    Science.gov (United States)

    Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

    2017-01-01

    Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not

  6. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

    International Nuclear Information System (INIS)

    Borgundvaag, B.; George, S.R.

    1985-01-01

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [ 3 H]-ATP to [ 3 H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC 50 values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC 50 values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D 2 dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table

  7. A novel bioassay for the activity determination of therapeutic human brain natriuretic peptide (BNP.

    Directory of Open Access Journals (Sweden)

    Lei Yu

    Full Text Available BACKGROUND: Recombinant human brain natriuretic peptide (rhBNP is an important peptide-based therapeutic drug indicated for the treatment of acute heart failure. Accurate determination of the potency of therapeutic rhBNP is crucial for the safety and efficacy of the drug. The current bioassay involves use of rabbit aortic strips, with experiments being complicated and time-consuming and markedly variable in results. Animal-less methods with better precision and accuracy should be explored. We have therefore developed an alternative cell-based assay, which relies on the ability of BNP to induce cGMP production in HEK293 cells expressing BNP receptor guanylyl cyclase-A. METHODOLOGY/PRINCIPAL FINDINGS: An alternative assay based on the measurement of BNP-induced cGMP production was developed. Specifically, the bioassay employs cells engineered to express BNP receptor guanylyl cyclase-A (GCA. Upon rhBNP stimulation, the levels of the second messager cGMP in these cells drastically increased and subsequently secreted into culture supernatants. The quantity of cGMP, which corresponds to the rhBNP activity, was determined using a competitive ELISA developed by us. Compared with the traditional assay, the novel cell-based assay demonstrated better reproducibility and precision. CONCLUSION/SIGNIFICANCE: The optimized cell-based assay is much simpler, more rapid and precise compared with the traditional assay using animal tissues. To our knowledge, this is the first report on a novel and viable alternative assay for rhBNP potency analysis.

  8. Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c.

    Science.gov (United States)

    Rotcheewaphan, Suwatchareeporn; Belisle, John T; Webb, Kristofor J; Kim, Hee-Jin; Spencer, John S; Borlee, Bradley R

    2016-09-01

    The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography-mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae.

  9. Indirect effect of ionizing radiation on adehylate cyclase activity of liver cells in rat embryos

    International Nuclear Information System (INIS)

    Slozhenikina, L.V.; Ushakova, T.E.; Mikhajlets, L.P.; Kuzin, A.M.

    1980-01-01

    A comparative study was made of the effect of ionizing radiation on basal and catecholamine-stimulating activity of adenylate cyclase in the liver of 20-day embroys under in vivo and in vitro conditions (a membrane fraction and plasma membranes). The authors discuss the share of the indirect effect of radiation in modifying the adenylate cyclase activity

  10. Comparison of the in vivo and in vitro activities of adenylate cyclase from Mycobacterium tuberculosis H37Ra(NCTC 7417)

    International Nuclear Information System (INIS)

    Padh, Harish; Venkitsubramanian, T.A.

    1979-01-01

    The incorporation of [ 14 C] adenine into the adenosine 3', 5'-monophosphate (cyclic AMP) fraction by whole cells of Mycobacterium tuberculosis was taken as a measure of the in vivo activity of adenylate cyclase. The in vivo activity of adenylate cyclase was significantly inhibited by glucose, thus suggesting that the low level of cyclic AMP in the presence of glucose is due to the inhibited synthesis of cyclic AMP. In vitro activity of adenylate cyclase had optimum pH of 8.5 and Km of 1.33 mM for ATP. Glucose and other sugars did not show significant inhibition of in vitro activity. The results suggest that the adenylate cyclase activity becomes less sensitive to glucose when the bacterial cells are disrupted, an analogy with eukaryotic adenylate cyclase which loses sensitivity to hormones when the cells are disrupted. (auth.)

  11. Multilevel control of glucose homeostasis by adenylyl cyclase 8

    NARCIS (Netherlands)

    Raoux, Matthieu; Vacher, Pierre; Papin, Julien; Picard, Alexandre; Kostrzewa, Elzbieta; Devin, Anne; Gaitan, Julien; Limon, Isabelle; Kas, Martien J.; Magnan, Christophe; Lang, Jochen

    2015-01-01

    Aims/hypothesis: Nutrient homeostasis requires integration of signals generated by glucose metabolism and hormones. Expression of the calcium-stimulated adenylyl cyclase ADCY8 is regulated by glucose and the enzyme is capable of integrating signals from multiple pathways. It may thus have an

  12. Toll-like Receptor 4 Signaling Confers Cardiac Protection Against Ischemic Injury via Inducible Nitric Oxide Synthase- and Soluble Guanylate Cyclase-dependent Mechanisms

    Science.gov (United States)

    Wang, E; Feng, Yan; Zhang, Ming; Zou, Lin; Li, Yan; Buys, Emmanuel S.; Huang, Peigen; Brouckaert, Peter; Chao, Wei

    2011-01-01

    Background Prior administration of a small dose of lipopolysaccharide confers a cardiac protection against ischemia-reperfusion injury. However, the signaling mechanisms that control the protection are incompletely understood. We tested the hypothesis that TLR4 mediates the ability of lipopolysaccharide to protect against cardiac ischemia-reperfusion injury through distinct intracellular pathways involving myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor protein inducing interferon-β–mediated transcription-factor (Trif), inducible nitric-oxide synthase (iNOS), and soluble guanylate cyclase (sGC). Methods Wild-type mice and the genetically modified mice, i.e., TLR4-deficient (TLR4-def), TLR2 knockout (TLR2−/−), MyD88−/−, Trif−/−, iNOS−/−, and sGCα1−/−, were treated with normal saline or 0.1 mg/kg of lipopolysaccharide, intraperitoneally. Twenty-four hours later, isolated hearts were perfused in a Langendorff apparatus and subsequently subjected to 30 min of global ischemia and reperfusion for up to 60 min. Left ventricular function and myocardial infarction sizes were examined. Results Compared to saline-treated mice, lipopolysaccharide-treated mice had markedly improved left ventricular developed pressure and dP/dtmax (P < 0.01) and reduced MI sizes (37.2 ± 3.4% vs. 19.8 ± 4.9%, P < 0.01) after ischemia-reperfusion. The cardiac protective effect of lipopolysaccharide was abolished in the TLR4-def and MyD88−/− mice, but remained intact in TLR2−/− or Trif−/− mice. iNOS−/− mice or wild-type mice treated with the iNOS inhibitor 1400W failed to respond to the TLR4-induced nitric oxide production and were not protected by the lipopolysaccharide preconditioning. While sGC 1−/− mice had robust nitric oxide production in response to lipopolysaccharide, they were not protected by the TLR4-elicited cardiac protection. Conclusions TLR4 activation confers a potent cardiac protection against ischemia

  13. Ionic imbalance and lack of effect of adjuvant treatment with methylene blue in the hamster model of leptospirosis

    Directory of Open Access Journals (Sweden)

    Cleiton Silva Santos

    2013-06-01

    Full Text Available Leptospirosis in humans usually involves hypokalaemia and hypomagnesaemia and the putative mechanism underlying such ionic imbalances may be related to nitric oxide (NO production. We previously demonstrated the correlation between serum levels of NO and the severity of renal disease in patients with severe leptospirosis. Methylene blue inhibits soluble guanylyl cyclase (downstream of the action of any NO synthase isoforms and was recently reported to have beneficial effects on clinical and experimental sepsis. We investigated the occurrence of serum ionic changes in experimental leptospirosis at various time points (4, 8, 16 and 28 days in a hamster model. We also determined the effect of methylene blue treatment when administered as an adjuvant therapy, combined with late initiation of standard antibiotic (ampicillin treatment. Hypokalaemia was not reproduced in this model: all of the groups developed increased levels of serum potassium (K. Furthermore, hypermagnesaemia, rather than magnesium (Mg depletion, was observed in this hamster model of acute infection. These findings may be associated with an accelerated progression to acute renal failure. Adjuvant treatment with methylene blue had no effect on survival or serum Mg and K levels during acute-phase leptospirosis in hamsters.

  14. Membrane Guanylate Cyclase catalytic Subdomain: Structure and Linkage with Calcium Sensors and Bicarbonate

    Directory of Open Access Journals (Sweden)

    Sarangan Ravichandran

    2017-06-01

    Full Text Available Membrane guanylate cyclase (MGC is a ubiquitous multi-switching cyclic GMP generating signaling machine linked with countless physiological processes. In mammals it is encoded by seven distinct homologous genes. It is a single transmembrane spanning multi-modular protein; composed of integrated blocks and existing in homo-dimeric form. Its core catalytic domain (CCD module is a common transduction center where all incoming signals are translated into the production of cyclic GMP, a cellular signal second messenger. Crystal structure of the MGC’s CCD does not exist and its precise identity is ill-defined. Here, we define it at a sub-molecular level for the phototransduction-linked MGC, the rod outer segment guanylate cyclase type 1, ROS-GC1. (1 The CCD is a conserved 145-residue structural unit, represented by the segment V820-P964. (2 It exists as a homo-dimer and contains seven conserved catalytic elements (CEs wedged into seven conserved motifs. (3 It also contains a conserved 21-residue neurocalcin δ-modulated structural domain, V836-L857. (4 Site-directed mutagenesis documents that each of the seven CEs governs the cyclase’s catalytic activity. (5 In contrast to the soluble and the bacterium MGC which use Mn2+-GTP substrate for catalysis, MGC CCD uses the natural Mg2+-GTP substrate. (6 Strikingly, the MGC CCD requires anchoring by the Transmembrane Domain (TMD to exhibit its major (∼92% catalytic activity; in isolated form the activity is only marginal. This feature is not linked with any unique sequence of the TMD; there is minimal conservation in TMD. Finally, (7 the seven CEs control each of four phototransduction pathways- -two Ca2+-sensor GCAPs-, one Ca2+-sensor, S100B-, and one bicarbonate-modulated. The findings disclose that the CCD of ROS-GC1 has built-in regulatory elements that control its signal translational activity. Due to conservation of these regulatory elements, it is proposed that these elements also control the

  15. Lycopene cyclase paralog CruP protects against reactive oxygen species in oxygenic photosynthetic organisms

    OpenAIRE

    Bradbury, Louis M. T.; Shumskaya, Maria; Tzfadia, Oren; Wu, Shi-Biao; Kennelly, Edward J.; Wurtzel, Eleanore T.

    2012-01-01

    In photosynthetic organisms, carotenoids serve essential roles in photosynthesis and photoprotection. A previous report designated CruP as a secondary lycopene cyclase involved in carotenoid biosynthesis [Maresca J, et al. (2007) Proc Natl Acad Sci USA 104:11784–11789]. However, we found that cruP KO or cruP overexpression plants do not exhibit correspondingly reduced or increased production of cyclized carotenoids, which would be expected if CruP was a lycopene cyclase. Instead, we show that...

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

    Science.gov (United States)

    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.

  17. Adenylate cyclase toxin-hemolysin relevance for pertussis vaccines

    Czech Academy of Sciences Publication Activity Database

    Šebo, Peter; Osička, Radim; Mašín, Jiří

    2014-01-01

    Roč. 13, č. 10 (2014), s. 1215-1227 ISSN 1476-0584 R&D Projects: GA ČR GA13-14547S; GA ČR(CZ) GAP302/11/0580; GA ČR GAP302/12/0460 Institutional support: RVO:61388971 Keywords : adenylate cyclase toxin * antigen delivery * Bordetella pertussis Subject RIV: EE - Microbiology, Virology Impact factor: 4.210, year: 2014

  18. Inhibitors of glutaminyl cyclases against Alzheimer´s disease

    Czech Academy of Sciences Publication Activity Database

    Kolenko, Petr; Koch, B.; Schilling, S.; Rahfeld, J.-U.; Demuth, H.-U.; Stubbs, M. T.

    2013-01-01

    Roč. 20, č. 1 (2013), s. 16 ISSN 1211-5894. [Discussions in Structural Molecular Biology /11./. 14.03.2013-16.03.2013, Nové Hrady] R&D Projects: GA MŠk EE2.3.30.0029 Institutional support: RVO:61389013 Keywords : glutaminyl cyclases * Alzheimer ´s disease Subject RIV: CE - Biochemistry

  19. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    International Nuclear Information System (INIS)

    Niles, L.P.; Hashemi, F.

    1990-01-01

    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, [ 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 [ 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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 β-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the β-adrenergic pathway, adenylate cyclase activity and β-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. β-Adrenergic receptors were identified in BAT using [ 125 I]iodocyanopindolol. Binding sites had the characteristics of mixed β 1 - and β 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 β-adrenergic receptor density due to a loss of the β 1 -adrenergic subtype. This BAT β-adrenergic receptor downregulation was tissue specific, since myocardial β-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 β-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability

  1. Methylene Blue for Vasoplegia When on Cardiopulmonary Bypass During Double-Lung Transplantation.

    Science.gov (United States)

    Carley, Michelle; Schaff, Jacob; Lai, Terrance; Poppers, Jeremy

    2015-10-15

    Vasoplegia syndrome, characterized by hypotension refractory to fluid resuscitation or high-dose vasopressors, low systemic vascular resistance, and normal-to-increased cardiac index, is associated with increased morbidity and mortality after cardiothoracic surgery. Methylene blue inhibits inducible nitric oxide synthase and guanylyl cyclase, and has been used to treat vasoplegia during cardiopulmonary bypass. However, because methylene blue is associated with increased pulmonary vascular resistance, its use in patients undergoing lung transplantion has been limited. Herein, we report the use of methylene blue to treat refractory vasoplegia during cardiopulmonary bypass in a patient undergoing double-lung transplantation.

  2. Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thaliana

    KAUST Repository

    Hussein, Rana

    2012-11-01

    Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.

  3. The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin

    Science.gov (United States)

    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

  4. Effects of PTH and Ca2+ on renal adenyl cyclase

    International Nuclear Information System (INIS)

    Nielsen, S.T.; Neuman, W.F.

    1978-01-01

    The effects of calcium ion on the adenylate cyclase system was studied in isolated, renal basal-lateral plasma membranes of the rat. Bovine parathyroid hormone (bPTH) and a guanyl triphosphate analogue, Gpp(NH)p were used to stimulate cyclase activity. Under conditions of maximal stimulation, calcium ions inhibited cyclic adenosine monophosphate (cAMP) formation, the formation rate falling exponentially with the calcium concentration. Fifty percent inhibition of either bPTH- or Gpp(NH)p-stimulated activity was given by approximately 50 μM Ca 2+ . Also the Hill coefficient for the inhibition was close to unity in both cases. The concentration of bPTH giving half-maximal stimulation of cAMP formation (1.8 x 10 -8 M) was unchanged by the presence of calcium. These data suggest that calcium acts at some point other than the initial hormone-receptor interaction, presumably decreasing the catalytic efficiency of the enzymic moiety of the membrane complex

  5. Bordetella adenylate cyclase toxin: a swift saboteur of host defense

    Czech Academy of Sciences Publication Activity Database

    Vojtová, Jana; Kamanová, Jana; Šebo, Peter

    2006-01-01

    Roč. 9, - (2006), s. 1-7 ISSN 1369-5274 R&D Projects: GA AV ČR IAA5020406; GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50200510 Keywords : cyaa * scanning electron microscopy * cyclase toxin Subject RIV: EE - Microbiology, Virology Impact factor: 7.445, year: 2006

  6. Activity Regulation by Heteromerization of Arabidopsis Allene Oxide Cyclase Family Members

    Czech Academy of Sciences Publication Activity Database

    Otto, M.; Naumann, Ch.; Brandt, W.; Wasternack, Claus; Hause, B.

    2016-01-01

    Roč. 5, č. 1 (2016), č. článku 3. ISSN 2223-7747 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Activity regulation * Arabidopsis allene oxide cyclase isoforms * Heteromerization Subject RIV: EB - Genetics ; Molecular Biology

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

    International Nuclear Information System (INIS)

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

    1985-01-01

    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 μM isoproterenol and 50 μM GTP-γ-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 μM GTP-γ-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 β-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

  8. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A; Turek, Ilona S.

    2017-01-01

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

  9. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A.

    2017-10-04

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

  10. Effects of stimulation of soluble guanylate cyclase on diabetic nephropathy in diabetic eNOS knockout mice on top of angiotensin II receptor blockade.

    Directory of Open Access Journals (Sweden)

    Ina M Ott

    Full Text Available The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN, is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy. Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d, riociguat (3 mg/kg/d, riociguat+telmisartan (3+1 mg/kg/d, and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071. Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg. Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002 reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h, whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h or riociguat (97.1±15.7 µg/24 h was not statistically significant. The combination treatment led to a significant (p<0.01 decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress. In conclusion, stimulation of sGC significantly reduced urinary

  11. Inhaled ammonium persulphate inhibits non-adrenergic, non-cholinergic relaxations in the guinea pig isolated trachea.

    Science.gov (United States)

    Dellabianca, A; Faniglione, M; De Angelis, S; Colucci, M; Cervio, M; Balestra, B; Tonini, S; Candura, S M

    2010-01-01

    Persulphates can act both as irritants and sensitizers in inducing occupational asthma. A dysfunction of nervous control regulating the airway tone has been hypothesized as a mechanism underlying bronchoconstriction in asthma. It was the aim of this study to investigate whether inhaled ammonium persulphate affects the non-adrenergic, non-cholinergic (NANC) inhibitory innervation, the cholinergic nerve-mediated contraction or the muscular response to the spasmogens, carbachol or histamine, in the guinea pig epithelium-free, isolated trachea. Male guinea pigs inhaled aerosols containing ammonium persulphate (10 mg/m(3) for 30 min for 5 days during 3 weeks). Control animals inhaled saline aerosol. NANC relaxations to electrical field stimulation at 3 Hz were evaluated in whole tracheal segments as intraluminal pressure changes. Drugs inactivating peptide transmission, nitric oxide synthase, carbon monoxide production by haem oxygenase-2 and soluble guanylyl cyclase were used to assess the involvement of various inhibitory neurotransmitters. Carbachol and histamine cumulative concentration-response curves were obtained. In both groups, nitric oxide and carbon monoxide participated to the same extent as inhibitory neurotransmitters. In exposed animals, the tracheal NANC relaxations were reduced to 45.9 +/- 12.1% (p guinea pig airways. This may represent a novel mechanism contributing to persulphate-induced asthma. Copyright 2009 S. Karger AG, Basel.

  12. l-Citrulline ameliorates cerebral blood flow during cortical spreading depression in rats: Involvement of nitric oxide- and prostanoids-mediated pathway

    Directory of Open Access Journals (Sweden)

    Yuki Kurauchi

    2017-03-01

    Full Text Available l-Citrulline is a potent precursor of l-arginine, and exerts beneficial effect on cardiovascular system via nitric oxide (NO production. Migraine is one of the most popular neurovascular disorder, and imbalance of cerebral blood flow (CBF observed in cortical spreading depression (CSD contributes to the mechanism of migraine aura. Here, we investigated the effect of l-citrulline on cardiovascular changes to KCl-induced CSD. in rats. Intravenous injection of l-citrulline prevented the decrease in CBF, monitored by laser Doppler flowmetry, without affecting mean arterial pressure and heart rate during CSD. Moreover, l-citrulline attenuated propagation velocity of CSD induced by KCl. The effect of l-citrulline on CBF change was prevented by l-NAME, an inhibitor of NO synthase, but not by indomethacin, an inhibitor of cyclooxygenase. On the other hand, attenuation effect of l-citrulline on CSD propagation velocity was prevented not only by l-NAME but also by indomethacin. In addition, propagation velocity of CSD was attenuated by intravenous injection of NOR3, a NO donor, which was diminished by ODQ, an inhibitor of soluble guanylyl cyclase. These results suggest that NO/cyclic GMP- and prostanoids-mediated pathway differently contribute to the effect of l-citrulline on the maintenance of CBF.

  13. Therapeutic Targeting of Redox Signaling in Myofibroblast Differentiation and Age-Related Fibrotic Disease

    Directory of Open Access Journals (Sweden)

    Natalie Sampson

    2012-01-01

    Full Text Available Myofibroblast activation plays a central role during normal wound healing. Whereas insufficient myofibroblast activation impairs wound healing, excessive myofibroblast activation promotes fibrosis in diverse tissues (including benign prostatic hyperplasia, BPH leading to organ dysfunction and also promotes a stromal response that supports tumor progression. The incidence of impaired wound healing, tissue fibrosis, BPH, and certain cancers strongly increases with age. This paper summarizes findings from in vitro fibroblast-to-myofibroblast differentiation systems that serve as cellular models to study fibrogenesis of diverse tissues. Supported by substantial in vivo data, a large body of evidence indicates that myofibroblast differentiation induced by the profibrotic cytokine transforming growth factor beta is driven by a prooxidant shift in redox homeostasis due to elevated production of NADPH oxidase 4 (NOX4-derived hydrogen peroxide and supported by concomitant decreases in nitric oxide/cGMP signaling and reactive oxygen species (ROS scavenging enzymes. Fibroblast-to-myofibroblast differentiation can be inhibited and reversed by restoring redox homeostasis using antioxidants or NOX4 inactivation as well as enhancing nitric oxide/cGMP signaling via activation of soluble guanylyl cyclases or inhibition of phosphodiesterases. Current evidence indicates the therapeutic potential of targeting the prooxidant shift in redox homeostasis for the treatment of age-related diseases associated with myofibroblast dysregulation.

  14. The NO signaling pathway differentially regulates KCC3a and KCC3b mRNA expression.

    Science.gov (United States)

    Di Fulvio, Mauricio; Lauf, Peter K; Adragna, Norma C

    2003-11-01

    Nitric oxide (NO) donors and protein kinase G (PKG) acutely up-regulate K-Cl cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in vascular smooth muscle cells (VSMCs). Here, we report the presence, relative abundance, and regulation by sodium nitroprusside (SNP) of the novel KCC3a and KCC3b mRNAs, in primary cultures of rat VSMCs. KCC3a and KCC3b mRNAs were expressed in an approximate 3:1 ratio, as determined by semiquantitative RT-PCR analysis. SNP as well as YC-1 and 8-Br-cGMP, a NO-independent stimulator of soluble guanylyl cyclase (sGC) and PKG, respectively, increased KCC3a and KCC3b mRNA expression by 2.5-fold and 8.1-fold in a time-dependent manner, following a differential kinetics. Stimulation of the NO/sGC/PKG signaling pathway with either SNP, YC-1, or 8-Br-cGMP decreased the KCC3a/KCC3b ratio from 3.0+/-0.4 to 0.9+/-0.1. This is the first report on a differential regulation by the NO/sGC/PKG signaling pathway of a cotransporter and of KCC3a and KCC3b mRNA expression.

  15. Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

    Science.gov (United States)

    Balfanz, Sabine; Ehling, Petra; Wachten, Sebastian; Jordan, Nadine; Erber, Joachim; Mujagic, Samir; Baumann, Arnd

    2012-06-01

    The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Adenylate cyclase regulation in intact cultured myocardial cells

    International Nuclear Information System (INIS)

    Marsh, J.D.; Roberts, D.J.

    1987-01-01

    To examine the coupling of cardiac cell-surface β-adrenergic receptors to adenylate cyclase activation and contractile response, the authors studied this receptor-effector response system in monolayers of spontaneously contracting chick embryo ventricular cells under physiological conditions. The hydrophilic ligand 3 H-CGP12177 identified uniformly high-agonist affinity β-adrenergic receptors. Isoproterenol-stimulated cyclic AMP (cAMP) accumulation with 50% effective concentration at (EC 50 ) = 12.1 nM and augmented contractile response with EC 50 = 6 nM under identical conditions. One micromolar isoproterenol induced receptor loss from the cell surface with t/sub 1/2/ = 13.2 min; under identical conditions cAMP content declined with t/sub 1/2/ = 13.5 min and contractile response with t/sub 1/2/ = 20.7 min. After agonist removal cAMP response recovered with t/sub 1/2/ = 15.7 min and receptors with t/sub 1/2/ = 24.7 min. Sixty minutes after agonist removal there was recovery of 52% of maximal cAMP responsiveness and 82% of the initial number of receptors; receptor occupancy was associated with 78% of initial contractile response. Agonist affinity for cell-surface receptors was changed only modestly by agonist exposure. They conclude that for this system there is relatively close coupling between high-affinity receptors, adenylate cyclase stimulation, and contractile response

  17. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    International Nuclear Information System (INIS)

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of [ 3 H]GDP binding to plasma membranes suggested a single high affinity site with a K d = 0.24 uM. Competition studies indicated that GTP γ S was 7-fold more potent than GDP β S. Bound GDP could be released by FSH in the presence of GTP γ S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP β S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP β S competitively inhibited GTP γ S-stimulated adenylate cyclase activity with a K i = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP γ S-bound form persisted even if GDP β S previously occupied all available binding sites. Two membrane proteins, M r = 43,000 and 48,000, were ADP·ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP γ S but not by GDP β S. The M r = 43,000 and 48,000 proteins represented variant forms of G S . A single protein of M r = 40,000 (G i ) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC 50 = 0.1 uM. The adenosine analog, N 6 ·phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin

  18. Irradiation inactivation studies of the dopamine D1 receptor and dopamine-stimulated adenylate cyclase in rat striatum

    International Nuclear Information System (INIS)

    Anderson, P.H.; Nielson, M.

    1987-01-01

    In frozen rat striatal tissue, exposed to 10 MeV electrons from a linear accelerator, the sizes of the dopamine (DA) D 1 receptor and the DA sensitive adenylate cyclase complex were determined using target size analysis. The number of D 1 receptors (labelled by [ 3 H]SCH 23390)declined monoexponentially with increasing radiation intensity, yielding a molecular weight (mol. wt.) of 80kDa. Also the activity of the catalytic unit (C) of the adenylate cyclase (as measured by forskolin stimulation), decreased monoexponentially however with a mol. wt. of 145 kDa. Both basal, DA- and flouride (F - ) stimulated activity declined in a concave downward fashion with a limiting mol. wt. of 134, 138 and 228 kDa respectively. It was estimated that the basal and DA - stimulated activity originated from an enzyme complex with a mol. wt. of 325 kDa a value close to the combined size of R G S + C. These data suggest that F - stimulation of the adenylate cyclase, which occurs by a G S activation, does not cause disassociation of G S into the α S and βγ subunits. Further, the AA-regulated adenylate cyclase apparently exists as a complex consisting of RG S and C; the mechanisms of hormonal activation is dissociation of C from this complex

  19. Effect of hypolipidemic drugs on basal and stimulated adenylate cyclase activity in tumor cells

    International Nuclear Information System (INIS)

    Bershtein, L.M.; Kovaleva, I.G.; Rozenberg, O.A.

    1986-01-01

    This paper studies adenylate cyclase acticvity in Ehrlich's ascites carcinoma (EAC) cells during administration of drugs with a hypolipidemic action. Seven to eight days before they were killed, male mice ingested the antidiabetic biguanide phenformin, and the phospholipid-containing preparation Essentiale in drinking water. The cAMP formed was isolated by chromatography on Silufol plates after incubation of the enzyme preparation with tritium-ATP, or was determined by the competitive binding method with protein. It is shown that despite the possible differences in the concrete mechanism of action of the hypolipidemic agents chosen for study on the cyclase system, the use of such agents, offers definite prospects for oriented modification of the hormone sensitivity of tumor cells

  20. Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.

    Science.gov (United States)

    Yang, Xinmei; Matsui, Takashi; Kodama, Takeshi; Mori, Takahiro; Zhou, Xiaoxi; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2016-03-01

    In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+β barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-β-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future. Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively. © 2016 Federation of European Biochemical Societies.

  1. The effects of sex and neonatal stress on pituitary adenylate cyclase-activating peptide expression.

    Science.gov (United States)

    Mosca, E V; Rousseau, J P; Gulemetova, R; Kinkead, R; Wilson, R J A

    2015-02-01

    What is the central question of this study? Does sex or neonatal stress affect the expression of pituitary adenylate cyclase-activating peptide or its receptors? What is the main finding and its importance? Neonatal-maternal separation stress has little long-lasting effect on the expression of pituitary adenylate cyclase-activating peptide or its receptors, but sex differences exist in these genes between males and females at baseline. Sex differences in classic stress hormones have been studied in depth, but pituitary adenylate cyclase-activating peptide (PACAP), recently identified as playing a critical role in the stress axes, has not. Here we studied whether baseline levels of PACAP differ between sexes in various stress-related tissues and whether neonatal-maternal separation stress has a sex-dependent effect on PACAP gene expression in stress pathways. Using quantitative RT-PCR, we found sex differences in PACAP and PACAP receptor gene expression in several respiratory and/or stress-related tissues, while neonatal-maternal separation stress did little to affect PACAP signalling in adult animals. We propose that sex differences in PACAP expression are likely to contribute to differences between males and females in responses to stress. © 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.

  2. Structure of glutaminyl cyclase from Drosophila melanogaster in space group I4

    Czech Academy of Sciences Publication Activity Database

    Kolenko, Petr; Koch, B.; Rahfeld, J.-U.; Schilling, S.; Demuth, H.-U.; Stubbs, M. T.

    2013-01-01

    Roč. 69, č. 4 (2013), s. 358-361 ISSN 1744-3091 R&D Projects: GA MŠk EE2.3.30.0029 Institutional support: RVO:61389013 Keywords : glutaminyl cyclases * Drosophila melanogaster * soaking Subject RIV: CE - Biochemistry Impact factor: 0.568, year: 2013

  3. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    Directory of Open Access Journals (Sweden)

    Ma’ayan Israeli

    2016-08-01

    Full Text Available 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.

  4. Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, transduces two independent signals, ANF and Ca2+

    Directory of Open Access Journals (Sweden)

    Teresa eDuda

    2014-03-01

    Full Text Available Atrial natriuretic factor receptor guanylate cyclase, ANF-RGC, was the first discovered member of the mammalian membrane guanylate cyclase family. The hallmark feature of the family is that a single protein contains both the site for recognition of the regulatory signal and the ability to transduce it into the production of the second messenger, cyclic GMP. For over two decades, the family has been classified into two subfamilies, the hormone receptor subfamily with ANF-RGC being its paramount member, and the Ca2+ modulated subfamily, which includes the rod outer segment guanylate cyclases, ROS-GC1 and 2, and the olfactory neuroepithelial guanylate cyclase, ONE-GC. ANF-RGC is the receptor and the signal transducer of the most hypotensive hormones, atrial natriuretic factor (ANF and B-type natriuretic peptide (BNP. After binding these hormones at the extracellular domain it, at its intracellular domain, signals activation of the C-terminal catalytic module and accelerates the production of cyclic GMP. Cyclic GMP then serves the second messenger role in biological responses of ANF and BNP such as natriuresis, diuresis, vasorelaxation and anti-proliferation. Very recently another modus operandi for ANF-RGC was revealed. Its crux is that ANF-RGC activity is also regulated by Ca2+. The Ca2+ sensor neurocalcin  mediates this signaling mechanism. Strikingly, the Ca2+ and ANF signaling mechanisms employ separate structural motifs of ANF-RGC in modulating its core catalytic domain in accelerating the production of cyclic GMP. In this review the biochemistry and physiology of these mechanisms with emphasis on cardiovascular regulation will be discussed.

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

    KAUST Repository

    Gehring, Christoph A.

    2010-06-25

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

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

    KAUST Repository

    Gehring, Christoph A

    2010-01-01

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

  7. In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, A; Bhattacharya, A K

    1988-06-01

    The incorporation of (/sup 14/C)adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with /sup 60/Co ..gamma..-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of ..gamma..-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after high doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m/sup -2/) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as ..gamma..-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells.

  8. In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli

    International Nuclear Information System (INIS)

    Chatterjee, A.; Bhattacharya, A.K.

    1988-01-01

    The incorporation of [ 14 C]adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with 60 Co γ-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of γ-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after high doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m -2 ) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as γ-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells. (author)

  9. In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli.

    Science.gov (United States)

    Chatterjee, A; Bhattacharya, A K

    1988-06-01

    The incorporation of [14C]adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with 60Co gamma-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of gamma-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after higher doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m-2) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as gamma-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells.

  10. Functional Lycopene Cyclase (CruA) in Cyanobacterium, Arthrospira platensis NIES-39, and its Role in Carotenoid Synthesis.

    Science.gov (United States)

    Sugiyama, Kenjiro; Ebisawa, Masashi; Yamada, Masaharu; Nagashima, Yoshiki; Suzuki, Hideyuki; Maoka, Takashi; Takaichi, Shinichi

    2017-04-01

    The genus Arthrospira is filamentous, non-nitrogen-fixing cyanobacteria that is commercially important. We identified the molecular structures of carotenoids in Arthrospira platensis NIES-39. The major carotenoid identified was β-carotene. In addition, the hydroxyl derivatives of β-cryptoxanthin and (3R,3'R)-zeaxanthin were also found to be present. The carotenoid glycosides were identified as (3R,2'S)-myxol 2'-methylpentoside and oscillol 2,2'-dimethylpentoside. The methylpentoside moiety was a mixture of fucoside and chinovoside in an approximate ratio of 1 : 4. Trace amounts of the ketocarotenoid 3'-hydroxyechinenone were also found. Three types of lycopene cyclases have been functionally confirmed in carotenogenesis organisms. In cyanobacteria, the functional lycopene cyclases (CrtL, CruA and CruP) have only been found in four species. In this study, we found that CruA exhibited lycopene cyclase activity in transformed Escherichia coli, which contains lycopene, but CruP exhibited no lycopene cyclase activity and crtL was absent. This is the third cyanobacterial species in which CruA activity has been confirmed. Neurosporene was not a substrate of CruA in E. coli, whereas lycopene cyclases of CrtY (bacteria), CrtL (plants) and CrtYB (fungi) have been reported to convert neurosporene to 7,8-dihydro-β-carotene. β-Carotene hydroxylase (CrtR) was found to convert β-carotene to zeaxanthin in transformed E. coli, which contains β-carotene. Among the β-carotene hydroxylases, bacterial CrtZ and eukaryotic CrtR and BCH have similarities, whereas cyanobacterial CrtR appears to belong to another clade. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the A. platensis NIES-39 genome, we propose a biosynthetic pathway for the carotenoids as well as the corresponding genes and enzymes. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved

  11. Identification of Adenyl Cyclase Activity in a Disease Resistance Protein in Arabidopsis thaliana

    KAUST Repository

    Hussein, Rana

    2012-01-01

    center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified

  12. Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.

    Science.gov (United States)

    Pastor-Soler, Nuria; Beaulieu, Valerie; Litvin, Tatiana N; Da Silva, Nicolas; Chen, Yanqiu; Brown, Dennis; Buck, Jochen; Levin, Lonny R; Breton, Sylvie

    2003-12-05

    Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.

  13. From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

    International Nuclear Information System (INIS)

    Lima, A.A.M.; Fonteles, M.C.

    2014-01-01

    The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia

  14. From Escherichia coli heat-stable enterotoxin to mammalian endogenous guanylin hormones

    Energy Technology Data Exchange (ETDEWEB)

    Lima, A.A.M. [Universidade Federal do Ceará, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Instituto de Biomedicina, Unidade de Pesquisas Clínicas, Fortaleza, CE, Brasil, Unidade de Pesquisas Clínicas, Instituto de Biomedicina, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil); Fonteles, M.C. [Universidade Federal do Ceará, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Instituto de Biomedicina, Unidade de Pesquisas Clínicas, Fortaleza, CE, Brasil, Unidade de Pesquisas Clínicas, Instituto de Biomedicina, Departamento de Fisiologia e Farmacologia, Escola de Medicina, Universidade Federal do Ceará, Fortaleza, CE (Brazil); Universidade Estadual do Ceará, Instituto de Ciências Biomédicas, Fortaleza, CE, Brasil, Instituto de Ciências Biomédicas, Universidade Estadual do Ceará, Fortaleza, CE (Brazil)

    2014-03-03

    The isolation of heat-stable enterotoxin (STa) from Escherichia coli and cholera toxin from Vibrio cholerae has increased our knowledge of specific mechanisms of action that could be used as pharmacological tools to understand the guanylyl cyclase-C and the adenylyl cyclase enzymatic systems. These discoveries have also been instrumental in increasing our understanding of the basic mechanisms that control the electrolyte and water balance in the gut, kidney, and urinary tracts under normal conditions and in disease. Herein, we review the evolution of genes of the guanylin family and STa genes from bacteria to fish and mammals. We also describe new developments and perspectives regarding these novel bacterial compounds and peptide hormones that act in electrolyte and water balance. The available data point toward new therapeutic perspectives for pathological features such as functional gastrointestinal disorders associated with constipation, colorectal cancer, cystic fibrosis, asthma, hypertension, gastrointestinal barrier function damage associated with enteropathy, enteric infection, malnutrition, satiety, food preferences, obesity, metabolic syndrome, and effects on behavior and brain disorders such as attention deficit, hyperactivity disorder, and schizophrenia.

  15. Multiple diguanylate cyclase-coordinated regulation of pyoverdine synthesis in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Chen, Yicai; Yuan, Mingjun; Mohanty, Anee

    2015-01-01

    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...

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

    International Nuclear Information System (INIS)

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

    2006-01-01

    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 1 2 1 2 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 M = 3.0 Å 3 Da −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

  17. Isotopically sensitive branching in the formation of cyclic monoterpenes: proof that (-)-alpha-pinene and (-)-beta-pinene are synthesized by the same monoterpene cyclase via deprotonation of a common intermediate

    International Nuclear Information System (INIS)

    Croteau, R.B.; Wheeler, C.J.; Cane, D.E.; Ebert, R.; Ha, H.J.

    1987-01-01

    To determine whether the bicyclic monoterpene olefins (-)-alpha-pinene and (-)-beta-pinene arise biosynthetically from the same monoterpene cyclase by alternate deprotonations of a common carbocationic intermediate, the product distributions arising from the acyclic precursor [10- 2 H 3 ,1- 3 H]geranyl pyrophosphate were compared with those resulting from incubation of [1-3H]geranyl pyrophosphate with (-)-pinene cyclase from Salvia officinalis. Alteration in proportions of the olefinic products generated by the partially purified pinene cyclase resulted from the suppression of the formation of (-)-beta-pinene (C10 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of (-)-alpha-pinene (C4 deprotonation). (-)-Pinene cyclase as well as (+)-pinene cyclase also exhibited a decrease in the proportion of the acyclic olefin myrcene generated from the deuteriated substrate, accompanied by a corresponding increase in the commitment to cyclized products. The observation of isotopically sensitive branching, in conjunction with quantitation of the magnitude of the secondary deuterium isotope effect on the overall rate of product formation by the (+)- and (-)-pinene cyclases as well as two other monoterpene cyclases from the same tissue, supports the biosynthetic origin of (-)-alpha-pinene and (-)-beta-pinene by alternative deprotonations of a common enzymatic intermediate. A biogenetic scheme consistent with these results is presented, and alternate proposals for the origin of the pinenes are addressed

  18. Platelet adenylyl cyclase activity as a biochemical trait marker for predisposition to alcoholism.

    NARCIS (Netherlands)

    Ratsma, J.E.; Gunning, W.B.; Leurs, R.; Schoffelmeer, A.N.M.

    1999-01-01

    Previous studies demonstrated a reduced G(s)-protein stimulated adenylyl cyclase activity in the brain and blood cells of alcoholics. We investigated this phenomenon in platelets of children of alcoholics (COA), i.e., of children at high risk for the acquisition of alcoholism and (as yet) not

  19. Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

    DEFF Research Database (Denmark)

    Wang, Yin-Qiu; Qian, Ya-Ping; Yang, Su

    2005-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a...

  20. Pituitary adenylate cyclase-activating polypeptide stimulates renin secretion via activation of PAC1 receptors

    DEFF Research Database (Denmark)

    Hautmann, Matthias; Friis, Ulla G; Desch, Michael

    2007-01-01

    Besides of its functional role in the nervous system, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of cardiovascular function. Therefore, PACAP is a potent vasodilator in several vascular beds, including the renal vasculature. Because...

  1. Quantification of potassium levels in cells treated with Bordetella adenylate cyclase toxin

    Czech Academy of Sciences Publication Activity Database

    Wald, Tomáš; Petry-Podgorska, Inga; Fišer, Radovan; Matoušek, Tomáš; Dědina, Jiří; Osička, Radim; Šebo, Peter; Mašín, Jiří

    2014-01-01

    Roč. 450, APR 2014 (2014), s. 57-62 ISSN 0003-2697 R&D Projects: GA ČR(CZ) GAP302/11/0580; GA ČR GA13-14547S; GA ČR GAP302/12/0460 Institutional support: RVO:61388971 ; RVO:68081715 Keywords : Potassium * Adenylate cyclase toxin * RTX Subject RIV: CE - Biochemistry Impact factor: 2.219, year: 2014

  2. Third Acivity of Bordetella Adenylate Cyclase (AC) Toxin-Hemolysin

    Czech Academy of Sciences Publication Activity Database

    Fišer, Radovan; Mašín, Jiří; Basler, Marek; Krůšek, Jan; Špuláková, V.; Konopásek, Ivo; Šebo, Peter

    2007-01-01

    Roč. 282, č. 5 (2007), s. 2808-2820 ISSN 0021-9258 R&D Projects: GA MŠk 1M0506; GA AV ČR IAA5020406 Grant - others:XE(XE) LSHB-CT-2003-503582; Univerzita Karlova(CZ) 146/2005/B-BIO Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50110509 Source of funding: R - rámcový projekt EK ; V - iné verejné zdroje Keywords : bordetella * adenylate cyclase toxin * enzymatic aktivity Subject RIV: EE - Microbiology, Virology Impact factor: 5.581, year: 2007

  3. [Construction of high-yield strain by optimizing lycopene cyclase for β-carotene production].

    Science.gov (United States)

    Jin, Yingfu; Han, Li; Zhang, Shasha; Li, Shizhong; Liu, Weifeng; Tao, Yong

    2017-11-25

    To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high β-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.

  4. Nitric oxide and catalase-sensitive relaxation by scutellarin in the mouse thoracic aorta.

    Science.gov (United States)

    Yang, Weimin; Lust, Robert M; Bofferding, April; Wingard, Christopher J

    2009-01-01

    The vascular activity of scutellarin (SCU), a flavonoid isolated from a Chinese traditional medicinal plant, was investigated in isolated thoracic aortic rings of mice. SCU-induced dose-dependent relaxation of phenylephrine (1 microM) stimulated contractions. This relaxation was reduced by endothelium removal, significantly reduced by both the nitric oxide synthase inhibitor (Nomega-nitro-L-arginine methylester, 300 microM) and slightly limited by the soluble guanylyl cyclase inhibitor (1 H-[1,2,4] oxidazolol [4,3-a] quinoxalin-1-one, 100 microM). The catalase inhibitor (3-amino-1,2,4-triazole, 50 mM) augmented the constriction and blocked the lowest SCU concentration relaxation, whereas catalase addition was without effect. Preincubation with 300 and 1000 microM SCU significantly suppressed the contractile dose-response to phenylephrine, causing both a significant rise in half maximal effective concentration and a decrease in the maximal developed force. Western blot analysis showed that SCU inhibition of contraction was independent of reductions in myosin light chain phosphorylation. These results suggested that SCU relaxation was predominantly endothelium dependent and likely involved the catalase-sensitive nitric oxide synthase signaling pathway, without loss of myosin phosphorylation. The potential clinical use of SCU may prove to be effective in increasing vasoreactivity, independently of smooth muscle contractile activity that is mediated by the 20-kDa myosin light chain phosphorylation.

  5. Intrinsic nitric oxide regulates the taste response of the sugar receptor cell in the blowfly, Phormia regina.

    Science.gov (United States)

    Murata, Yoshihiro; Mashiko, Masashi; Ozaki, Mamiko; Amakawa, Taisaku; Nakamura, Tadashi

    2004-01-01

    The taste organ in insects is a hair-shaped taste sensory unit having four functionally differentiated contact chemoreceptor cells. In the blowfly, Phormia regina, cGMP has been suggested to be a second messenger for the sugar receptor cell. Generally, cGMP is produced by membranous or soluble guanylyl cyclase (sGC), which can be activated by nitric oxide (NO). In the present paper, we electrophysiologically showed that an NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO), an NO donor, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC 7) or an NO synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) specifically affected the response in the sugar receptor cell, but not in other receptor cells. PTIO, when introduced into the receptor cells in a sensillum aided by sodium deoxycholate (DOC, pH 7.2), depressed the response of sugar receptor cells to sucrose but did not affect those of the salt or water receptor cells. NOC 7, given extracellularly, latently induced the response of sugar receptor cells; and L-NAME, when introduced into the receptor cells, depressed the response of sugar receptor cells. The results clearly suggest that NO, which may be produced by intrinsic NOS in sugar receptor cells, participates in the transduction cascade of these cells in blowfly.

  6. Nitric oxide synthesis and biological functions of nitric oxide released from ruthenium compounds

    Directory of Open Access Journals (Sweden)

    A.C. Pereira

    2011-09-01

    Full Text Available During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP production, which in turn leads to protein kinase G (PKG activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca2+ signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.

  7. PPARα-Independent Arterial Smooth Muscle Relaxant Effects of PPARα Agonists

    Directory of Open Access Journals (Sweden)

    Neerupma Silswal

    2012-01-01

    Full Text Available We sought to determine direct vascular effects of peroxisome proliferator-activated receptor alpha (PPARα agonists using isolated mouse aortas and middle cerebral arteries (MCAs. The PPARα agonists GW7647, WY14643, and gemfibrozil acutely relaxed aortas held under isometric tension and dilated pressurized MCAs with the following order of potency: GW7647≫WY14643>gemfibrozil. Responses were endothelium-independent, and the use of PPARα deficient mice demonstrated that responses were also PPARα-independent. Pretreating arteries with high extracellular K+ attenuated PPARα agonist-mediated relaxations in the aorta, but not in the MCA. In the aorta, the ATP sensitive potassium (KATP channel blocker glibenclamide also impaired relaxations whereas the other K+ channel inhibitors, 4-aminopyridine and Iberiotoxin, had no effect. In aortas, GW7647 and WY14643 elevated cGMP levels by stimulating soluble guanylyl cyclase (sGC, and inhibition of sGC with ODQ blunted relaxations to PPARα agonists. In the MCA, dilations were inhibited by the protein kinase C (PKC activator, phorbol 12,13-dibutyrate, and also by ODQ. Our results demonstrated acute, nonreceptor-mediated relaxant effects of PPARα agonists on smooth muscle of mouse arteries. Responses to PPARα agonists in the aorta involved KATP channels and sGC, whereas in the MCA the PKC and sGC pathways also appeared to contribute to the response.

  8. Spironolactone treatment attenuates vascular dysfunction in type 2 diabetic mice by decreasing oxidative stress and restoring NO/GC signaling

    Directory of Open Access Journals (Sweden)

    Marcondes Alves Barbosa Da Silva

    2015-10-01

    Full Text Available Type 2 diabetes (DM2 increases the risk of cardiovascular disease. Aldosterone, which has pro-oxidative and pro-inflammatory effects in the cardiovascular system, is positively regulated in DM2. We assessed whether blockade of mineralocorticoid receptors (MR with spironolactone decreases ROS-associated vascular dysfunction and improves vascular NO signaling in diabetes. Leptin receptor knockout [LepRdb/LepRdb (db/db] mice, a model of DM2, and their counterpart controls [LepRdb/LepR+, (db/+ mice] received spironolactone (50 mg/kg body weight/day or vehicle (ethanol 1% via oral per gavage for 6 weeks. Spironolactone treatment abolished the endothelial dysfunction and increased endothelial nitric oxide synthase (eNOS phosphorylation (Ser1177, determined by acetylcholine-induced relaxation and Western Blot analysis, respectively. MR antagonist therapy also abrogated augmented ROS-generation in aorta from diabetic mice, determined by lucigenin luminescence assay. Spironolactone treatment increased superoxide dismutase-1 (SOD1 and catalase expression, improved sodium nitroprusside (SNP and BAY 41-2272-induced relaxation, as well as increased soluble guanylyl cyclase (sGC subunit β protein expression in arteries from db/db mice. Our results demonstrate that spironolactone decreases diabetes-associated vascular oxidative stress and prevents vascular dysfunction through processes involving increased expression of antioxidant enzymes and sGC. These findings further elucidate redox-sensitive mechanisms whereby spironolactone protects against vascular injury in diabetes.

  9. Effect of an Ethanol Extract of Scutellaria baicalensis on Relaxation in Corpus Cavernosum Smooth Muscle

    Directory of Open Access Journals (Sweden)

    Xiang Li

    2012-01-01

    Full Text Available Aims of study. The aim of the present study was to investigate whether an ethanol extract of Scutellaria baicalensis (ESB relaxes penile corpus cavernosum muscle in organ bath experiments. Materials and methods. Changes in tension of cavernous smooth muscle strips were determined by penile strip chamber model and in penile perfusion model. Isolated endothelium-intact rabbit corpus cavernosum was precontracted with phenylephrine (PE and then treated with ESB. Results. ESB relaxed penile smooth muscle in a dose-dependent manner, and this was inhibited by pre-treatment with NG-nitro-l-arginine methyl ester (l-NAME, a nitric oxide (NO synthase inhibitor, and 1H-[1, 2, 4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ, a soluble guanylyl cyclase (sGC inhibitor. ESB-induced relaxation was significantly attenuated by pretreatment with tetraethylammonium (TEA, a nonselective K+ channel blocker, and charybdotoxin, a selective Ca2+-dependent K+ channel inhibitor. ESB increased the cGMP levels of rabbit corpus cavernosum in a concentration-dependent manner without changes in cAMP levels. In a perfusion model of penile tissue, ESB also relaxed penile corpus cavernosum smooth muscle in a dose-dependent manner. Conclusion. Taken together, these results suggest that ESB relaxed rabbit cavernous smooth muscle via the NO/cGMP system and Ca2+-sensitive K+ channels in the corpus cavernosum.

  10. NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells.

    Science.gov (United States)

    Di Fulvio, Mauricio; Lauf, Peter K; Shah, Shalin; Adragna, Norma C

    2003-05-01

    Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.

  11. Nitric oxide signaling pathway regulates potassium chloride cotransporter-1 mRNA expression in vascular smooth muscle cells.

    Science.gov (United States)

    Di Fulvio, M; Lauf, P K; Adragna, N C

    2001-11-30

    Rat vascular smooth muscle cells (VSMCs) express at least two mRNAs for K-Cl cotransporters (KCC): KCC1 and KCC3. cGMP-dependent protein kinase I regulates KCC3 mRNA expression in these cells. Here, we show evidence implicating the nitric oxide (NO)/cGMP signaling pathway in the expression of KCC1 mRNA, considered to be the major cell volume regulator. VSMCs, expressing soluble guanylyl cyclase (sGC) and PKG-I isoforms showed a time- and concentration-dependent increase in KCC1 mRNA levels after treatment with sodium nitroprusside as demonstrated by semiquantitative RT-PCR. sGC-dependent regulation of KCC1 mRNA expression was confirmed using YC-1, a NO-independent sGC stimulator. The sGC inhibitor LY83583 blocked the effects of sodium nitroprusside and YC-1. Moreover, 8-Br-cGMP increased KCC1 mRNA expression in a concentration- and time-dependent fashion. The 8-Br-cGMP effect was partially blocked by KT5823 but not by actinomycin D. However, actinomycin D and cycloheximide increased basal KCC1 mRNA in an additive manner, suggesting different mechanisms of action for both drugs. These findings suggest that in VSMCs, the NO/cGMP-signaling pathway participates in KCC1 mRNA regulation at the post-transcriptional level.

  12. Initiation of proteolysis of yeast fructose-1,6-bisphosphatase by pH-control of adenylate cyclase

    International Nuclear Information System (INIS)

    Holzer, H.; Purwin, C.; Pohlig, G.; Scheffers, W.A.; Nicolay, K.

    1986-01-01

    Addition of fermentable sugars or uncouplers such as CCCP to resting yeast cells grown on glucose initiates phosphorylation of fructose-1,6-bisphosphatase (FBPase). There is good evidence that phosphorylation marks FBPase for proteolytic degradation. 31 P-NMR measurements of the cytosolic pH of yeast cells demonstrated a decrease of the cytosolic pH from 7.0 to 6.5 after addition of glucose or CCCP to starved yeast. Activity of adenylate cyclase in permeabilized yeast cells increases 2-3-fold when the pH is lowered from 7.0 to 6.5. It is concluded that pH controlled activation of adenylate cyclase causes the previously described increase in cyclic AMP which leads to phosphorylation of FBPase and finally to proteolysis of FBPase

  13. Identification and Characterization of Novel Plant Adenylate Cyclases – The Arabidopsis Thaliana Potassium Uptake Permeases

    KAUST Repository

    Al-Younis, Inas

    2018-01-01

    Adenylyl Cyclases (ACs) catalyze the formation of the key universal second messenger adenosine 3’, 5’-cyclic monophosphate (cAMP) from adenosine 5’- triphosphate. Cyclic AMP participates in several signal transduction pathways and is present

  14. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

    Directory of Open Access Journals (Sweden)

    César Martín

    Full Text Available Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  15. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

    Science.gov (United States)

    Martín, César; Uribe, Kepa B; Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2011-02-23

    Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT) that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin) family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  16. Adenylate cyclase activity in fish gills in relation to salt adaptation

    International Nuclear Information System (INIS)

    Guibbolini, M.E.; Lahlou, B.

    1987-01-01

    The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of [α- 32 P] - ATP into [α- 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 1 . These results are interpreted in relation to the general process of osmoregulation. 27 references, 6 figures

  17. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB. 20

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB.20

    International Nuclear Information System (INIS)

    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 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 [ 3 H]serotonin, [ 3 H]lysergic acid diethylamide or [ 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

  19. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    Science.gov (United States)

    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.

  20. Bicarbonate-responsive “soluble” adenylyl cyclase defines a nuclear cAMP microdomain

    Science.gov (United States)

    Zippin, Jonathan H.; Farrell, Jeanne; Huron, David; Kamenetsky, Margarita; Hess, Kenneth C.; Fischman, Donald A.; Levin, Lonny R.; Buck, Jochen

    2004-01-01

    Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation. PMID:14769862

  1. Liaison of 3H 5-HT and adenyl cyclasic activation induced by the 5-HT in preparations of brain glial membranes

    International Nuclear Information System (INIS)

    Fillion, Gilles; Beaudoin, Dominique; Rousselle, J.-C.; Jacob, Joseph

    1980-01-01

    Purified glial membrane preparations have been isolated from horse brain striatum. Tritiated 5-HT bound to these membranes with a high affinity (K(D)=10 nM); the corresponding bindings is reversible and appears specific of the serotoninergic structure. In parallel, 5-HT activates an adenylate cyclase with a low affinity (K(D)=1 μM). The sites involved in this binding and in this adenylate cyclase activation appear different from the serotoninergic sites reported in the neuronal membrane preparations [fr

  2. Genomic variants in the ASS1 gene, involved in the nitric oxide biosynthesis and signaling pathway, predict hydroxyurea treatment efficacy in compound sickle cell disease/β-thalassemia patients.

    Science.gov (United States)

    Chalikiopoulou, Constantina; Tavianatou, Anastasia-Gerasimoula; Sgourou, Argyro; Kourakli, Alexandra; Kelepouri, Dimitra; Chrysanthakopoulou, Maria; Kanelaki, Vasiliki-Kaliopi; Mourdoukoutas, Evangelos; Siamoglou, Stavroula; John, Anne; Symeonidis, Argyris; Ali, Bassam R; Katsila, Theodora; Papachatzopoulou, Adamantia; Patrinos, George P

    2016-03-01

    Hemoglobinopathies exhibit a remarkable phenotypic diversity that restricts any safe association between molecular pathology and clinical outcomes. Herein, we explored the role of genes involved in the nitric oxide biosynthesis and signaling pathway, implicated in the increase of fetal hemoglobin levels and response to hydroxyurea treatment, in 119 Hellenic patients with β-type hemoglobinopathies. We show that two ASS1 genomic variants (namely, rs10901080 and rs10793902) can serve as pharmacogenomic biomarkers to predict hydroxyurea treatment efficacy in sickle cell disease/β-thalassemia compound heterozygous patients. These markers may exert their effect by inducing nitric oxide biosynthesis, either via altering splicing and/or miRNA binding, as predicted by in silico analysis, and ultimately, increase γ-globin levels, via guanylyl cyclase targeting.

  3. Investigation of the pathophysiological mechanisms of migraine attacks induced by pituitary adenylate cyclase-activating polypeptide-38

    DEFF Research Database (Denmark)

    Amin, Faisal Mohammad; Hougaard, Anders; Schytz, Henrik W

    2014-01-01

    Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) and vasoactive intestinal polypeptide are structurally and functionally closely related but show differences in migraine-inducing properties. Mechanisms responsible for the difference in migraine induction are unknown. Here, for the ...

  4. Adenylyl Cyclase Signaling in the Developing Chick Heart: The Deranging Effect of Antiarrhythmic Drugs

    Czech Academy of Sciences Publication Activity Database

    Hejnová, L.; Hahnová, K.; Kočková, Radka; Svatůňková, Jarmila; Sedmera, David; Novotný, J.

    2014-01-01

    Roč. 2014, č. 2014 (2014), s. 463123 ISSN 2314-6133 R&D Projects: GA ČR(CZ) GAP302/11/1308 Institutional support: RVO:67985823 Keywords : embryo nic heart * embryo toxicity * adenylyl cyclase * G protein * beta-blocking agents Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 1.579, year: 2014

  5. Adenylate cyclase regulation in the spermatogenic cell plasma membrane: Modulating effects of TPA and TCDD

    International Nuclear Information System (INIS)

    Beebe, L.E.

    1989-01-01

    This research was designed to compare the effects of TPA, a phorbol ester, and TCDD in a spermatogenic cell population, a target of TCDD toxicity. Membrane-bound adenylate cyclase activity was used an index of membrane function, and was quantified by the amount of 32 P-cAMP formed from 32 P-ATP following chromatographic separation. Exposure to male germ cells in-vitro to TPA and TCDD followed by direct measurement of enzyme activity was used to investigate the potential of each agent to perturb membrane function. TPA and TCDD consistently inhibited adenylate cyclase activity at the levels of G s -catalytic unit coupling and hormone-receptor activation, as measured by the stimulation of enzyme activity by concomitant addition of forskolin and GTP and FSH and GTP, respectively. The effect on coupling required at least 60 minutes of exposure to TPA or TCDD. Concentration-response curves demonstrated a progressive desensitization with increasing TPA concentration, while TCDD exhibited consistent inhibition over the same concentration range

  6. Design and Synthesis of Fluorescent Acyclic Nucleoside Phosphonates as Potent Inhibitors of Bacterial Adenylate Cyclases

    Czech Academy of Sciences Publication Activity Database

    Břehová, Petra; Šmídková, Markéta; Skácel, Jan; Dračínský, Martin; Mertlíková-Kaiserová, Helena; Velasquez, M. P. S.; Watts, V. J.; Janeba, Zlatko

    2016-01-01

    Roč. 11, č. 22 (2016), s. 2534-2546 ISSN 1860-7179 R&D Projects: GA MV VG20102015046; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : adenylate cyclase toxin * acyclic nucleoside phosphonates * anthranilic acid Subject RIV: CC - Organic Chemistry Impact factor: 3.225, year: 2016

  7. Adenylate Cyclase Toxin Subverts Phagocyte Function by RhoA Inhibition and Unproductive Ruffling

    Czech Academy of Sciences Publication Activity Database

    Kamanová, Jana; Kofroňová, Olga; Mašín, Jiří; Genth, H.; Vojtová, Jana; Linhartová, Irena; Benada, Oldřich; Just, I.; Šebo, Peter

    2008-01-01

    Roč. 181, č. 8 (2008), s. 5587-5597 ISSN 0022-1767 R&D Projects: GA MŠk 1M0506; GA MŠk 2B06161; GA ČR GA310/08/0447 Grant - others:XE(XE) LSHB-CT-2003-503582 Institutional research plan: CEZ:AV0Z50200510 Keywords : bordetella * adenylate cyclase toxin * rhoa Subject RIV: EC - Immunology Impact factor: 6.000, year: 2008

  8. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    Science.gov (United States)

    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. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  9. Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

    Science.gov (United States)

    Simko, Veronika; Iuliano, Filippo; Sevcikova, Andrea; Labudova, Martina; Barathova, Monika; Radvak, Peter; Pastorekova, Silvia; Pastorek, Jaromir; Csaderova, Lucia

    2017-08-31

    Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

  10. Cellular targets of nitric oxide in the hippocampus.

    Directory of Open Access Journals (Sweden)

    Katalin Bartus

    Full Text Available In the hippocampus, as in many other CNS areas, nitric oxide (NO participates in synaptic plasticity, manifested as changes in pre- and/or postsynaptic function. While it is known that these changes are brought about by cGMP following activation of guanylyl cyclase-coupled NO receptors attempts to locate cGMP by immunocytochemistry in hippocampal slices in response to NO have failed to detect the cGMP elevation where expected, i.e. in the pyramidal neurones. Instead, astrocytes, unidentified varicose fibres and GABA-ergic nerve terminals are reported to be the prominent NO targets, raising the possibility that NO acts indirectly via other cells. We have re-investigated the distribution of cGMP generated in response to endogenous and exogenous NO in hippocampal slices using immunohistochemistry and new conditions designed to optimise cGMP accumulation and, hence, its detectability. The conditions included use of tissue from the developing rat hippocampus, a potent inhibitor of phosphodiesterase-2, and an allosteric enhancer of the NO-receptive guanylyl cyclase. Under these conditions, cGMP was formed in response to endogenous NO and was found in a population of pyramidal cell somata in area CA3 and subiculum as well as in structures described previously. The additional presence of exogenous NO resulted in hippocampal cGMP reaching the highest level recorded for brain tissue (1700 pmol/mg protein and in cGMP immunolabelling throughout the pyramidal cell layer. Populations of axons and interneurones were also stained. According with these results, immunohistochemistry for the common NO receptor β1-subunit indicated widespread expression. A similar staining pattern for the α1-subunit with an antibody used previously in the hippocampus and elsewhere, however, proved to be artefactual. The results indicate that the targets of NO in the hippocampus are more varied and extensive than previous evidence had suggested and, in particular, that the

  11. Different strictuctural requirements for adenylate cyclase toxin interactions with erythrocyte and liposome membranes

    Czech Academy of Sciences Publication Activity Database

    Mašín, Jiří; Konopásek, I.; Svobodová, J.; Šebo, Peter

    2004-01-01

    Roč. 1660, - (2004), s. 144-154 ISSN 0005-2736 R&D Projects: GA AV ČR IPP1050128; GA AV ČR IAA5020907 Grant - others:GA Howard Hughes Medical Institut(US) 55000334; GA(XE) QLK2-CT-1999-00556 Institutional research plan: CEZ:AV0Z5020903 Keywords : bordetella pertussis * adenylate cyclase toxin * membrane interaction Subject RIV: EE - Microbiology, Virology Impact factor: 3.441, year: 2004

  12. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

    Czech Academy of Sciences Publication Activity Database

    Osička, Radim; Osičková, Adriana; Hasan, Shakir; Bumba, Ladislav; Černý, Jiří; Šebo, Peter

    2015-01-01

    Roč. 4, DEC 9 (2015) ISSN 2050-084X R&D Projects: GA ČR(CZ) GAP302/11/0580; GA ČR(CZ) GA15-11851S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 ; RVO:86652036 Keywords : E. coli * adenylate cyclase toxin * biochemistry Subject RIV: CE - Biochemistry Impact factor: 8.282, year: 2015

  13. Synthesis of alpha-Branched Acyclic Nucleoside Phosphonates as Potential Inhibitors of Bacterial Adenylate Cyclases

    Czech Academy of Sciences Publication Activity Database

    Frydrych, Jan; Skácel, Jan; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Gnanasekaran, Ramachandran; Lepšík, Martin; Soto-Velasquez, M.; Watts, V. J.; Janeba, Zlatko

    2018-01-01

    Roč. 13, č. 2 (2018), s. 199-206 ISSN 1860-7179 R&D Projects: GA MV VG20102015046; GA ČR(CZ) GBP208/12/G016; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * adenylate cyclase toxin * bisamidates * Bordetella pertussis * prodrugs Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 3.225, year: 2016

  14. Magnesium Lithospermate B, an Active Extract of Salvia miltiorrhiza, Mediates sGC/cGMP/PKG Translocation in Experimental Vasospasm

    Directory of Open Access Journals (Sweden)

    Chih-Zen Chang

    2014-01-01

    Full Text Available Background. Soluble guanylyl cyclases (sGCs and Ras homolog gene family, member A (rhoA/Ras homolog gene family kinase(rho-kinase plays a role in vascular smooth muscle relaxation in subarachnoid hemorrhage (SAH. It is of interest to examine the effect of MLB on rhoA/ROCK and sGC/cGMP/PKG expression. Methods. A rodent SAH model was employed. Tissue samples were for sGCα1, sGCβ1, PKG, rhoA, ROCK (Western blot, and cGMP (ELISA measurement. Results. MLB morphologically improved convolution of the internal elastic lamina, distortion of endothelial wall, and necrosis of the smooth muscle in the SAH rats. Expressed cGMP, sGCα1, sGCβ1, and PKG in the SAH groups were reduced (P<0.01, and MLB precondition significantly induced cGMP, sGCα1, sGCβ1, and PKG. L-NAME reversed the vasodilation effect of MLB, reduced the bioexpression of PKG and cGMP (P<0.01, and tends to reduce sGCα1 level and induce rhoA, ROCK level in MLB precondition + SAH groups. Conclusion. These results demonstrate that sGC/cGMP/PKG and NO/ET pathways play pivotal roles in SAH-induced vasospasm. Through activating sGC/cGMP/PKG pathway and partially by inactivating rho-kinase in a NO-dependent mechanism, MLB shows promise to be an effective strategy for the treatment of this disease entity.

  15. Melanopsin mediates light-dependent relaxation in blood vessels.

    Science.gov (United States)

    Sikka, Gautam; Hussmann, G Patrick; Pandey, Deepesh; Cao, Suyi; Hori, Daijiro; Park, Jong Taek; Steppan, Jochen; Kim, Jae Hyung; Barodka, Viachaslau; Myers, Allen C; Santhanam, Lakshmi; Nyhan, Daniel; Halushka, Marc K; Koehler, Raymond C; Snyder, Solomon H; Shimoda, Larissa A; Berkowitz, Dan E

    2014-12-16

    Melanopsin (opsin4; Opn4), a non-image-forming opsin, has been linked to a number of behavioral responses to light, including circadian photo-entrainment, light suppression of activity in nocturnal animals, and alertness in diurnal animals. We report a physiological role for Opn4 in regulating blood vessel function, particularly in the context of photorelaxation. Using PCR, we demonstrate that Opn4 (a classic G protein-coupled receptor) is expressed in blood vessels. Force-tension myography demonstrates that vessels from Opn4(-/-) mice fail to display photorelaxation, which is also inhibited by an Opn4-specific small-molecule inhibitor. The vasorelaxation is wavelength-specific, with a maximal response at ∼430-460 nm. Photorelaxation does not involve endothelial-, nitric oxide-, carbon monoxide-, or cytochrome p450-derived vasoactive prostanoid signaling but is associated with vascular hyperpolarization, as shown by intracellular membrane potential measurements. Signaling is both soluble guanylyl cyclase- and phosphodiesterase 6-dependent but protein kinase G-independent. β-Adrenergic receptor kinase 1 (βARK 1 or GRK2) mediates desensitization of photorelaxation, which is greatly reduced by GRK2 inhibitors. Blue light (455 nM) regulates tail artery vasoreactivity ex vivo and tail blood blood flow in vivo, supporting a potential physiological role for this signaling system. This endogenous opsin-mediated, light-activated molecular switch for vasorelaxation might be harnessed for therapy in diseases in which altered vasoreactivity is a significant pathophysiologic contributor.

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

    NARCIS (Netherlands)

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

    1986-01-01

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

  17. The plant natriuretic peptide receptor is a guanylyl cyclase and enables cGMP-dependent signaling

    KAUST Repository

    Turek, Ilona; Gehring, Christoph A

    2016-01-01

    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

  18. Adenylyl cyclase type 9 gene polymorphisms are associated with asthma and allergy in Brazilian children.

    Science.gov (United States)

    Teixeira, Helena M P; Alcantara-Neves, Neuza M; Barreto, Maurício; Figueiredo, Camila A; Costa, Ryan S

    2017-02-01

    Asthma is a chronic inflammatory disease of the respiratory tract. This heterogeneous disease is caused by the interaction of interindividual genetic variability and environmental factors. The gene adenylyl cyclase type 9 (ADCY9) encodes a protein called adenylyl cyclase (AC), responsible for producing the second messenger cyclic AMP (cAMP). cAMP is produced by T regulatory cells and is involved in the down-regulation of T effector cells. Failures in cAMP production may be related to an imbalance in the regulatory immune response, leading to immune-mediated diseases, such as allergic disorders. The aim of this study was to investigate how polymorphisms in the ADCY9 are associated with asthma and allergic markers. The study comprised 1309 subjects from the SCAALA (Social Changes Asthma and Allergy in Latin America) program. Genotyping was accomplished using the Illumina 2.5 Human Omni bead chip. Logistic regression was used to assess the association between allergy markers and ADCY9 variation in PLINK 1.07 software with adjustments for sex, age, helminth infection and ancestry markers. The ADCY9 candidate gene was associated with different phenotypes, such as asthma, specific IgE, skin prick test, and cytokine production. Among 133 markers analyzed, 29 SNPs where associated with asthma and allergic markers in silico analysis revealed the functional impact of the 6 SNPs on ADCY9 expression. It can be concluded that polymorphisms in the ADCY9 gene are significantly associated with asthma and/or allergy markers. We believe that such polymorphisms may lead to increased expression of adenylyl cyclase with a consequent increase in immunoregulatory activity. Therefore, these SNPs may offer an impact on the occurrence of these conditions in admixture population from countries such as Brazil. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. A concise discussion of the regulatory role of cGMP kinase I in cardiac physiology and pathology.

    Science.gov (United States)

    Hofmann, Franz

    2018-06-22

    The underlying cause of cardiac hypertrophy, fibrosis, and heart failure has been investigated in great detail using different mouse models. These studies indicated that cGMP and cGMP-dependent protein kinase type I (cGKI) may ameliorate these negative phenotypes in the adult heart. Recently, evidence has been published that cardiac mitochondrial BKCa channels are a target for cGKI and that activation of mitoBKCa channels may cause some of the positive effects of conditioning in ischemia/reperfusion injury. It will be pointed out that most studies could not present convincing evidence that it is the cGMP level and the activity cGKI in specific cardiac cells that reduces hypertrophy or heart failure. However, anti-fibrotic compounds stimulating nitric oxide-sensitive guanylyl cyclase may be an upcoming therapy for abnormal cardiac remodeling.

  20. Immunochemical similarity of GTP-binding proteins from different systems

    International Nuclear Information System (INIS)

    Kalinina, S.N.

    1986-01-01

    It was found that antibodies against the GTP-binding proteins of bovine retinal photoreceptor membranes blocked the inhibitory effect of estradiol on phosphodiesterase from rat and human uterine cytosol and prevented the cumulative effect of catecholamines and guanylyl-5'-imidodiphosphate on rat skeletal muscle adenylate cyclase. It was established by means of double radial immunodiffusion that these antibodies form a precipitating complex with purified bovine brain tubulin as well as with retinal preparations obtained from eyes of the bull, pig, rat, frog, some species of fish, and one reptile species. Bands of precipitation were not observed with these antibodies when retinal preparations from invertebrates (squid and octopus) were used as the antigens. The antibodies obtained interacted with the α- and β-subunits of GTP-binding proteins from bovine retinal photoreceptor membranes

  1. Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

    Directory of Open Access Journals (Sweden)

    Catalina Sanz

    Full Text Available Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.

  2. Interaction of Bordetella adenylate cyclase toxin with complement receptor 3 involves multivalent glycan binding

    Czech Academy of Sciences Publication Activity Database

    Hasan, Shakir; Osičková, Adriana; Bumba, Ladislav; Novák, Petr; Šebo, Peter; Osička, Radim

    2015-01-01

    Roč. 589, č. 3 (2015), s. 374-379 ISSN 0014-5793 R&D Projects: GA ČR(CZ) GAP302/11/0580; GA ČR(CZ) GA15-09157S; GA ČR(CZ) GA15-11851S Institutional support: RVO:61388971 Keywords : Adenylate cyclase toxin * CD11b/CD18 * Complement receptor type 3 Subject RIV: CE - Biochemistry Impact factor: 3.519, year: 2015

  3. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    International Nuclear Information System (INIS)

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1987-01-01

    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 [ 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 [ 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 β-adrenergic receptor in rat heart

  4. Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.

    Science.gov (United States)

    Condie, R; Herring, A; Koh, W S; Lee, M; Kaminski, N E

    1996-05-31

    Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

  5. G-protein-mediated interconversions of cell-surface cAMP receptors and their involvement in excitation and desensitization of guanylate cyclase in Dictyostelium discoideum

    International Nuclear Information System (INIS)

    van Haastert, P.J.; de Wit, R.J.; Janssens, P.M.; Kesbeke, F.; DeGoede, J.

    1986-01-01

    In Dictyostelium discoideum cells, extracellular cAMP induces the rapid (within 2 s) activation of guanylate cyclase, which is followed by complete desensitization after about 10 s. cAMP binding to these cells is heterogeneous, showing a subclass of fast dissociating sites coupled to adenylate cyclase (A-sites) and a subclass of slowly dissociating sites coupled to guanylate cyclase (B-sites). The kinetics of the B-sites were further investigated on a seconds time scale. Statistical analysis of the association of [ 3 H]cAMP to the B-sites and dissociation of the complex revealed that the receptor can exist in three states which interconvert according to the following scheme. cAMP binds to the BF-state (off-rate 2.5 s) which rapidly (t1/2 = 3 s) converts to the BS-state (off-rate 15 s) and subsequently (without a detectable delay) into the BSS-state (off-rate 150 s). In membranes, both the BS- and BSS-states are converted to the BF-state by GTP and GDP, suggesting the involvement of a G-protein. Densensitized cells show a 80% reduction of the formation of the BSS-state, but no reduction of the BF- or BS-state. These data are combined into a model in which the transitions of the B-sites are mediated by a G-protein; activation of the G-protein and guanylate cyclase is associated with the transition of the BS- to the BSS-state of the receptor, whereas desensitization is associated with the inhibition of this transition

  6. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    International Nuclear Information System (INIS)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L.

    2014-01-01

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R h ) and reduced thermal stability in the mutant complex. Taken together

  7. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Finley, Natosha L., E-mail: finleynl@miamioh.edu [Department of Microbiology, Miami University, Oxford, OH 45056 (United States); Cell, Molecular, and Structural Biology Program, Miami University, Oxford, OH 45056 (United States)

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  8. Alteration in adenylate cyclase response to aminergic stimulation following neonatal x-irradiation

    International Nuclear Information System (INIS)

    Chronister, R.B.; Palmer, G.C.; Gerbrandt, L.

    1980-01-01

    X-irradiation of the rat neonatal hippocampus produces severe alterations in the architectonic features of the mature hippocampus. The most prominent alteration is a marked depletion of the granule cells of the dentate gyrus, with a subsequent realignment of CA 4 cells. The present data also show that norepinephrine (NE), dopamine and histamine stimulation of adenylate cyclase activity is severely attenuated in the hippocampi of irradiated animals. This failure suggests that the NE fibers of irradiated subjects, although normal in content of NE, are not functional in some of their NE-effector actions

  9. Human glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site

    Directory of Open Access Journals (Sweden)

    Misquitta Stephanie A

    2004-02-01

    Full Text Available Abstract Background Glutaminyl cyclase (QC forms the pyroglutamyl residue at the amino terminus of numerous secretory peptides and proteins. We previously proposed the mammalian QC has some features in common with zinc aminopeptidases. We now have generated a structural model for human QC based on the aminopeptidase fold (pdb code 1AMP and mutated the apparent active site residues to assess their role in QC catalysis. Results The structural model proposed here for human QC, deposited in the protein databank as 1MOI, is supported by a variety of fold prediction programs, by the circular dichroism spectrum, and by the presence of the disulfide. Mutagenesis of the six active site residues present in both 1AMP and QC reveal essential roles for the two histidines (140 and 330, QC numbering and the two glutamates (201 and 202, while the two aspartates (159 and 248 appear to play no catalytic role. ICP-MS analysis shows less than stoichiometric zinc (0.3:1 in the purified enzyme. Conclusions We conclude that human pituitary glutaminyl cyclase and bacterial zinc aminopeptidase share a common fold and active site residues. In contrast to the aminopeptidase, however, QC does not appear to require zinc for enzymatic activity.

  10. Catecholamine-induced desensitization of adenylate cyclase coupled β-adrenergic receptors in turkey erythrocytes: evidence for a two-step mechanism

    International Nuclear Information System (INIS)

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

    1987-01-01

    Preincubation of turkey erythrocytes with isoproterenol is associated with (1) 50-60% attenuation of agonist-stimulated adenylate cyclase activity, (2) altered mobility of the β-adrenergic receptor on sodium dodecyl sulfate-polyacrylamide gels, and (3) increased phosphorylation of the β-adrenergic receptor. Using a low-cross-linked polyacrylamide gel, the β-adrenergic receptor protein from isoproterenol-desensitized cells, labeled with 32 P or with the photoaffinity label 125 I-(p-azidobenzyl)carazolol, can be resolved into a doublet (M/sub r/ similarly ordered 37,000 and M/sub r/ similarly ordered 41,000) as compared to a single M/sub r/ similarly ordered 37,000 β-adrenergic receptor protein from control erythrocytes. The appearance of the doublet was dependent on the concentration of agonist used to desensitize the cells. Incubation of erythrocytes with dibutyryl-cAMP did not promote formation of the doublet but decreased agonist-stimulated adenylate cyclase activity 40-50%. Limited-digestion peptide maps of 32 P-labeled β-adrenergic receptors using papain revealed a unique phosphopeptide in the larger molecular weight band (M/sub r/ similarly ordered 41,000) of the doublet from the agonist-desensitized preparation that was absent in the peptide maps of the smaller band (M/sub r/ similarly ordered 37,000), as well as control or dibutyryl-cAMP-desensitized receptor. These data provide evidence that maximal agonist-induced desensitization of adenylate cyclase coupled β-adrenergic receptors in turkey erythrocytes occurs by a two-step mechanism

  11. Transgenic rescue of defective Cd36 enhances myocardial adenylyl cyclase signaling in spontaneously hypertensive rats

    Czech Academy of Sciences Publication Activity Database

    Klevstig, M.; Manakov, D.; Kašparová, D.; Brabcová, I.; Papoušek, František; Žurmanová, J.; Zídek, Václav; Šilhavý, Jan; Neckář, Jan; Pravenec, Michal; Kolář, František; Nováková, O.; Novotný, J.

    2013-01-01

    Roč. 465, č. 10 (2013), s. 1477-1486 ISSN 0031-6768 R&D Projects: GA MŠk(CZ) LL1204; GA AV ČR(CZ) IAAX01110901; GA ČR(CZ) GAP303/10/0505 Institutional support: RVO:67985823 Keywords : SHR rats * Cd36 * heart * beta-Adrenergic receptors * Adenylyl cyclase * Protein kinase A Subject RIV: ED - Physiology Impact factor: 3.073, year: 2013

  12. Alkaline pH sensor molecules.

    Science.gov (United States)

    Murayama, Takashi; Maruyama, Ichiro N

    2015-11-01

    Animals can survive only within a narrow pH range. This requires continual monitoring of environmental and body-fluid pH. Although a variety of acidic pH sensor molecules have been reported, alkaline pH sensor function is not well understood. This Review describes neuronal alkaline pH sensors, grouped according to whether they monitor extracellular or intracellular alkaline pH. Extracellular sensors include the receptor-type guanylyl cyclase, the insulin receptor-related receptor, ligand-gated Cl- channels, connexin hemichannels, two-pore-domain K+ channels, and transient receptor potential (TRP) channels. Intracellular sensors include TRP channels and gap junction channels. Identification of molecular mechanisms underlying alkaline pH sensing is crucial for understanding how animals respond to environmental alkaline pH and how body-fluid pH is maintained within a narrow range. © 2015 Wiley Periodicals, Inc.

  13. Segments Crucial for Membrane Translocation and Pore-forming Activity of Bordetella Adenylate Cyclase Toxin

    Czech Academy of Sciences Publication Activity Database

    Basler, Marek; Knapp, O.; Mašín, Jiří; Fišer, R.; Maier, E.; Benz, R.; Šebo, Peter; Osička, Radim

    2007-01-01

    Roč. 282, č. 17 (2007), s. 12419-12429 ISSN 0021-9258 R&D Projects: GA MŠk 1M0506; GA AV ČR IAA5020406 Grant - others:XE(XE) European Union 6th FP contract LSHB-CT-2003-503582 THERAVAC Institutional research plan: CEZ:AV0Z50200510 Source of funding: R - rámcový projekt EK Keywords : bordetella * adenylate cyclase toxin * ac membrane translocation Subject RIV: EE - Microbiology, Virology Impact factor: 5.581, year: 2007

  14. Plutonium solubilities

    International Nuclear Information System (INIS)

    Puigdomnech, I.; Bruno, J.

    1991-02-01

    Thermochemical data has been selected for plutonium oxide, hydroxide, carbonate and phosphate equilibria. Equilibrium constants have been evaluated in the temperature range 0 to 300 degrees C at a pressure of 1 bar to T≤100 degrees C and at the steam saturated pressure at higher temperatures. Measured solubilities of plutonium that are reported in the literature for laboratory experiments have been collected. Solubility data on oxides, hydroxides, carbonates and phosphates have been selected. No solubility data were found at temperatures higher than 60 degrees C. The literature solubility data have been compared with plutonium solubilities calculated with the EQ3/6 geochemical modelling programs, using the selected thermodynamic data for plutonium. (authors)

  15. Effects of sevoflurane on adenylate cyclase and phosphodiesterases activity in brain of rats

    International Nuclear Information System (INIS)

    Feng Changdong; Yang Jianping; Dai Tijun

    2009-01-01

    Objective: To investigate the effects of sevoflurane on c adenylate cyclase (AC) and phosphodiesterases (PDE) activity in the cerebrocortex, hippocampus and brain stem of rats, and to examine the role of cAMP in sevoflurane anesthesia. Methods: Fourty SD rats were delaminately designed and allocated randomly to 5 groups inhaling 1.5% sevoflurane i.e., no recovery (recovery group, n=8) and one hour after righting reflexrecovery (aware group, n=8). The brain tissues were rapidly dissected into cerebrocortex and hippocampus and brain stem.Then the adenylate cyclase and phosphodiesterases activity were assessed. Results: So far as the activity of AC is concerned, compared with the control group, the activity of AC in the cerebrocortex, hippocampus and brain stem brain stem of induction group and anesthesia group, the cerebrocortex, and hippocampus in the recovery group were significantly increased; compared with those in the anesthesia group, the activity of AC in the cerebrocortex, hippocampus and brain stem of aware group were significantly decreased (P<0.05); For the activity of PDE, compared with the control group, the activity of PDE in the cerebrocortex, hippocampus and brain stem in the induction group and anesthesia group was significantly decreased, compared with that in anesthesia group, the activity of PDE in the cerebrocortex, hippocampus and brain stem of recovery group and aware group was significantly increased (P<0.05). Conclusion: cAMP may play an important role in sevoflurane anesthesia. (authors)

  16. Heterosubtypic protection against influenza A induced by adenylate cyclase toxoids delivering conserved HA2 subunit of hemagglutinin

    Czech Academy of Sciences Publication Activity Database

    Staneková, Z.; Adkins, Irena; Kosová, Martina; Janulíková, J.; Šebo, Peter; Varečková, E.

    2013-01-01

    Roč. 97, č. 1 (2013), s. 24-35 ISSN 0166-3542 R&D Projects: GA ČR GA310/08/0447; GA ČR GP310/09/P582 Institutional support: RVO:61388971 Keywords : Bordetella adenylate cyclase toxoid * Influenza A infection * Cross-protection Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.434, year: 2013

  17. Amidate Prodrugs of 9-[2-(Phosphonomethoxy)Ethyl]Adenine as Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Šmídková, Markéta; Dvořáková, Alexandra; Tloušťová, Eva; Česnek, Michal; Janeba, Zlatko; Mertlíková-Kaiserová, Helena

    2014-01-01

    Roč. 58, č. 2 (2014), s. 664-671 ISSN 0066-4804 R&D Projects: GA MV VG20102015046 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 Keywords : Bordetella pertussis * adenylate cyclase toxin * ACT * inhibitors * PMEA * amidate prodrugs Subject RIV: CC - Organic Chemistry Impact factor: 4.476, year: 2014

  18. Amidate prodrugs of 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA) as inhibitors of adenylate cyclase toxin from Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Šmídková, Markéta; Dvořáková, Alexandra; Tloušťová, Eva; Česnek, Michal; Janeba, Zlatko; Mertlíková-Kaiserová, Helena

    2014-01-01

    Roč. 281, Suppl S1 (2014), s. 729 ISSN 1742-464X. [FEBS EMBO 2014 Conference. 30.08.2014-04.09.2014, Paris] R&D Projects: GA MŠk LO1302; GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : Bordetella pertussis * adenylyl cyclase toxin * inhibitors Subject RIV: CE - Biochemistry

  19. Nitric oxide amplifies the rat electroretinogram.

    Science.gov (United States)

    Vielma, Alex; Delgado, Luz; Elgueta, Claudio; Osorio, Rodrigo; Palacios, Adrián G; Schmachtenberg, Oliver

    2010-11-01

    It is well established that nitric oxide (NO) participates in retinal signal processing through stimulation of its receptor enzyme, soluble guanylyl cyclase (sGC). However, under pathological conditions such as uveoretinitis, diabetic or ischemic retinopathy, elevated NO concentrations may cause protein S-nitrosation and peroxynitrite formation in the retina, promoting cellular injury and apoptosis. Previous electroretinogram (ERG) studies demonstrated deleterious effects of NO on the retinal light response, but showed no evidence for a role in normal signal processing. To better understand the function of NO in ocular physiology, we investigated the effects of exogenous NO, produced by NO donors with different release kinetics, on the flash ERG of the rat. Within a limited concentration range, NO strongly amplified ERG a- and b-waves, oscillatory potentials, and the scotopic threshold response. Amplification exceeded 100% under dark adaptation, whereas the photopic ERG and the isolated cone response were increased by less than 50%. Blocking photoreceptor-bipolar cell synapses by AP-4 demonstrated a significant increase of the isolated a-wave by NO, and modeling the ERG generator PIII supported photoreceptors as primary NO targets. The sGC inhibitors ODQ and NS2028 did not reduce NO-dependent ERG amplification, ruling out an involvement of the classical NO effector cyclic GMP. Using immunohistochemistry, we show that illumination and exogenous NO altered the S-nitrosation level of the photoreceptor layer, suggesting that direct protein modifications caused by elevated levels of NO may be responsible for the observed phenomenon. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. Ethanol extract of seeds of Oenothera odorata induces vasorelaxation via endothelium-dependent NO-cGMP signaling through activation of Akt-eNOS-sGC pathway.

    Science.gov (United States)

    Kim, Hye Yoom; Oh, Hyuncheol; Li, Xiang; Cho, Kyung Woo; Kang, Dae Gill; Lee, Ho Sub

    2011-01-27

    The vasorelaxant effect of ethanol extract of seeds of Oenothera odorata (Onagraceae) (one species of evening primroses) (ESOO) and its mechanisms involved were defined. Changes in vascular tension, guanosine 3',5'-cyclic monophosphate (cGMP) levels, and Akt expression were measured in carotid arterial rings from rats. Seeds of Oenothera odorata were extracted with ethanol (94%) and the extract was filtered, concentrated and stored at -70°C. ESOO relaxed endothelium-intact, but not endothelium-denuded, carotid arterial rings in a concentration-dependent manner. Similarly, ESOO increased cGMP levels of the carotid arterial rings. Pretreatment of endothelium-intact arterial rings with L-NAME, an inhibitor of nitric oxide synthase (NOS), or ODQ, an inhibitor of soluble guanylyl cyclase (sGC), blocked the ESOO-induced vasorelaxation and increase in cGMP levels. Nominally Ca(2+)-free but not L-typed Ca(2+) channel inhibition attenuated the ESOO-induced vasorelaxation. Thapsigargin, Gd(3+), and 2-aminoethyl diphenylborinate, modulators of store-operated Ca(2+) entry (SOCE), significantly attenuated the ESOO-induced vasorelaxation and increase in cGMP levels. Further, wortmannin, an inhibitor of Akt, attenuated the ESOO-induced vasorelaxation and increases in cGMP levels and phosphorylated Akt2 expression. K(+) channel blockade with TEA, 4-aminopyridine, and glibenclamide attenuated the ESOO-induced vascular relaxation. Taken together, the present study demonstrates that ESOO relaxes vascular smooth muscle via endothelium-dependent NO-cGMP signaling through activation of the Akt-eNOS-sGC pathway. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  1. Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes

    Czech Academy of Sciences Publication Activity Database

    Novák, Jakub; Černý, Ondřej; Osičková, Adriana; Linhartová, Irena; Mašín, Jiří; Bumba, Ladislav; Šebo, Peter; Osička, Radim

    2017-01-01

    Roč. 9, č. 10 (2017), s. 1-28, č. článku 300. E-ISSN 2072-6651 R&D Projects: GA ČR GA15-09157S; GA ČR(CZ) GA16-05919S; GA MŠk(CZ) LM2015064; GA MZd(CZ) NV16-28126A Institutional support: RVO:61388971 Keywords : adenylate cyclase toxin * Bordetella * cAMP Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.030, year: 2016

  2. Issues concerning the determination of solubility products of sparingly soluble crystalline solids. Solubility of HfO2(cr)

    International Nuclear Information System (INIS)

    Rai, Dhanpat; Kitamura, Akira; Rosso, Kevin M.; Sasaki, Takayuki; Kobayashi, Taishi

    2016-01-01

    Solubility studies were conducted with HfO 2 (cr) solid as a function HCl and ionic strength ranging from 2.0 to 0.004 mol kg -1 . These studies involved (1) using two different amounts of the solid phase, (2) acid washing the bulk solid phase, (3) preheating the solid phase to 1400 C, and (4) heating amorphous HfO 2 (am) suspensions to 90 C to ascertain whether the HfO 2 (am) converts to HfO 2 (cr) and to determine the solubility from the oversaturation direction. Based on the results of these treatments it is concluded that the HfO 2 (cr) contains a small fraction of less crystalline, but not amorphous, material [HfO 2 (lcr)] and this, rather than the HfO 2 (cr), is the solubility-controlling phase in the range of experimental variables investigated in this study. The solubility data are interpreted using both the Pitzer and SIT models and they provide log 10 K 0 values of -(59.75±0.35) and -(59.48±0.41), respectively, for the solubility product of HfO 2 (lcr)[HfO 2 (lcr) + 2H 2 O ↔ Hf 4+ + 4OH - ]. The log 10 of the solubility product of HfO 2 (cr) is estimated to be < -63. The observation of a small fraction of less crystalline higher solubility material is consistent with the general picture that mineral surfaces are often structurally and/or compositionally imperfect leading to a higher solubility than the bulk crystalline solid. This study stresses the urgent need, during interpretation of solubility data, of taking precautions to make certain that the observed solubility behavior for sparingly-soluble solids is assigned to the proper solid phase.

  3. Tocopherol synthesis from homogentisate in Capsicum anuum L. (yellow pepper) chromoplast membranes: evidence for tocopherol cyclase.

    OpenAIRE

    Arango, Y; Heise, K P

    1998-01-01

    The present study shows for the first time appreciable tocopherol cyclase activities in plastidial membrane preparations of Capsicum annuum L. (yellow pepper) fruits. When chromoplast membranes from yellow peppers were incubated with [3H]homogentisate and phytyl pyrophosphate under strictly reducing conditions, all biosynthesis precursors were labelled. The main labelling was found in gamma-tocopherol. These observations contradict the hypothesis that assigns a rate-limiting function to tocop...

  4. Characterization of beta-adrenergic receptors and adenylate cyclase activity in rat brown fat

    International Nuclear Information System (INIS)

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

    1986-01-01

    Catecholamines stimulate thermogenesis in rat brown fat through a mechanism which involves binding to the beta-adrenergic receptor (BAR), stimulation of adenylate cyclase (AC) and culminating with uncoupling of mitochondrial respiration from ATP synthesis. The authors characterized BAR, AC and cytochrome (cyt) c oxidase in CDF (F-344) interscapular brown fat. Scatchard analysis of [ 125 ]Iodopindolol binding yields a straight line consistent with a single class of antagonist binding sites with 41.8 +/- 12.0 fmol BAR/mg protein and a K/sub d/ of 118 +/- 15 pM. Binding was both specific and stereospecific. Competition with 1-propranolol (K/sub d/ = 6.7 nM) was 15 times more potent than d-propranolol (K/sub d/ = 103 nM). Competition with isoproterenol (K/sub d/ = 79 nM) was 10 times more potent than epinephrine (K/sub d/ = 820 nM) which was 35 times more potent than norepinephrine (K/sub d/ = 2.9 x 10 -5 M) suggesting predominate beta 2 -type BAR. Cyt c oxidase activity was assessed in brown fat mitochrondrial preparations. The ratio of BAR to cyt c activity was 959 +/- 275 nmol BAR/mol cyc c/min. Isoproterenol (0.1 mM) stimulated AC activity was 24 times GTP (0.1 mM) stimulated AC (98.5 vs 40.7 pmol cAMP/min/mg). NaF-stimulated AC was nine times basal activity (90.5 vs 11.3 pmol cAMP/min/mg). These data demonstrate the presence of a beta- 2 -type BAR coupled to adenylate cyclase in rat brown fat

  5. Docosahexaenoic acid alters Gsα localization in lipid raft and potentiates adenylate cyclase.

    Science.gov (United States)

    Zhu, Zhuoran; Tan, Zhoubin; Li, Yan; Luo, Hongyan; Hu, Xinwu; Tang, Ming; Hescheler, Jürgen; Mu, Yangling; Zhang, Lanqiu

    2015-01-01

    Supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA), recently has become popular for the amelioration of depression; however the molecular mechanism of DHA action remains unclear. The aim of this study was to investigate the mechanism underlying the antidepressant effect of DHA by evaluating Gsα localization in lipid raft and the activity of adenylate cyclase in an in vitro glioma cell model. Lipid raft fractions from C6 glioma cells treated chronically with DHA were isolated by sucrose gradient ultracentrifugation. The content of Gsα in lipid raft was analyzed by immunoblotting and colocalization of Gsα with lipid raft was subjected to confocal microscopic analysis. The intracellular cyclic adenosine monophosphate (cAMP) level was determined by cAMP immunoassay kit. DHA decreased the amount of Gsα in lipid raft, whereas whole cell lysate Gsα was not changed. Confocal microscopic analysis demonstrated that colocalization of Gsα with lipid raft was decreased, whereas DHA increased intracellular cAMP accumulation in a dose-dependent manner. Interestingly, we found that DHA increased the lipid raft level, instead of disrupting it. The results of this study suggest that DHA may exert its antidepressant effect by translocating Gsα from lipid raft and potentiating the activity of adenylate cyclase. Importantly, the reduced Gsα in lipid raft by DHA is independent of disruption of lipid raft. Overall, the study provides partial preclinical evidence supporting a safe and effective therapy using DHA for depression. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. An adenylyl cyclase like-9 gene (NlAC9) influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

    Science.gov (United States)

    Ge, LinQuan; Gu, HaoTian; Huang, Bo; Song, Qisheng; Stanley, David; Liu, Fang; Yang, Guo-Qing; Wu, Jin-Cai

    2017-01-01

    The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC) conversion of adenosine triphosphate (ATP) to 3', 5'-cyclic AMP (cAMP) and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in some very small pest insects, such as the brown planthopper (BPH), Nilaparvata lugens Stål. BPH is a destructive pest responsible for tremendous crop losses in rice cropping systems. We are investigating the potentials of novel pest management technologies from RNA interference perspective. Based on analysis of transcriptomic data, the BPH AC like-9 gene (NlAC9) was up-regulated in post-mating females, which led us to pose the hypothesis that NlAC9 is a target gene that would lead to reduced BPH fitness and populations. Targeting NlAC9 led to substantially decreased soluble ovarian protein content, yeast-like symbiont abundance, and vitellogenin gene expression, accompanied with stunted ovarian development and body size. Eggs laid were decreased and oviposition period shortened. Taken together, our findings indicated that NlAC9 exerted pronounced effects on female fecundity, growth and longevity, which strongly supports our hypothesis.

  7. An adenylyl cyclase like-9 gene (NlAC9 influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål (Hemiptera: Delphacidae.

    Directory of Open Access Journals (Sweden)

    LinQuan Ge

    Full Text Available The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC conversion of adenosine triphosphate (ATP to 3', 5'-cyclic AMP (cAMP and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in some very small pest insects, such as the brown planthopper (BPH, Nilaparvata lugens Stål. BPH is a destructive pest responsible for tremendous crop losses in rice cropping systems. We are investigating the potentials of novel pest management technologies from RNA interference perspective. Based on analysis of transcriptomic data, the BPH AC like-9 gene (NlAC9 was up-regulated in post-mating females, which led us to pose the hypothesis that NlAC9 is a target gene that would lead to reduced BPH fitness and populations. Targeting NlAC9 led to substantially decreased soluble ovarian protein content, yeast-like symbiont abundance, and vitellogenin gene expression, accompanied with stunted ovarian development and body size. Eggs laid were decreased and oviposition period shortened. Taken together, our findings indicated that NlAC9 exerted pronounced effects on female fecundity, growth and longevity, which strongly supports our hypothesis.

  8. Pore formation by the Bordetella adenylate cyclase toxin in lipid bilayer membranes: Role of voltage and pH

    Czech Academy of Sciences Publication Activity Database

    Knapp, O.; Maier, E.; Mašín, Jiří; Šebo, Peter; Benz, R.

    2008-01-01

    Roč. 1778, č. 1 (2008), s. 260-269 ISSN 0005-2736 R&D Projects: GA AV ČR(CZ) IAA5020406 Grant - others:XE(XE) QLK2-CT-1999-00556 Institutional research plan: CEZ:AV0Z50200510 Keywords : adenylate cyclase toxin * act * voltage Subject RIV: EE - Microbiology, Virology Impact factor: 4.180, year: 2008

  9. Adenylate Cyclases of Trypanosoma brucei, Environmental Sensors and Controllers of Host Innate Immune Response.

    Science.gov (United States)

    Salmon, Didier

    2018-04-25

    Trypanosoma brucei , etiological agent of Sleeping Sickness in Africa, is the prototype of African trypanosomes, protozoan extracellular flagellate parasites transmitted by saliva ( Salivaria ). In these parasites the molecular controls of the cell cycle and environmental sensing are elaborate and concentrated at the flagellum. Genomic analyses suggest that these parasites appear to differ considerably from the host in signaling mechanisms, with the exception of receptor-type adenylate cyclases (AC) that are topologically similar to receptor-type guanylate cyclase (GC) of higher eukaryotes but control a new class of cAMP targets of unknown function, the cAMP response proteins (CARPs), rather than the classical protein kinase A cAMP effector (PKA). T. brucei possesses a large polymorphic family of ACs, mainly associated with the flagellar membrane, and these are involved in inhibition of the innate immune response of the host prior to the massive release of immunomodulatory factors at the first peak of parasitemia. Recent evidence suggests that in T. brucei several insect-specific AC isoforms are involved in social motility, whereas only a few AC isoforms are involved in cytokinesis control of bloodstream forms, attesting that a complex signaling pathway is required for environmental sensing. In this review, after a general update on cAMP signaling pathway and the multiple roles of cAMP, I summarize the existing knowledge of the mechanisms by which pathogenic microorganisms modulate cAMP levels to escape immune defense.

  10. Adenylate Cyclases of Trypanosoma brucei, Environmental Sensors and Controllers of Host Innate Immune Response

    Directory of Open Access Journals (Sweden)

    Didier Salmon

    2018-04-01

    Full Text Available Trypanosoma brucei, etiological agent of Sleeping Sickness in Africa, is the prototype of African trypanosomes, protozoan extracellular flagellate parasites transmitted by saliva (Salivaria. In these parasites the molecular controls of the cell cycle and environmental sensing are elaborate and concentrated at the flagellum. Genomic analyses suggest that these parasites appear to differ considerably from the host in signaling mechanisms, with the exception of receptor-type adenylate cyclases (AC that are topologically similar to receptor-type guanylate cyclase (GC of higher eukaryotes but control a new class of cAMP targets of unknown function, the cAMP response proteins (CARPs, rather than the classical protein kinase A cAMP effector (PKA. T. brucei possesses a large polymorphic family of ACs, mainly associated with the flagellar membrane, and these are involved in inhibition of the innate immune response of the host prior to the massive release of immunomodulatory factors at the first peak of parasitemia. Recent evidence suggests that in T. brucei several insect-specific AC isoforms are involved in social motility, whereas only a few AC isoforms are involved in cytokinesis control of bloodstream forms, attesting that a complex signaling pathway is required for environmental sensing. In this review, after a general update on cAMP signaling pathway and the multiple roles of cAMP, I summarize the existing knowledge of the mechanisms by which pathogenic microorganisms modulate cAMP levels to escape immune defense.

  11. The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Sekiya, M.; Frohlich, E.D.; Cole, F.E. (Alton Ochsner Medical Foundation, New Orleans, LA (USA))

    1991-01-01

    In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

  12. Molecular determinants of Guanylate Cyclase Activating Protein subcellular distribution in photoreceptor cells of the retina.

    Science.gov (United States)

    López-Begines, Santiago; Plana-Bonamaisó, Anna; Méndez, Ana

    2018-02-13

    Retinal guanylate cyclase (RetGC) and guanylate cyclase activating proteins (GCAPs) play an important role during the light response in photoreceptor cells. Mutations in these proteins are linked to distinct forms of blindness. RetGC and GCAPs exert their role at the ciliary outer segment where phototransduction takes place. We investigated the mechanisms governing GCAP1 and GCAP2 distribution to rod outer segments by expressing selected GCAP1 and GCAP2 mutants as transient transgenes in the rods of GCAP1/2 double knockout mice. We show that precluding GCAP1 direct binding to RetGC (K23D/GCAP1) prevented its distribution to rod outer segments, while preventing GCAP1 activation of RetGC post-binding (W94A/GCAP1) did not. We infer that GCAP1 translocation to the outer segment strongly depends on GCAP1 binding affinity for RetGC, which points to GCAP1 requirement to bind to RetGC to be transported. We gain further insight into the distinctive regulatory steps of GCAP2 distribution, by showing that a phosphomimic at position 201 is sufficient to retain GCAP2 at proximal compartments; and that the bovine equivalent to blindness-causative mutation G157R/GCAP2 results in enhanced phosphorylation in vitro and significant retention at the inner segment in vivo, as likely contributing factors to the pathophysiology.

  13. Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture

    International Nuclear Information System (INIS)

    Cronin, M.J.; Evans, W.S.; Rogol, A.D.; Weiss, A.A.; Thorner, M.O.; Orth, D.N.; Nicholson, W.E.; Yasumoto, T.; Hewlett, E.L.

    1986-01-01

    Bordetella pertussis synthesis a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changes the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplified the secretory response to hypophysiotrophic hormones. The authors conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is the response that explains the subsequent acceleration of hormone release

  14. YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan (China); Tang, Ming-Chi [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Kuo, Liang-Mou [Department of General Surgery, Chang Gung Memorial Hospital at Chia-Yi, Taiwan (China); Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China)

    2012-04-15

    Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation.

  15. Issues concerning the determination of solubility products of sparingly soluble crystalline solids. Solubility of HfO{sub 2}(cr)

    Energy Technology Data Exchange (ETDEWEB)

    Rai, Dhanpat [Rai Enviro-Chem, LLC, Yachats, OR (United States); Kitamura, Akira [Japan Atomic Energy Agency, Ibaraki (Japan); Rosso, Kevin M. [Pacific Northwest National Laboratory, Richland, WA (United States); Sasaki, Takayuki; Kobayashi, Taishi [Kyoto Univ. (Japan)

    2016-11-01

    Solubility studies were conducted with HfO{sub 2}(cr) solid as a function HCl and ionic strength ranging from 2.0 to 0.004 mol kg{sup -1}. These studies involved (1) using two different amounts of the solid phase, (2) acid washing the bulk solid phase, (3) preheating the solid phase to 1400 C, and (4) heating amorphous HfO{sub 2}(am) suspensions to 90 C to ascertain whether the HfO{sub 2}(am) converts to HfO{sub 2}(cr) and to determine the solubility from the oversaturation direction. Based on the results of these treatments it is concluded that the HfO{sub 2}(cr) contains a small fraction of less crystalline, but not amorphous, material [HfO{sub 2}(lcr)] and this, rather than the HfO{sub 2}(cr), is the solubility-controlling phase in the range of experimental variables investigated in this study. The solubility data are interpreted using both the Pitzer and SIT models and they provide log{sub 10} K{sup 0} values of -(59.75±0.35) and -(59.48±0.41), respectively, for the solubility product of HfO{sub 2}(lcr)[HfO{sub 2}(lcr) + 2H{sub 2}O ↔ Hf{sup 4+} + 4OH{sup -}]. The log{sub 10} of the solubility product of HfO{sub 2}(cr) is estimated to be < -63. The observation of a small fraction of less crystalline higher solubility material is consistent with the general picture that mineral surfaces are often structurally and/or compositionally imperfect leading to a higher solubility than the bulk crystalline solid. This study stresses the urgent need, during interpretation of solubility data, of taking precautions to make certain that the observed solubility behavior for sparingly-soluble solids is assigned to the proper solid phase.

  16. Mice Overexpressing Type 1 Adenylyl Cyclase Show Enhanced Spatial Memory Flexibility in the Absence of Intact Synaptic Long-Term Depression

    Science.gov (United States)

    Zhang, Ming; Wang, Hongbing

    2013-01-01

    There is significant interest in understanding the contribution of intracellular signaling and synaptic substrates to memory flexibility, which involves new learning and suppression of obsolete memory. Here, we report that enhancement of Ca[superscript 2+]-stimulated cAMP signaling by overexpressing type 1 adenylyl cyclase (AC1) facilitated…

  17. High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

    Science.gov (United States)

    Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

    2014-01-17

    The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

  18. Effect of different forms of adenylate cyclase toxin of Bordetella pertussis on protection afforded by an acellular pertussis vaccine in a murine model.

    Science.gov (United States)

    Cheung, Gordon Y C; Xing, Dorothy; Prior, Sandra; Corbel, Michael J; Parton, Roger; Coote, John G

    2006-12-01

    Four recombinant forms of the cell-invasive adenylate cyclase toxin (CyaA) of Bordetella pertussis were compared for the ability to enhance protection against B. pertussis in mice when coadministered with an acellular pertussis vaccine (ACV). The four forms were as follows: fully functional CyaA, a CyaA form lacking adenylate cyclase enzymatic activity (CyaA*), and the nonacylated forms of these toxins, i.e., proCyaA and proCyaA*, respectively. None of these forms alone conferred significant (P > 0.05) protection against B. pertussis in a murine intranasal challenge model. Mice immunized with ACV alone showed significant (P protection was only significant (P protection provided by CyaA* was due to an augmentation of both Th1 and Th2 immune responses to B. pertussis antigens.

  19. Gas solubilities widespread applications

    CERN Document Server

    Gerrard, William

    1980-01-01

    Gas Solubilities: Widespread Applications discusses several topics concerning the various applications of gas solubilities. The first chapter of the book reviews Henr's law, while the second chapter covers the effect of temperature on gas solubility. The third chapter discusses the various gases used by Horiuti, and the following chapters evaluate the data on sulfur dioxide, chlorine data, and solubility data for hydrogen sulfide. Chapter 7 concerns itself with solubility of radon, thoron, and actinon. Chapter 8 tackles the solubilities of diborane and the gaseous hydrides of groups IV, V, and

  20. Phenylalanine 445 within oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae influences C-Ring cyclization and deprotonation reactions.

    Science.gov (United States)

    Wu, Tung-Kung; Liu, Yuan-Ting; Chiu, Feng-Hsuan; Chang, Cheng-Hsiang

    2006-10-12

    [reaction: see text] We describe the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase Phe445 site-saturated mutants that generate truncated tricyclic and altered deprotonation product profiles. Among these mutants, only polar side-chain group substitutions genetically complemented yeast viability and produced spatially related product diversity, supporting the Johnson model that cation-pi interactions between a carbocationic intermediate and an enzyme can be replaced by an electrostatic or polar side chain to stabilize the cationic intermediate, but with product differentiation.

  1. Pituitary adenylate cyclase-activating polypeptide: occurrence and relaxant effect in female genital tract

    DEFF Research Database (Denmark)

    Steenstrup, B R; Alm, P; Hannibal, J

    1995-01-01

    The distribution, localization, and smooth muscle effects of pituitary adenylate cyclase-activating polypeptide (PACAP) were studied in the human female genital tract. The concentrations of PACAP-38 and PACAP-27 were measured by radioimmunoassays, and both peptides were found throughout the genital...... was observed. The findings suggest a smooth muscle regulatory role of PACAP in the human female reproductive tract....... tract. The highest concentrations of PACAP-38 were detected in the ovary, the upper part of vagina, and the perineum. The concentrations of PACAP-27 were generally low, in some regions below the detection limit and in other regions 1 to 5% of the PACAP-38 concentrations. Immunocytochemistry revealed...

  2. Lycopene cyclase paralog CruP protects against reactive oxygen species in oxygenic photosynthetic organisms.

    Science.gov (United States)

    Bradbury, Louis M T; Shumskaya, Maria; Tzfadia, Oren; Wu, Shi-Biao; Kennelly, Edward J; Wurtzel, Eleanore T

    2012-07-03

    In photosynthetic organisms, carotenoids serve essential roles in photosynthesis and photoprotection. A previous report designated CruP as a secondary lycopene cyclase involved in carotenoid biosynthesis [Maresca J, et al. (2007) Proc Natl Acad Sci USA 104:11784-11789]. However, we found that cruP KO or cruP overexpression plants do not exhibit correspondingly reduced or increased production of cyclized carotenoids, which would be expected if CruP was a lycopene cyclase. Instead, we show that CruP aids in preventing accumulation of reactive oxygen species (ROS), thereby reducing accumulation of β-carotene-5,6-epoxide, a ROS-catalyzed autoxidation product, and inhibiting accumulation of anthocyanins, which are known chemical indicators of ROS. Plants with a nonfunctional cruP accumulate substantially higher levels of ROS and β-carotene-5,6-epoxide in green tissues. Plants overexpressing cruP show reduced levels of ROS, β-carotene-5,6-epoxide, and anthocyanins. The observed up-regulation of cruP transcripts under photoinhibitory and lipid peroxidation-inducing conditions, such as high light stress, cold stress, anoxia, and low levels of CO(2), fits with a role for CruP in mitigating the effects of ROS. Phylogenetic distribution of CruP in prokaryotes showed that the gene is only present in cyanobacteria that live in habitats characterized by large variation in temperature and inorganic carbon availability. Therefore, CruP represents a unique target for developing resilient plants and algae needed to supply food and biofuels in the face of global climate change.

  3. Pituitary adenylate cyclase activating polypeptide induces vascular relaxation and inhibits non-vascular smooth muscle activity in the rabbit female genital tract

    DEFF Research Database (Denmark)

    Steenstrup, B R; Ottesen, B; Jørgensen, M

    1994-01-01

    In vitro effects of two bioactive forms of pituitary adenylate cyclase activating polypeptide (PACAP): PACAP-38 and PACAP-27 were studied on rabbit vascular and non-vascular smooth muscle. Segments of the ovarian artery and muscle strips from the fallopian tube were used. Two series of experiment...

  4. Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Česnek, Michal; Jansa, Petr; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Brust, T. F.; Pávek, P.; Trejtnar, F.; Watts, V. J.; Janeba, Zlatko

    2015-01-01

    Roč. 10, č. 8 (2015), s. 1351-1364 ISSN 1860-7179 R&D Projects: GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : adenylate cyclase toxin * bisamidates * Bordetella pertussis * nucleosides * phosphonates Subject RIV: CC - Organic Chemistry Impact factor: 2.980, year: 2015

  5. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the circulation after sumatriptan

    DEFF Research Database (Denmark)

    Hansen, Jakob Møller; Fahrenkrug, Jan; Petersen, Jesper Troensegaard

    2013-01-01

    The origin of migraine pain is still elusive, but increasingly researchers focus on the neuropeptides in the perivascular space of cranial vessels as important mediators of nociceptive input during migraine attacks. The parasympathetic neurotransmitters, pituitary adenylate cyclase activating...... peptide-38 (PACAP38) and vasoactive intestinal peptide (VIP) may be released from parasympathetic fibres and activate sensory nerve fibres during migraine attacks. Triptans are effective and well tolerated in acute migraine management but the exact mechanism of action is still debated. Triptans might...

  6. Activity of adenylate cyclase in plasma membranes of pulmonary tissue remote times following nonlethal gamma-irradiation of rats

    International Nuclear Information System (INIS)

    Slozhenkina, L.V.; Ruda, V.P.; Ushakova, T.E.; Kuzin, A.M.

    1990-01-01

    Basal and stimulated activity of adenylate cyclase (cyclizing ATP-pyrophosphate lyase, E.C. 4.6.1.1., AC) in plasma membranes of pumonary tissye was being studied during a year after fractionated irradiation of rats (2 Gyx3). Basal and hormone-stimulated activity of AC was shown to vary significantly from normal 6 and 12 months after irradiation. The exposed membranes responded differently to AC activation by isoproterenol and F -

  7. Evidence for a possible role for nitric oxide in the modulation of heart activity in Achatina fulica and Helix aspersa.

    Science.gov (United States)

    White, A R; Curtis, S A; Walker, R J

    2004-02-01

    The effects of nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine, S-nitroso-l-glutathione, sodium nitroprusside and sodium nitrite were investigated on the activity of the isolated hearts of Achatina fulica and Helix aspersa. NO donors inhibited heart activity in a concentration-dependent manner. The only exception was sodium nitroprusside, which excited H. aspersa heart. The inhibitory effects of these NO donors were reduced by the NO scavenger, methylene blue, the guanylyl cyclase inhibitor, 1H-(1,2,4) Oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), and potentiated by 8-Br-cGMP and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Acetylcholine also inhibited the heart activity, and this inhibition was reduced by methylene blue and ODQ. Positive NADPH-diaphorase staining was located in the outer pericardial layer of the heart of A. fulica. The present results provide evidence that NO may modulate the activity of gastropod hearts, and this modulation may modify the inhibitory action of acetylcholine on heart activity.

  8. BAY 41-2272 Treatment Improves Acetylcholine-Induced Aortic Relaxation in L-NAME Hypertensive Rats.

    Science.gov (United States)

    Prawez, Shahid; Ahanger, Azad Ahmad; Singh, Thakur Uttam; Mishra, Santosh Kumar; Sarkar, Souvendra Nath; Kumar, Dinesh

    2016-12-01

    Hypertension, an emerging problem of recent era, and many pathophysiological factors are participating to produce the disease. Nitric oxide (NO) is an important constituent to ameliorate hypertensive condition. Inhibition of endogenous NO synthase by L-N G -Nitroarginine methyl ester (L-NAME) was responsible for generating hypertension in rats. BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine), a soluble guanylyl cyclase activator, restricts rise of blood pressure and shows cardioprotective activity. The aim of the present study was to analyze effect of short-term BAY 41-2272 treatment on blood pressure and vascular function. Male Wistar rats were randomly divided into three groups such as control (group-A), hypertensive (group-B), and BAY 41-2272-treated hypertensive (group-C) rats. Normal saline was administered intramuscularly to control rats for last 3 days (days 40, 41, and 42) of total 42 days treatment, whereas rats of group-B and group-C were treated with L-NAME hydrochloride in drinking water at 50 mg/kg body weight daily for 42 days. Also, normal saline and BAY 41-2272 were administered for last 3 days at two different dosages at 1 and 3 mg/kg body weight/day intramuscularly to group-B and group-C rats, respectively. Administration of BAY 41-2272 for 3 days was not sufficient enough to decrease mean arterial pressure of hypertensive rats significantly. BAY at both the treatment dosages significantly ameliorate acetylcholine-induced maximal aortic relaxation compared with BAY-untreated hypertensive rats. Findings of the present study indicate that even shorter period of BAY 41-2272 treatment (3 days) improves vascular relaxation.

  9. Nitric oxide donors enhance the frequency dependence of dopamine release in nucleus accumbens.

    Science.gov (United States)

    Hartung, Henrike; Threlfell, Sarah; Cragg, Stephanie J

    2011-08-01

    Dopamine (DA) neurotransmission in the nucleus accumbens (NAc) is critically involved in normal as well as maladaptive motivated behaviors including drug addiction. Whether the striatal neuromodulator nitric oxide (NO) influences DA release in NAc is unknown. We investigated whether exogenous NO modulates DA transmission in NAc core and how this interaction varies depending on the frequency of presynaptic activation. We detected DA with cyclic voltammetry at carbon-fiber microelectrodes in mouse NAc in slices following stimuli spanning a full range of DA neuron firing frequencies (1-100 Hz). NO donors 3-morpholinosydnonimine hydrochloride (SIN-1) or z-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate (PAPA/NONOate) enhanced DA release with increasing stimulus frequency. This NO-mediated enhancement of frequency sensitivity of DA release was not prevented by inhibition of soluble guanylyl cyclase (sGC), DA transporters, or large conductance Ca(2+)-activated K(+) channels, and did not require glutamatergic or GABAergic input. However, experiments to identify whether frequency-dependent NO effects were mediated via changes in powerful acetylcholine-DA interactions revealed multiple components to NO modulation of DA release. In the presence of a nicotinic receptor antagonist (dihydro-β-erythroidine), NO donors increased DA release in a frequency-independent manner. These data suggest that NO in the NAc can modulate DA release through multiple GC-independent neuronal mechanisms whose net outcome varies depending on the activity in DA neurons and accumbal cholinergic interneurons. In the presence of accumbal acetylcholine, NO promotes the sensitivity of DA release to presynaptic activation, but with reduced acetylcholine input, NO will promote DA release in an activity-independent manner through a direct action on dopaminergic terminals.

  10. Role of the NO/sGC/PKG signaling pathway of hippocampal CA1 in morphine-induced reward memory.

    Science.gov (United States)

    Shen, Fang; Li, Yi-Jing; Shou, Xiao-Jing; Cui, Cai-Lian

    2012-09-01

    Evidence suggests that the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP dependent protein kinase (PKG) signaling pathway plays a key role in memory processing, but the actual participation of this signaling cascade in the hippocampal CA1 during morphine-induced reward memory remains unknown. In this study, we investigated the role of the NO/sGC/PKG signaling pathway in the CA1 on morphine-induced reward memory using a conditioned place preference (CPP) paradigm. We found that rats receiving an intraperitoneal (i.p.) injection of 4mg/kg morphine exhibited CPP, whereas rats treated with only 0.2mg/kg morphine failed to produce CPP. Intra-CA1 injection of the neuronal NO synthase (nNOS) inhibitor 7-NI, the sGC inhibitor ODQ or the PKG inhibitor Rp-8-Br-PET-cGMPS had no effect on the acquisition of CPP by 4mg/kg morphine. Intra-CA1 injection of 7-NI blocked the consolidation of CPP induced by 4mg/kg morphine, and this amnesic effect of 7-NI was mimicked by ODQ and Rp-8-Br-PET-cGMPS. Intra-CA1 injection of the NOS substrate L-arg or the sGC activator YC-1 with an ineffective dose of morphine (0.2mg/kg, i.p.) elicited CPP. This response induced by L-arg or YC-1 was reversed by pre-microinjection of Rp-8-Br-PET-cGMPS in the CA1. These results indicated that the activation of the NO/sGC/PKG signaling pathway in the CA1 is necessary for the consolidation of morphine-related reward memory. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Organic Nitrate Therapy, Nitrate Tolerance, and Nitrate-Induced Endothelial Dysfunction: Emphasis on Redox Biology and Oxidative Stress.

    Science.gov (United States)

    Daiber, Andreas; Münzel, Thomas

    2015-10-10

    Organic nitrates, such as nitroglycerin (GTN), isosorbide-5-mononitrate and isosorbide dinitrate, and pentaerithrityl tetranitrate (PETN), when given acutely, have potent vasodilator effects improving symptoms in patients with acute and chronic congestive heart failure, stable coronary artery disease, acute coronary syndromes, or arterial hypertension. The mechanisms underlying vasodilation include the release of •NO or a related compound in response to intracellular bioactivation (for GTN, the mitochondrial aldehyde dehydrogenase [ALDH-2]) and activation of the enzyme, soluble guanylyl cyclase. Increasing cyclic guanosine-3',-5'-monophosphate (cGMP) levels lead to an activation of the cGMP-dependent kinase I, thereby causing the relaxation of the vascular smooth muscle by decreasing intracellular calcium concentrations. The hemodynamic and anti-ischemic effects of organic nitrates are rapidly lost upon long-term (low-dose) administration due to the rapid development of tolerance and endothelial dysfunction, which is in most cases linked to increased intracellular oxidative stress. Enzymatic sources of reactive oxygen species under nitrate therapy include mitochondria, NADPH oxidases, and an uncoupled •NO synthase. Acute high-dose challenges with organic nitrates cause a similar loss of potency (tachyphylaxis), but with distinct pathomechanism. The differences among organic nitrates are highlighted regarding their potency to induce oxidative stress and subsequent tolerance and endothelial dysfunction. We also address pleiotropic effects of organic nitrates, for example, their capacity to stimulate antioxidant pathways like those demonstrated for PETN, all of which may prevent adverse effects in response to long-term therapy. Based on these considerations, we will discuss and present some preclinical data on how the nitrate of the future should be designed.

  12. Nitric oxide in the nervous system: biochemical, developmental, and neurobiological aspects.

    Science.gov (United States)

    Cossenza, Marcelo; Socodato, Renato; Portugal, Camila C; Domith, Ivan C L; Gladulich, Luis F H; Encarnação, Thaísa G; Calaza, Karin C; Mendonça, Henrique R; Campello-Costa, Paula; Paes-de-Carvalho, Roberto

    2014-01-01

    Nitric oxide (NO) is a very reactive molecule, and its short half-life would make it virtually invisible until its discovery. NO activates soluble guanylyl cyclase (sGC), increasing 3',5'-cyclic guanosine monophosphate levels to activate PKGs. Although NO triggers several phosphorylation cascades due to its ability to react with Fe II in heme-containing proteins such as sGC, it also promotes a selective posttranslational modification in cysteine residues by S-nitrosylation, impacting on protein function, stability, and allocation. In the central nervous system (CNS), NO synthesis usually requires a functional coupling of nitric oxide synthase I (NOS I) and proteins such as NMDA receptors or carboxyl-terminal PDZ ligand of NOS (CAPON), which is critical for specificity and triggering of selected pathways. NO also modulates CREB (cAMP-responsive element-binding protein), ERK, AKT, and Src, with important implications for nerve cell survival and differentiation. Differences in the regulation of neuronal death or survival by NO may be explained by several mechanisms involving localization of NOS isoforms, amount of NO being produced or protein sets being modulated. A number of studies show that NO regulates neurotransmitter release and different aspects of synaptic dynamics, such as differentiation of synaptic specializations, microtubule dynamics, architecture of synaptic protein organization, and modulation of synaptic efficacy. NO has also been associated with synaptogenesis or synapse elimination, and it is required for long-term synaptic modifications taking place in axons or dendrites. In spite of tremendous advances in the knowledge of NO biological effects, a full description of its role in the CNS is far from being completely elucidated. © 2014 Elsevier Inc. All rights reserved.

  13. Pharmacological characterization of a novel phosphodiesterase type 5 (PDE5) inhibitor lodenafil carbonate on human and rabbit corpus cavernosum.

    Science.gov (United States)

    Toque, Haroldo A; Teixeira, Cleber E; Lorenzetti, Raquel; Okuyama, Cristina E; Antunes, Edson; De Nucci, Gilberto

    2008-09-04

    Nitrergic nerves and endothelial cells release nitric oxide (NO) in the corpus cavernosum, a key mediator that stimulates soluble guanylyl cyclase to increase cGMP levels causing penile erection. Phosphodiesterase 5 (PDE5) inhibitors, such as sildenafil, prolong the NO effects by inhibiting cGMP breakdown. Here, we report a novel PDE5 inhibitor, lodenafil carbonate, (Bis-(2-{4-[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-benzenesulfonyl]piperazin-1-yl}-ethyl)carbonate) that is a dimer of lodenafil. We therefore aimed to compare the effects of sildenafil, lodenafil and lodenafil carbonate on in vitro human and rabbit cavernosal relaxations, activity of crude PDE extracts from human platelets, as well as stability and metabolic studies in rat, dog and human plasma. Pharmacokinetic evaluations after intravenous and oral administration were performed in male beagles. Functional experiments were conducted using organ bath techniques. Pharmacokinetics was studied in beagles by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), following oral or intravascular administration. All PDE5 inhibitors tested concentration-dependently relaxed (0.001-100 microM) phenylephrine-precontracted rabbit and human corpus cavernosum. The cavernosal relaxations evoked by either acetylcholine (0.01-100 microM) or electrical field stimulation (EFS, 1-20 Hz) were markedly potentiated by sildenafil, lodenafil and lodenafil carbonate. Lodenafil carbonate was more potent to inhibit the cGMP hydrolysis in PDE extracts compared with lodenafil and sildenafil. Following intravascular and single oral administration of lodenafil carbonate, only lodenafil and norlodenafil were detected in vivo. These results indicate that lodenafil carbonate works as a prodrug, being lodenafil the active moiety of lodenafil carbonate.

  14. Signaling through cGMP-dependent protein kinase I in the amygdala is critical for auditory-cued fear memory and long-term potentiation.

    Science.gov (United States)

    Paul, Cindy; Schöberl, Florian; Weinmeister, Pascal; Micale, Vincenzo; Wotjak, Carsten T; Hofmann, Franz; Kleppisch, Thomas

    2008-12-24

    Long-term potentiation (LTP) of inputs relaying sensory information from cortical and thalamic neurons to principal neurons in the lateral amygdala (LA) is thought to serve as a cellular mechanism for associative fear learning. Nitric oxide (NO), a messenger molecule widely implicated in synaptic plasticity and behavior, has been shown to enhance LTP in the LA as well as consolidation of associative fear memory. Additional evidence suggests that NO-induced enhancement of LTP and amygdala-dependent learning requires signaling through soluble guanylyl cyclase (sGC) and cGMP-dependent protein kinase (cGK). Mammals possess two genes for cGK: the prkg1 gene gives rise to the cGK type I isoforms, cGKIalpha and cGKIbeta, and the prkg2 gene encodes the cGK type II. Reportedly, both cGKI and cGKII are expressed in the amygdala, and cGKII is involved in controlling anxiety-like behavior. Because selective pharmacological tools for individual cGK isoforms are lacking, we used different knock-out mouse models to examine the function of cGKI and cGKII for LTP in the LA and pavlovian fear conditioning. We found robust expression of the cGKI specifically in the LA with cGKIbeta as the prevailing isoform. We further show a marked reduction of LTP at both thalamic and cortical inputs to the LA and a selective impairment of auditory-cued fear memory in cGKI-deficient mutants. In contrast, cGKII null mutants lack these phenotypes. Our data suggest a function of cGKI, likely the beta isoform, in the LA, supporting synaptic plasticity and consolidation of fear memory.

  15. Organic Nitrate Therapy, Nitrate Tolerance, and Nitrate-Induced Endothelial Dysfunction: Emphasis on Redox Biology and Oxidative Stress

    Science.gov (United States)

    2015-01-01

    Abstract Organic nitrates, such as nitroglycerin (GTN), isosorbide-5-mononitrate and isosorbide dinitrate, and pentaerithrityl tetranitrate (PETN), when given acutely, have potent vasodilator effects improving symptoms in patients with acute and chronic congestive heart failure, stable coronary artery disease, acute coronary syndromes, or arterial hypertension. The mechanisms underlying vasodilation include the release of •NO or a related compound in response to intracellular bioactivation (for GTN, the mitochondrial aldehyde dehydrogenase [ALDH-2]) and activation of the enzyme, soluble guanylyl cyclase. Increasing cyclic guanosine-3′,-5′-monophosphate (cGMP) levels lead to an activation of the cGMP-dependent kinase I, thereby causing the relaxation of the vascular smooth muscle by decreasing intracellular calcium concentrations. The hemodynamic and anti-ischemic effects of organic nitrates are rapidly lost upon long-term (low-dose) administration due to the rapid development of tolerance and endothelial dysfunction, which is in most cases linked to increased intracellular oxidative stress. Enzymatic sources of reactive oxygen species under nitrate therapy include mitochondria, NADPH oxidases, and an uncoupled •NO synthase. Acute high-dose challenges with organic nitrates cause a similar loss of potency (tachyphylaxis), but with distinct pathomechanism. The differences among organic nitrates are highlighted regarding their potency to induce oxidative stress and subsequent tolerance and endothelial dysfunction. We also address pleiotropic effects of organic nitrates, for example, their capacity to stimulate antioxidant pathways like those demonstrated for PETN, all of which may prevent adverse effects in response to long-term therapy. Based on these considerations, we will discuss and present some preclinical data on how the nitrate of the future should be designed. Antioxid. Redox Signal. 23, 899–942. PMID:26261901

  16. Disease: H01396 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available pha1beta1 soluble guanylate cyclase, the major nitric oxide receptor, leads to moyamoya and achalasia. ... JOURNAL ... Am J Hum Genet 94:385-94 (2014) DOI:10.1016/j.ajhg.2014.01.018

  17. Neptunium (IV) oxalate solubility

    International Nuclear Information System (INIS)

    Luerkens, D.W.

    1983-07-01

    The equilibrium solubility of neptunium (IV) oxalate in nitric/oxalic acid solutions was determined at 22 0 C, 45 0 C, and 60 0 C. The concentrations of nitric/oxalic acid solutions represented a wide range of free oxalate ion concentration. A mathematical solubility model was developed which is based on the formation of the known complexes of neptunium (IV) oxalate. the solubility model uses a simplified concentration parameter which is proportional to the free oxalate ion concentration. The solubility model can be used to estimate the equilibrium solubility of neptunium (IV) oxalate over a wide range of oxalic and nitric acid concentrations at each temperature

  18. Investigation of cAMP microdomains as a path to novel cancer diagnostics.

    Science.gov (United States)

    Desman, Garrett; Waintraub, Caren; Zippin, Jonathan H

    2014-12-01

    Understanding of cAMP signaling has greatly improved over the past decade. The advent of live cell imaging techniques and more specific pharmacologic modulators has led to an improved understanding of the intricacies by which cAMP is able to modulate such a wide variety of cellular pathways. It is now appreciated that cAMP is able to activate multiple effector proteins at distinct areas in the cell leading to the activation of very different downstream targets. The investigation of signaling proteins in cancer is a common route to the development of diagnostic tools, prognostic tools, and/or therapeutic targets, and in this review we highlight how investigation of cAMP signaling microdomains driven by the soluble adenylyl cyclase in different cancers has led to the development of a novel cancer biomarker. Antibodies directed against the soluble adenylyl cyclase (sAC) are highly specific markers for melanoma especially for lentigo maligna melanoma and are being described as "second generation" cancer diagnostics, which are diagnostics that determine the 'state' of a cell and not just identify the cell type. Due to the wide presence of cAMP signaling pathways in cancer, we predict that further investigation of both sAC and other cAMP microdomains will lead to additional cancer biomarkers. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Uranium solubility and solubility controls in selected Needle's Eye groundwaters

    International Nuclear Information System (INIS)

    Falck, W.E.; Hooker, P.J.

    1991-01-01

    The solubility control of uranium in selected groundwater samples from the cliff and sediments at the Needle's Eye natural analogue site is investigated using the speciation code PHREEQE and the CHEMVAL thermodynamic database (release 3). Alkali-earth bearing uranyl carbonate secondary minerals are likely to exert influence on the solubility . Other candidates are UO 2 and arsenates, depending on the prevailing redox conditions. In the absence of literature data, solubility products for important arsenates have been estimated from analogy with other arsenates and phosphates. Phosphates themselves are unlikely to exert control owing to their comparatively high solubilities. The influence of seawater flooding into the sediments is also discussed. The importance of uranyl arsenates in the retardation of uranium in shallow sediments has been demonstrated in theory, but there are some significant gaps in the thermodynamic databases used. (author)

  20. Thyroid-stimulating immunoglobulins in Hashimoto's thyroiditis measured by radioreceptor assay and adenylate cyclase stimulation and their relationship to HLA-D alleles

    International Nuclear Information System (INIS)

    Bliddal, H.; Bech, K.; Feldt-Rasmussen, U.; Thomsen, M.; Ryder, L.P.; Hansen, J.M.; Siersbaek-Nielsen, K.; Friis, T.

    1982-01-01

    The relationship between thyroid-stimulating immunoglobulins, measured by both radioreceptor assay and adenylate cyclase stimulation, and the HLA alleles was studied in 41 patients with Hashimoto's thyroiditis. TSH binding-inhibiting immunoglobulins (TBII) were detected in 9 (22%) patients, and human thyroid adenylate cyclase-stimulating immunoglobulins (HTACS) were found in 21 (51%) patients. Only 2 patients were positive in both assays, and an inverse relationship was observed between TBII and HTACS. In the 21 HTACS-positive patients, HLA-Dw5 was found in 1 subject, compared to 8 of the 20 HTACS-negative patients (P < 0.01), while 4 of the 9 TBII-positive patients had HLA-Dw5 compared to 5 of the 32 TBII-negative subjects (P = 0.09).No significant relations were observed between the presence of HTACS or TBII and HLA-Dw3 or HLA-B8. It is concluded that TBII and HTACS are produced independently in Hashimoto's thyroiditis, and that the production of these autoantibodies seems to be related to the HLA-D region in this disease

  1. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.; Kwezi, Lusisizwe; Wheeler, Janet I.; Gehring, Christoph A

    2012-01-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  2. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.

    2012-02-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  3. Effect of cardiopulmonary bypass on beta adrenergic receptor-adenylate cyclase system on surfaces of peripheral lymphocytes.

    Science.gov (United States)

    Luo, A; Tian, Y; Jin, S

    2000-01-01

    The experimental results showed that the level of CAMP, the ratio of cAPM to cGMP, IL-2R expression and IL-2 production in vitro in lymphocytes immediate and 2 weeks after cardiopulmonary bypass (CPB) were significantly lower than those before anesthetics in the patients undergoing cardiac surgery with CPB. These findings suggested that CPB could cause serious damage to adrenergic beta receptor-adenylate cyclase system on circulating lymphocytes surfaces, which might be one of the mechanisms resulting in immunosuppression after open heart surgery with CPB.

  4. Cyclic guanosine monophosphate in the regulation of the cell function

    Directory of Open Access Journals (Sweden)

    Małgorzata Zbrojkiewicz

    2016-12-01

    Full Text Available Intracellular concentration of cGMP depends on the activity of guanylate cyclase, responsible for its synthesis, on the activity of cyclic nucleotide degrading enzymes - phosphodiesterases (PDEs. There are two forms of guanylate cyclase: the membrane-bound cyclase and the soluble form. The physiological activators of the membrane guanylate cyclase are natriuretic peptides (NPs, and of the cytosolic guanylate cyclase - nitric oxide (NO and carbon monoxide (CO. Intracellular cGMP signaling pathways arise from its direct effect on the activity of G protein kinases, phosphodiesterases and cyclic nucleotide dependent cation channels. It has been shown in recent years that cGMP can also affect other signal pathways in cell signaling activity involving Wnt proteins and sex hormones. The increased interest in the research on the role of cGMP, resulted also in the discovery of its role in the regulation of phototransduction in the eye, neurotransmission, calcium homeostasis, platelet aggregation, heartbeat, bone remodeling, lipid metabolism and the activity of the cation channels. Better understanding of the mechanisms of action of cGMP in the regulation of cell function can create new opportunities for the cGMP affecting drugs use in the pharmacotherapy.

  5. Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

    Penzkofer, A., E-mail: alfons.penzkofer@physik.uni-regensburg.de [Fakultät für Physik, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg (Germany); Tanwar, M.; Veetil, S.K.; Kateriya, S. [Department of Biochemistry, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021 (India); Stierl, M.; Hegemann, P. [Institut für Biologie/Experimentelle Biophysik, Humboldt Universität zu Berlin, Invalidenstrasse 42, D-10115 Berlin (Germany)

    2013-09-23

    Highlights: • Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity. • Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2. • Photo-induced Tyr–Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2. • Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2. • Two NgPAC2 conformations with fast and slow photo-induced electron transfer. - Abstract: The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction.

  6. Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

    International Nuclear Information System (INIS)

    Penzkofer, A.; Tanwar, M.; Veetil, S.K.; Kateriya, S.; Stierl, M.; Hegemann, P.

    2013-01-01

    Highlights: • Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity. • Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2. • Photo-induced Tyr–Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2. • Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2. • Two NgPAC2 conformations with fast and slow photo-induced electron transfer. - Abstract: The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr–Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction

  7. Hydrogen solubility measurements of analyzed tall oil fractions and a solubility model

    International Nuclear Information System (INIS)

    Uusi-Kyyny, Petri; Pakkanen, Minna; Linnekoski, Juha; Alopaeus, Ville

    2017-01-01

    Highlights: • Hydrogen solubility was measured in four tall oil fractions between 373 and 597 K. • Continuous flow synthetic isothermal and isobaric method was used. • A Henry’s law model was developed for the distilled tall oil fractions. • The complex composition of the samples was analyzed and is presented. - Abstract: Knowledge of hydrogen solubility in tall oil fractions is important for designing hydrotreatment processes of these complex nonedible biobased materials. Unfortunately measurements of hydrogen solubility into these fractions are missing in the literature. This work reports hydrogen solubility measured in four tall oil fractions between 373 and 597 K and at pressures from 5 to 10 MPa. Three of the fractions were distilled tall oil fractions their resin acids contents are respectively 2, 20 and 23 in mass-%. Additionally one fraction was a crude tall oil (CTO) sample containing sterols as the main neutral fraction. Measurements were performed using a continuous flow synthetic isothermal and isobaric method based on the visual observation of the bubble point. Composition of the flow was changed step-wise for the bubble point composition determination. We assume that the tall oil fractions did not react during measurements, based on the composition analysis performed before and after the measurements. Additionally the densities of the fractions were measured at atmospheric pressure from 293.15 to 323.15 K. A Henry’s law model was developed for the distilled tall oil fractions describing the solubility with an absolute average deviation of 2.1%. Inputs of the solubility model are temperature, total pressure and the density of the oil at 323.15 K. The solubility of hydrogen in the CTO sample can be described with the developed model with an absolute average deviation of 3.4%. The solubility of hydrogen increases both with increasing pressure and/or increasing temperature. The more dense fractions of the tall oil exhibit lower hydrogen

  8. An adenylyl cyclase gene (NlAC9) influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

    Science.gov (United States)

    The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC) conversion of adenosine triphosphate (ATP) to 3', 5'-cyclic AMP (cAMP) and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in som...

  9. Pure Phase Solubility Limits: LANL

    International Nuclear Information System (INIS)

    C. Stockman

    2001-01-01

    The natural and engineered system at Yucca Mountain (YM) defines the site-specific conditions under which one must determine to what extent the engineered and the natural geochemical barriers will prevent the release of radioactive material from the repository. Most important mechanisms for retention or enhancement of radionuclide transport include precipitation or co-precipitation of radionuclide-bearing solid phases (solubility limits), complexation in solution, sorption onto surfaces, colloid formation, and diffusion. There may be many scenarios that could affect the near-field environment, creating chemical conditions more aggressive than the conditions presented by the unperturbed system (such as pH changes beyond the range of 6 to 9 or significant changes in the ionic strength of infiltrated waters). For an extended period of time, the near-field water composition may be quite different and more extreme in pH, ionic strength, and CO 2 partial pressure (or carbonate concentration) than waters at some distance from the repository. Reducing conditions, high pH (up to 11), and low carbonate concentration may be present in the near-field after reaction of infiltrating groundwater with engineered barrier systems, such as cementitious materials. In the far-field, conditions are controlled by the rock-mass buffer providing a near-neutral, oxidizing, low-ionic-strength environment that controls radionuclide solubility limits and sorption capacities. There is the need for characterization of variable chemical conditions that affect solubility, speciation, and sorption reactions. Modeling of the groundwater chemistry is required and leads to an understanding of solubility and speciation of the important radionuclides. Because experimental studies cannot be performed under the numerous potential chemical conditions, solubility limitations must rely on geochemical modeling of the radionuclide's chemistry. Fundamental thermodynamic properties, such as solubility products

  10. Pure Phase Solubility Limits: LANL

    Energy Technology Data Exchange (ETDEWEB)

    C. Stockman

    2001-01-26

    The natural and engineered system at Yucca Mountain (YM) defines the site-specific conditions under which one must determine to what extent the engineered and the natural geochemical barriers will prevent the release of radioactive material from the repository. Most important mechanisms for retention or enhancement of radionuclide transport include precipitation or co-precipitation of radionuclide-bearing solid phases (solubility limits), complexation in solution, sorption onto surfaces, colloid formation, and diffusion. There may be many scenarios that could affect the near-field environment, creating chemical conditions more aggressive than the conditions presented by the unperturbed system (such as pH changes beyond the range of 6 to 9 or significant changes in the ionic strength of infiltrated waters). For an extended period of time, the near-field water composition may be quite different and more extreme in pH, ionic strength, and CO{sub 2} partial pressure (or carbonate concentration) than waters at some distance from the repository. Reducing conditions, high pH (up to 11), and low carbonate concentration may be present in the near-field after reaction of infiltrating groundwater with engineered barrier systems, such as cementitious materials. In the far-field, conditions are controlled by the rock-mass buffer providing a near-neutral, oxidizing, low-ionic-strength environment that controls radionuclide solubility limits and sorption capacities. There is the need for characterization of variable chemical conditions that affect solubility, speciation, and sorption reactions. Modeling of the groundwater chemistry is required and leads to an understanding of solubility and speciation of the important radionuclides. Because experimental studies cannot be performed under the numerous potential chemical conditions, solubility limitations must rely on geochemical modeling of the radionuclide's chemistry. Fundamental thermodynamic properties, such as solubility

  11. Effect of cyclodextrin complexation on the aqueous solubility and solubility/dose ratio of praziquantel.

    Science.gov (United States)

    Maragos, Stratos; Archontaki, Helen; Macheras, Panos; Valsami, Georgia

    2009-01-01

    Praziquantel (PZQ), the primary drug of choice in the treatment of schistosomiasis, is a highly lipophilic drug that possesses high permeability and low aqueous solubility and is, therefore, classified as a Class II drug according to the Biopharmaceutics Classification System (BCS). In this work, beta-cyclodextrin (beta-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) were used in order to determine whether increasing the aqueous solubility of a drug by complexation with CDs, a BCS-Class II compound like PZQ could behave as BCS-Class I (highly soluble/highly permeable) drug. Phase solubility and the kneading and lyophilization techniques were used for inclusion complex preparation; solubility was determined by UV spectroscopy. The ability of the water soluble polymer polyvinylpyrolidone (PVP) to increase the complexation and solubilization efficiency of beta-CD and HP-beta-CD for PZQ was examined. Results showed significant improvement of PZQ solubility in the presence of both cyclodextrins but no additional effect in the presence of PVP. The solubility/dose ratios values of PZQ-cyclodextrin complexes calculated considering the low (150 mg) and the high dose (600 mg) of PZQ, used in practice, indicate that PZQ complexation with CDs may result in drug dosage forms that would behave as a BCS-Class I depending on the administered dose.

  12. Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants.

    Science.gov (United States)

    Kühnel, Lisa C; Nestl, Bettina M; Hauer, Bernhard

    2017-11-16

    Squalene-hopene cyclases (SHCs) catalyze the polycyclization of squalene into a mixture of hopene and hopanol. Recently, amino-acid residues lining the catalytic cavity of the SHC from Alicyclobacillus acidocaldarius were replaced by small and large hydrophobic amino acids. The alteration of leucine 607 to phenylalanine resulted in increased enzymatic activity towards the formation of an intermolecular farnesyl-farnesyl ether product from farnesol. Furthermore, the addition of small-chain alcohols acting as nucleophiles led to the formation of non-natural ether-linked terpenoids and, thus, to significant alteration of the product pattern relative to that obtained with the wild type. It is proposed that the mutation of leucine at position 607 may facilitate premature quenching of the intermediate by small alcohol nucleophiles. This mutagenesis-based study opens the field for further intermolecular bond-forming reactions and the generation of non-natural products. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Oral formulation strategies to improve solubility of poorly water-soluble drugs.

    Science.gov (United States)

    Singh, Abhishek; Worku, Zelalem Ayenew; Van den Mooter, Guy

    2011-10-01

    In the past two decades, there has been a spiraling increase in the complexity and specificity of drug-receptor targets. It is possible to design drugs for these diverse targets with advances in combinatorial chemistry and high throughput screening. Unfortunately, but not entirely unexpectedly, these advances have been accompanied by an increase in the structural complexity and a decrease in the solubility of the active pharmaceutical ingredient. Therefore, the importance of formulation strategies to improve the solubility of poorly water-soluble drugs is inevitable, thus making it crucial to understand and explore the recent trends. Drug delivery systems (DDS), such as solid dispersions, soluble complexes, self-emulsifying drug delivery systems (SEDDS), nanocrystals and mesoporous inorganic carriers, are discussed briefly in this review, along with examples of marketed products. This article provides the reader with a concise overview of currently relevant formulation strategies and proposes anticipated future trends. Today, the pharmaceutical industry has at its disposal a series of reliable and scalable formulation strategies for poorly soluble drugs. However, due to a lack of understanding of the basic physical chemistry behind these strategies, formulation development is still driven by trial and error.

  14. The solubility-permeability interplay and its implications in formulation design and development for poorly soluble drugs.

    Science.gov (United States)

    Dahan, Arik; Miller, Jonathan M

    2012-06-01

    While each of the two key parameters of oral drug absorption, the solubility and the permeability, has been comprehensively studied separately, the relationship and interplay between the two have been largely ignored. For instance, when formulating a low-solubility drug using various solubilization techniques: what are we doing to the apparent permeability when we increase the solubility? Permeability is equal to the drug's diffusion coefficient through the membrane times the membrane/aqueous partition coefficient divided by the membrane thickness. The direct correlation between the intestinal permeability and the membrane/aqueous partitioning, which in turn is dependent on the drug's apparent solubility in the GI milieu, suggests that the solubility and the permeability are closely associated, exhibiting a certain interplay between them, and the current view of treating the one irrespectively of the other may not be sufficient. In this paper, we describe the research that has been done thus far, and present new data, to shed light on this solubility-permeability interplay. It has been shown that decreased apparent permeability accompanies the solubility increase when using different solubilization methods. Overall, the weight of the evidence indicates that the solubility-permeability interplay cannot be ignored when using solubility-enabling formulations; looking solely at the solubility enhancement that the formulation enables may be misleading with regards to predicting the resulting absorption, and hence, the solubility-permeability interplay must be taken into account to strike the optimal solubility-permeability balance, in order to maximize the overall absorption.

  15. Physiological desensitization of carbohydrate permeases and adenylate cyclase to regulation by the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli and Salmonella typhimurium. Involvement of adenosine cyclic 3',5'-phosphate and inducer.

    Science.gov (United States)

    Saier, M H; Keeler, D K; Feucht, B U

    1982-03-10

    Adenylate cyclase and a number of carbohydrate transport systems are subject to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. These sensitive carbohydrate transport systems are desensitized to regulation by the phosphotransferase system, and adenylate cyclase is deactivated when cells are grown in medium containing cyclic AMP. These effects are specific for cyclic AMP and are potentiated by the genetic loss of cyclic AMP phosphodiesterase. Inclusion in the growth medium of an inducer of a sensitive transport system also promotes desensitization of that particular transport system. Inducer-promoted desensitization is specific for the particular target transport system, while cyclic AMP-promoted desensitization is general and affects several systems. Desensitization of the permeases to regulation, and inactivation of adenylate cyclase, are slow processes which are blocked by chloramphenicol and are therefore presumably dependent on protein synthesis. Several sugar substrates of the phosphotransferase system are capable of regulating the sensitive carbohydrate transport systems. The evidence suggests that desensitization to this regulation does not result from a direct effect on the functioning of Enzyme I, a small heat-stable protein of the phosphotransferase system, HPr, or an Enzyme II of the phosphotransferase system, but specifically uncouples the permease systems from regulation.

  16. Head-To-Head Comparison of Different Solubility-Enabling Formulations of Etoposide and Their Consequent Solubility-Permeability Interplay.

    Science.gov (United States)

    Beig, Avital; Miller, Jonathan M; Lindley, David; Carr, Robert A; Zocharski, Philip; Agbaria, Riad; Dahan, Arik

    2015-09-01

    The purpose of this study was to conduct a head-to-head comparison of different solubility-enabling formulations, and their consequent solubility-permeability interplay. The low-solubility anticancer drug etoposide was formulated in several strengths of four solubility-enabling formulations: hydroxypropyl-β-cyclodextrin, the cosolvent polyethylene glycol 400 (PEG-400), the surfactant sodium lauryl sulfate, and an amorphous solid dispersion formulation. The ability of these formulations to increase the solubility of etoposide was investigated, followed by permeability studies using the parallel artificial membrane permeability assay (PAMPA) and examination of the consequent solubility-permeability interplay. All formulations significantly increased etoposide's apparent solubility. The cyclodextrin-, surfactant-, and cosolvent-based formulations resulted in a concomitant decreased permeability that could be modeled directly from the proportional increase in the apparent solubility. On the contrary, etoposide permeability remained constant when using the ASD formulation, irrespective of the increased apparent solubility provided by the formulation. In conclusion, supersaturation resulting from the amorphous form overcomes the solubility-permeability tradeoff associated with other formulation techniques. Accounting for the solubility-permeability interplay may allow to develop better solubility-enabling formulations, thereby maximizing the overall absorption of lipophilic orally administered drugs. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  17. Isolation and functional characterization of Lycopene β-cyclase (CYC-B promoter from Solanum habrochaites

    Directory of Open Access Journals (Sweden)

    Chinnusamy Viswanathan

    2010-04-01

    Full Text Available Abstract 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

  18. Indomethacin solubility estimation in 1,4-dioxane + water mixtures by the extended hildebrand solubility approach

    Directory of Open Access Journals (Sweden)

    Miller A Ruidiaz

    2011-09-01

    Full Text Available Extended Hildebrand Solubility Approach (EHSA was successfully applied to evaluate the solubility of Indomethacin in 1,4-dioxane + water mixtures at 298.15 K. An acceptable correlation-performance of EHSA was found by using a regular polynomial model in order four of the W interaction parameter vs. solubility parameter of the mixtures (overall deviation was 8.9%. Although the mean deviation obtained was similar to that obtained directly by means of an empiric regression of the experimental solubility vs. mixtures solubility parameters, the advantages of EHSA are evident because it requires physicochemical properties easily available for drugs.

  19. Solubility behavior and biopharmaceutical classification of novel high-solubility ciprofloxacin and norfloxacin pharmaceutical derivatives.

    Science.gov (United States)

    Breda, Susana A; Jimenez-Kairuz, Alvaro F; Manzo, Ruben H; Olivera, María E

    2009-04-17

    The hydrochlorides of the 1:3 aluminum:norfloxacin and aluminum:ciprofloxacin complexes were characterized according to the Biopharmaceutics Classification System (BCS) premises in comparison with their parent compounds. The pH-solubility profiles of the complexes were experimentally determined at 25 and 37 degrees C in the range of pH 1-8 and compared to that of uncomplexed norfloxacin and ciprofloxacin. Both complexes are clearly more soluble than the antibiotics themselves, even at the lowest solubility pHs. The increase in solubility was ascribed to the species controlling solubility, which were analyzed in the solid phases at equilibrium at selected pHs. Additionally, permeability was set as low, based on data reported in the scientific literature regarding oral bioavailability, intestinal and cell cultures permeabilities and also considering the influence of stoichiometric amounts of aluminum. The complexes fulfill the BCS criterion to be classified as class 3 compounds (high solubility/low permeability). Instead, the active pharmaceutical ingredients (APIs) currently used in solid dosage forms, norfloxacin and ciprofloxacin hydrochloride, proved to be BCS class 4 (low solubility/low permeability). The solubility improvement turns the complexes as potential biowaiver candidates from the scientific point of view and may be a good way for developing more dose-efficient formulations. An immediate release tablet showing very rapid dissolution was obtained. Its dissolution profile was compared to that of the commercial ciprofloxacin hydrochloride tablets allowing to dissolution of the complete dose at a critical pH such as 6.8.

  20. cDNA cloning of a novel gene codifying for the enzyme lycopene β-cyclase from Ficus carica and its expression in Escherichia coli.

    Science.gov (United States)

    Araya-Garay, José Miguel; Feijoo-Siota, Lucía; Veiga-Crespo, Patricia; Villa, Tomás González

    2011-11-01

    Lycopene beta-cyclase (β-LCY) is the key enzyme that modifies the linear lycopene molecule into cyclic β-carotene, an indispensable carotenoid of the photosynthetic apparatus and an important source of vitamin A in human and animal nutrition. Owing to its antioxidant activity, it is commercially used in the cosmetic and pharmaceutical industries, as well as an additive in foodstuffs. Therefore, β-carotene has a large share of the carotenoidic market. In this study, we used reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE)-PCR to obtain and clone a cDNA copy of the gene Lyc-β from Ficus carica (Lyc-β Fc), which codes for the enzyme lycopene β-cyclase (β-LCY). Expression of this gene in Escherichia coli produced a single polypeptide of 56 kDa of weight, containing 496 amino acids, that was able to cycle both ends of the lycopene chain. Amino acid analysis revealed that the protein contained several conserved plant cyclase motifs. β-LCY activity was revealed by heterologous complementation analysis, with lycopene being converted to β-carotene as a result of the enzyme's action. The β-LCY activity of the expressed protein was confirmed by high-performance liquid chromatography (HPLC) identification of the β-carotene. The lycopene to β-carotene conversion rate was 90%. The experiments carried out in this work showed that β-LYC is the enzyme responsible for converting lycopene, an acyclic carotene, to β-carotene, a bicyclic carotene in F. carica. Therefore, by cloning and expressing β-LCY in E. coli, we have obtained a new gene for β-carotene production or as part of the biosynthetic pathway of astaxanthin. So far, this is the first and only gene of the carotenoid pathway identified in F. carica. © Springer-Verlag 2011

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

    Science.gov (United States)

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

    2016-04-01

    In bacteria, proteins containing GGDEF domains are involved in production of the second messenger c-di-GMP. Here we report that the cdgA gene encoding diguanylate cyclase A (CdgA) is involved in biofilm formation and exopolysaccharide (EPS) production in Azospirillum brasilense Sp7. Biofilm quantification using crystal violet staining revealed that inactivation of cdgA decreased biofilm formation. In addition, confocal laser scanning microscopy analysis of green-fluorescent protein-labeled bacteria showed that, during static growth, the biofilms had differential levels of development: bacteria harboring a cdgA mutation exhibited biofilms with considerably reduced thickness compared with those of the wild-type Sp7 strain. Moreover, DNA-specific staining and treatment with DNase I, and epifluorescence studies demonstrated that extracellular DNA and EPS are components of the biofilm matrix in Azospirillum. After expression and purification of the CdgA protein, diguanylate cyclase activity was detected. The enzymatic activity of CdgA-producing cyclic c-di-GMP was determined using GTP as a substrate and flavin adenine dinucleotide (FAD(+)) and Mg(2)(+) as cofactors. Together, our results revealed that A. brasilense possesses a functional c-di-GMP biosynthesis pathway. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  2. Thyroid-stimulating immunoglobulins in Hashimoto's thyroiditis measured by radioreceptor assay and adenylate cyclase stimulation and their relationship to HLA-D alleles

    Energy Technology Data Exchange (ETDEWEB)

    Bliddal, H. (Frederiksberg Hospital, Copenhagen, Denmark); Bech, K.; Feldt-Rasmussen, U.; Thomsen, M.; Ryder, L.P.; Hansen, J.M.; Siersbaek-Nielsen, K.; Friis, T.

    1982-11-01

    The relationship between thyroid-stimulating immunoglobulins, measured by both radioreceptor assay and adenylate cyclase stimulation, and the HLA alleles was studied in 41 patients with Hashimoto's thyroiditis. TSH binding-inhibiting immunoglobulins (TBII) were detected in 9 (22%) patients, and human thyroid adenylate cyclase-stimulating immunoglobulins (HTACS) were found in 21 (51%) patients. Only 2 patients were positive in both assays, and an inverse relationship was observed between TBII and HTACS. In the 21 HTACS-positive patients, HLA-Dw5 was found in 1 subject, compared to 8 of the 20 HTACS-negative patients (P < 0.01), while 4 of the 9 TBII-positive patients had HLA-Dw5 compared to 5 of the 32 TBII-negative subjects (P = 0.09).No significant relations were observed between the presence of HTACS or TBII and HLA-Dw3 or HLA-B8. It is concluded that TBII and HTACS are produced independently in Hashimoto's thyroiditis, and that the production of these autoantibodies seems to be related to the HLA-D region in this disease.

  3. Roles of Protein Kinase A and Adenylate Cyclase in Light-Modulated Cellulase Regulation in Trichoderma reesei

    Science.gov (United States)

    Schuster, André; Tisch, Doris; Seidl-Seiboth, Verena; Kubicek, Christian P.

    2012-01-01

    The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process. PMID:22286997

  4. Participation of the NO/cGMP/K+ATP pathway in the antinociception induced by Walker tumor bearing in rats

    International Nuclear Information System (INIS)

    Barbosa, A.L.R.; Pinheiro, C.A.; Oliveira, G.J.; Torres, J.N.L.; Moraes, M.O.; Ribeiro, R.A.; Vale, M.L.; Souza, M.H.L.P.

    2012-01-01

    Implantation of Walker 256 tumor decreases acute systemic inflammation in rats. Inflammatory hyperalgesia is one of the most important events of acute inflammation. The L-arginine/NO/cGMP/K + ATP pathway has been proposed as the mechanism of peripheral antinociception mediated by several drugs and physical exercise. The objective of this study was to investigate a possible involvement of the NO/cGMP/K + ATP pathway in antinociception induced in Walker 256 tumor-bearing male Wistar rats (180-220 g). The groups consisted of 5-6 animals. Mechanical inflammatory hypernociception was evaluated using an electronic version of the von Frey test. Walker tumor (4th and 7th day post-implantation) reduced prostaglandin E 2 - (PGE 2 , 400 ng/paw; 50 µL; intraplantar injection) and carrageenan-induced hypernociception (500 µg/paw; 100 µL; intraplantar injection). Walker tumor-induced analgesia was reversed (99.3% for carrageenan and 77.2% for PGE 2 ) by a selective inhibitor of nitric oxide synthase (L-NAME; 90 mg/kg, ip) and L-arginine (200 mg/kg, ip), which prevented (80% for carrageenan and 65% for PGE 2 ) the effect of L-NAME. Treatment with the soluble guanylyl cyclase inhibitor ODQ (100% for carrageenan and 95% for PGE 2 ; 8 µg/paw) and the ATP-sensitive K + channel (KATP) blocker glibenclamide (87.5% for carrageenan and 100% for PGE 2 ; 160 µg/paw) reversed the antinociceptive effect of tumor bearing in a statistically significant manner (P < 0.05). The present study confirmed an intrinsic peripheral antinociceptive effect of Walker tumor bearing in rats. This antinociceptive effect seemed to be mediated by activation of the NO/cGMP pathway followed by the opening of KATP channels

  5. Endothelium-Dependent Relaxation Effect of Apocynum venetum Leaf Extract via Src/PI3K/Akt Signalling Pathway

    Directory of Open Access Journals (Sweden)

    Yeh Siang Lau

    2015-06-01

    Full Text Available Botanical herbs are consumed globally not only as an essential diet but also as medicines or as functional/recreational food supplements. The extract of the Apocynum venetum leaves (AVLE, also known as Luobuma, exerts its antihypertensive effect via dilating the blood vessels in an endothelium- and concentration-dependent manner with optimal effect seen at as low as 10 µg/mL. A commercial Luoboma “antihypertensive tea” is available commercially in the western province of China. The present study seeks to investigate the underlying cellular mechanisms of the nitric oxide (NO-releasing property of AVLE in rat aortas and human umbilical vein endothelial cells (HUVECs. Endothelium-dependent relaxation induced by AVLE was assessed in organ chambers in the presence or absence of polyethyleneglycol catalase (PP2, 20 µM; inhibitor of Src kinase, wortmannin (30 nM and LY294002 (20 µM; PI3 (phosphatidylinositol3-Kinase inhibitor, NG-nitro-l-arginine (L-NAME, 100 µM; endothelial NO synthase inhibitor (eNOS and ODQ (1 µM; soluble guanylyl cyclase inhibitor. Total nitrite and nitrate (NOx level and protein expression of p-Akt and p-eNOS were measured. AVLE-induced endothelium-dependent relaxation was reduced by PP2, wortmannin and LY294002 and abolished by L-NAME and ODQ. AVLE significantly increased total NOx level in rat aortas and in HUVECs compared to control. It also instigated phosphorylation of Akt and eNOS in cultured HUVECs in a concentration-dependent manner and this was markedly suppressed by PP2, wortmannin and LY294002. AVLE also inhibited superoxide generated from both NADPH oxidase and xanthine/xanthine oxidase system. Taken together, AVLE causes endothelium-dependent NO mediated relaxations of rat aortas through Src/PI3K/Akt dependent NO signalling pathway and possesses superoxide scavenging activity.

  6. Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen

    Science.gov (United States)

    Quinteros, Fernanda A.; Duvilanski, Beatriz H.; Cabilla, Jimena P.

    2016-01-01

    Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2) are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS) activity and expression and may thereby modulate the production of nitric oxide (NO), an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX) as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC) expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS) and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary. PMID:27611913

  7. Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen.

    Directory of Open Access Journals (Sweden)

    Sonia A Ronchetti

    Full Text Available Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2 are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS activity and expression and may thereby modulate the production of nitric oxide (NO, an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary.

  8. In vivo xenoestrogenic actions of cadmium and arsenic in anterior pituitary and uterus.

    Science.gov (United States)

    Ronchetti, Sonia A; Novack, Gisela V; Bianchi, María S; Crocco, Melisa C; Duvilanski, Beatriz H; Cabilla, Jimena P

    2016-07-01

    Cadmium (Cd) and arsenic (iAs) are toxic metals ubiquitously present in the environment. Both pollutants exert nonmonotonic dose responses, being mostly cytotoxic at high concentrations but mimicking estrogen (E2) effects at low doses. Xenoestrogenic activity of Cd and iAs has been demonstrated in different hormone-dependent tumor cell lines; however, their actions in vivo remain largely unknown. Here, we investigated whether in vivo administration of low doses of Cd and iAs through drinking water would display xenoestrogenic effects in the anterior pituitary gland and uterus of ovariectomized rats. Cd (1ppm) and iAs (0.1ppm) exposure increased the wet weight of anterior pituitary gland and uterus and induced proestrus- and estrus-like vaginal smears. Both metals stimulate cell proliferation of these tissues as they increased the expression of proliferation markers. More importantly, they augmented soluble guanylyl cyclase α1 subunit expression, which has been linked to hormone-dependent tumor progression. Also, Cd and iAs modified protein levels of full-length estrogen receptor α and its truncated variants in an E2-like manner. Anterior pituitary hormone secretion was differentially affected by both metals. Luteinizing hormone synthesis and release were strongly diminished after Cd exposure and only mildly reduced by iAs. Both metals were able to increase prolactin synthesis, although only iAs augmented serum prolactin levels. This study shows for the first time that Cd and iAs exert strong xenoestrogenic effects on anterior pituitary gland at low doses. The differences between Cd and iAs E2-like behavior indicate that other Cd- and iAs-specific mechanisms could be involved. Altogether, these results contribute to the knowledge of reproductive disorders associated with Cd and iAs environmental contamination. © 2016 Society for Reproduction and Fertility.

  9. Differential Regulation of cGMP Signaling in Human Melanoma Cells at Altered Gravity: Simulated Microgravity Down-Regulates Cancer-Related Gene Expression and Motility

    Science.gov (United States)

    Ivanova, Krassimira; Eiermann, Peter; Tsiockas, Wasiliki; Hemmersbach, Ruth; Gerzer, Rupert

    2018-03-01

    Altered gravity is known to affect cellular function by changes in gene expression and cellular signaling. The intracellular signaling molecule cyclic guanosine-3',5'-monophosphate (cGMP), a product of guanylyl cyclases (GC), e.g., the nitric oxide (NO)-sensitive soluble GC (sGC) or natriuretic peptide-activated GC (GC-A/GC-B), is involved in melanocyte response to environmental stress. NO-sGC-cGMP signaling is operational in human melanocytes and non-metastatic melanoma cells, whereas up-regulated expression of GC-A/GC-B and inducible NO synthase (iNOS) are found in metastatic melanoma cells, the deadliest skin cancer. Here, we investigated the effects of altered gravity on the mRNA expression of NOS isoforms, sGC, GC-A/GC-B and multidrug resistance-associated proteins 4/5 (MRP4/MRP5) as selective cGMP exporters in human melanoma cells with different metastatic potential and pigmentation. A specific centrifuge (DLR, Cologne Germany) was used to generate hypergravity (5 g for 24 h) and a fast-rotating 2-D clinostat (60 rpm) to simulate microgravity values ≤ 0.012 g for 24 h. The results demonstrate that hypergravity up-regulates the endothelial NOS-sGC-MRP4/MRP5 pathway in non-metastatic melanoma cells, but down-regulates it in simulated microgravity when compared to 1 g. Additionally, the suppression of sGC expression and activity has been suggested to correlate inversely to tumor aggressiveness. Finally, hypergravity is ineffective in highly metastatic melanoma cells, whereas simulated microgravity down-regulates predominantly the expression of the cancer-related genes iNOS and GC-A/GC-B (shown additionally on protein levels) as well as motility in comparison to 1 g. The results suggest that future studies in real microgravity can benefit from considering GC-cGMP signaling as possible factor for melanocyte transformation.

  10. Nitric Oxide Plays a Key Role in Ovariectomy-Induced Apoptosis in Anterior Pituitary: Interplay between Nitric Oxide Pathway and Estrogen.

    Science.gov (United States)

    Ronchetti, Sonia A; Machiavelli, Leticia I; Quinteros, Fernanda A; Duvilanski, Beatriz H; Cabilla, Jimena P

    2016-01-01

    Changes in the estrogenic status produce deep changes in pituitary physiology, mainly because estrogens (E2) are one of the main regulators of pituitary cell population. Also, E2 negatively regulate pituitary neuronal nitric oxide synthase (nNOS) activity and expression and may thereby modulate the production of nitric oxide (NO), an important regulator of cell death and survival. Little is known about how ovary ablation affects anterior pituitary cell remodelling and molecular mechanisms that regulate this process have not yet been elucidated. In this work we used freshly dispersed anterior pituitaries as well as cell cultures from ovariectomized female rats in order to study whether E2 deficiency induces apoptosis in the anterior pituitary cells, the role of NO in this process and effects of E2 on the NO pathway. Our results showed that cell activity gradually decreases after ovariectomy (OVX) as a consequence of cell death, which is completely prevented by a pan-caspase inhibitor. Furthermore, there is an increase of fragmented nuclei and DNA cleavage thereby presenting the first direct evidence of the existence of apoptosis in the anterior pituitary gland after OVX. NO production and soluble guanylyl cyclase (sGC) expression in anterior pituitary cells increased concomitantly to the apoptosis. Inhibition of both, NO synthase (NOS) and sGC activities prevented the drop of cell viability after OVX, showing for the first time that increased NO levels and sGC activity observed post-OVX play a key role in the induction of apoptosis. Conversely, E2 and prolactin treatments decreased nNOS expression and activity in pituitary cells from OVX rats in a time- and E2 receptor-dependent manner, thus suggesting interplay between NO and E2 pathways in anterior pituitary.

  11. Expression of a hyperactive androgen receptor leads to androgen-independent growth of prostate cancer cells.

    Science.gov (United States)

    Hsieh, Chen-Lin; Cai, Changmeng; Giwa, Ahmed; Bivins, Aaronica; Chen, Shao-Yong; Sabry, Dina; Govardhan, Kumara; Shemshedini, Lirim

    2008-07-01

    Cellular changes that affect the androgen receptor (AR) can cause prostate cancer to transition from androgen dependent to androgen independent, which is usually lethal. One common change in prostate tumors is overexpression of the AR, which has been shown to lead to androgen-independent growth of prostate cancer cells. This led us to hypothesize that expression of a hyperactive AR would be sufficient for androgen-independent growth of prostate cancer cells. To test this hypothesis, stable lune cancer prostate (LNCaP) cell lines were generated, which express a virion phosphoprotein (VP)16-AR hybrid protein that contains full-length AR fused to the strong viral transcriptional activation domain VP16. This fusion protein elicited as much as a 20-fold stronger transcriptional activity than the natural AR. Stable expression of VP16-AR in LNCaP cells yielded androgen-independent cell proliferation, while under the same growth conditions the parental LNCaP cells exhibited only androgen-dependent growth. These results show that expression of a hyperactive AR is sufficient for androgen-independent growth of prostate cancer cells. To study the molecular basis of this enhanced growth, we measured the expression of soluble guanylyl cyclase-alpha1 (sGCalpha1), a subunit of the sGC, an androgen-regulated gene that has been shown to be involved in prostate cancer cell growth. Interestingly, the expression of sGCalpha1 is androgen independent in VP16-AR-expressing cells, in contrast to its androgen-induced expression in control LNCaP cells. RNA(I)-dependent inhibition of sGCalpha1 expression resulted in significantly reduced proliferation of VP16-AR cells, implicating an important role for sGCalpha1 in the androgen-independent growth of these cells.

  12. cIMP synthesized by sGC as a mediator of hypoxic contraction of coronary arteries.

    Science.gov (United States)

    Chen, Zhengju; Zhang, Xu; Ying, Lei; Dou, Dou; Li, Yanhui; Bai, Yun; Liu, Juan; Liu, Limei; Feng, Han; Yu, Xiaoxing; Leung, Susan Wai-Sum; Vanhoutte, Paul M; Gao, Yuansheng

    2014-08-01

    cGMP is considered the only mediator synthesized by soluble guanylyl cyclase (sGC) in response to nitric oxide (NO). However, purified sGC can synthesize several other cyclic nucleotides, including inosine 3',5'-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension. Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy. Rho kinase (ROCK) activity was determined by measuring the phosphorylation of myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by hypoxia in arteries with, but not in those without, endothelium [except if treated with diethylenetriamine (DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Hypoxia (Po2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by inosine 5'-triphosphate, the precursor for cIMP. The augmentation of contraction caused by hypoxia or cIMP was accompanied by increased phosphorylation of myosin phosphatase target subunit 1 at Thr(853), which was prevented by the ROCK inhibitor Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK. Copyright © 2014 the American Physiological Society.

  13. Argon solubility in liquid steel

    NARCIS (Netherlands)

    Boom, R; Dankert, O; Van Veen, A; Kamperman, AA

    2000-01-01

    Experiments have been performed to establish the solubility of argon in liquid interstitial-free steel. The solubility appears to be lower than 0.1 at ppb, The results are in line with argon solubilities reported in the literature on liquid iron. Semiempirical theories and calculations based on the

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

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1987-01-01

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

  15. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    International Nuclear Information System (INIS)

    Murayama, T.; Ui, M.

    1985-01-01

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45 Ca 2+ uptake into the cell monolayer, and (f) increased 86 Rb + uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca 2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca 2+ -mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca 2+ gating

  16. Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

    Science.gov (United States)

    Navarro, Gemma; Cordomí, Arnau; Casadó-Anguera, Verónica; Moreno, Estefanía; Cai, Ning-Sheng; Cortés, Antoni; Canela, Enric I; Dessauer, Carmen W; Casadó, Vicent; Pardo, Leonardo; Lluís, Carme; Ferré, Sergi

    2018-03-28

    G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A 2A receptor and dopamine D 2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.

  17. Inhibitors for human glutaminyl cyclase by structure based design and bioisosteric replacement.

    Science.gov (United States)

    Buchholz, Mirko; Hamann, Antje; Aust, Susanne; Brandt, Wolfgang; Böhme, Livia; Hoffmann, Torsten; Schilling, Stephan; Demuth, Hans-Ulrich; Heiser, Ulrich

    2009-11-26

    The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimer's disease. The hallmark of this principle is the prevention of the formation of Abeta(3,11(pE)-40,42), as these Abeta-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Abeta-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Abeta(3,11(pE)-40,42) formation.

  18. Adenyl cyclase activator forskolin protects against Huntington's disease-like neurodegenerative disorders

    Directory of Open Access Journals (Sweden)

    Sidharth Mehan

    2017-01-01

    Full Text Available Long term suppression of succinate dehydrogenase by selective inhibitor 3-nitropropionic acid has been used in rodents to model Huntington's disease where mitochondrial dysfunction and oxidative damages are primary pathological hallmarks for neuronal damage. Improvements in learning and memory abilities, recovery of energy levels, and reduction of excitotoxicity damage can be achieved through activation of Adenyl cyclase enzyme by a specific phytochemical forskolin. In this study, intraperitoneal administration of 10 mg/kg 3-nitropropionic acid for 15 days in rats notably reduced body weight, worsened motor cocordination (grip strength, beam crossing task, locomotor activity, resulted in learning and memory deficits, greatly increased acetylcholinesterase, lactate dehydrogenase, nitrite, and malondialdehyde levels, obviously decreased adenosine triphosphate, succinate dehydrogenase, superoxide dismutase, catalase, and reduced glutathione levels in the striatum, cortex and hippocampus. Intragastric administration of forskolin at 10, 20, 30 mg/kg dose-dependently reversed these behavioral, biochemical and pathological changes caused by 3-nitropropionic acid. These results suggest that forskolin exhibits neuroprotective effects on 3-nitropropionic acid-induced Huntington's disease-like neurodegeneration.

  19. Students’ misconceptions on solubility equilibrium

    Science.gov (United States)

    Setiowati, H.; Utomo, S. B.; Ashadi

    2018-05-01

    This study investigated the students’ misconceptions of the solubility equilibrium. The participants of the study consisted of 164 students who were in the science class of second year high school. Instrument used is two-tier diagnostic test consisting of 15 items. Responses were marked and coded into four categories: understanding, misconception, understand little without misconception, and not understanding. Semi-structured interviews were carried out with 45 students according to their written responses which reflected different perspectives, to obtain a more elaborated source of data. Data collected from multiple methods were analyzed qualitatively and quantitatively. Based on the data analysis showed that the students misconceptions in all areas in solubility equilibrium. They had more misconceptions such as in the relation of solubility and solubility product, common-ion effect and pH in solubility, and precipitation concept.

  20. Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: heterologous expression and evidence for diverse and multiple catalytic functions of C(50) carotenoid cyclases.

    Science.gov (United States)

    Netzer, Roman; Stafsnes, Marit H; Andreassen, Trygve; Goksøyr, Audun; Bruheim, Per; Brautaset, Trygve

    2010-11-01

    We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

  1. Differences between high-affinity forskolin binding sites in dopamine-riche and other regions of rat brain

    International Nuclear Information System (INIS)

    Poat, J.A.; Cripps, H.E.; Iversen, L.L.

    1988-01-01

    Forskolin labelled with [ 3 H] bound to high- and low-affinity sites in the rat brain. The high-affinity site was discretely located, with highest densities in the striatum, nucleus accumbens, olfactory tubercule, substantia nigra, hippocampus, and the molecular layers of the cerebellum. This site did not correlate well with the distribution of adenylate cyclase. The high-affinity striatal binding site may be associated with a stimulatory guanine nucleotide-binding protein. Thus, the number of sites was increased by the addition of Mg 2+ and guanylyl imidodiphosphate. Cholera toxin stereotaxically injected into rat striatum increased the number of binding sites, and no further increase was noted following the subsequent addition of guanyl nucleotide. High-affinity forskolin binding sites in non-dopamine-rich brain areas (hippocampus and cerebullum) were modulated in a qualitatively different manner by guanyl nucleotides. In these areas the number of binding sites was significantly reduced by the addition of guanyl nucleotide. These results suggest that forskolin may have a potential role in identifying different functional/structural guanine nucleotide-binding proteins

  2. The two parallel photocycles of the Chlamydomonas sensory photoreceptor histidine kinase rhodopsin 1.

    Science.gov (United States)

    Luck, Meike; Hegemann, Peter

    2017-10-01

    Histidine kinase rhodopsins (HKRs) belong to a class of unexplored sensory photoreceptors that share a similar modular architecture. The light sensing rhodopsin domain is covalently linked to signal-transducing modules and in some cases to a C-terminal guanylyl-cyclase effector. In spite of their wide distribution in unicellular organisms, very little is known about their physiological role and mechanistic functioning. We investigated the photochemical properties of the recombinant rhodopsin-fragment of Cr-HKR1 originating from Chlamydomonas reinhardtii. Our spectroscopic studies revealed an unusual thermal stability of the photoproducts with the deprotonated retinal Schiff base (RSB). Upon UV-irradiation these Rh-UV states with maximal absorbance in the UVA-region (Rh-UV) photochemically convert to stable blue light absorbing rhodopsin (Rh-Bl) with protonated chromophore. The heterogeneity of the sample is based on two parallel photocycles with the chromophore in C 15 =N-syn- or -anti-configuration. This report represents an attempt to decipher the underlying reaction schemes and interconversions of the two coexisting photocycles. Copyright © 2017 Elsevier GmbH. All rights reserved.

  3. The Solubility Parameters of Ionic Liquids

    Science.gov (United States)

    Marciniak, Andrzej

    2010-01-01

    The Hildebrand’s solubility parameters have been calculated for 18 ionic liquids from the inverse gas chromatography measurements of the activity coefficients at infinite dilution. Retention data were used for the calculation. The solubility parameters are helpful for the prediction of the solubility in the binary solvent mixtures. From the solubility parameters, the standard enthalpies of vaporization of ionic liquids were estimated. PMID:20559495

  4. The Solubility Parameters of Ionic Liquids

    Directory of Open Access Journals (Sweden)

    Andrzej Marciniak

    2010-04-01

    Full Text Available The Hildebrand’s solubility parameters have been calculated for 18 ionic liquids from the inverse gas chromatography measurements of the activity coefficients at infinite dilution. Retention data were used for the calculation. The solubility parameters are helpful for the prediction of the solubility in the binary solvent mixtures. From the solubility parameters, the standard enthalpies of vaporization of ionic liquids were estimated.

  5. Solubility of sparingly soluble drug derivatives of anthranilic acid.

    Science.gov (United States)

    Domańska, Urszula; Pobudkowska, Aneta; Pelczarska, Aleksandra

    2011-03-24

    This work is a continuation of our systematic study of the solubility of pharmaceuticals (Pharms). All substances here are derivatives of anthranilic acid, and have an anti-inflammatory direction of action (niflumic acid, flufenamic acid, and diclofenac sodium). The basic thermal properties of pure Pharms, i.e., melting and glass-transition temperatures as well as the enthalpy of melting, have been measured with the differential scanning microcalorimetry technique (DSC). Molar volumes have been calculated with the Barton group contribution method. The equilibrium mole fraction solubilities of three pharmaceuticals were measured in a range of temperatures from 285 to 355 K in three important solvents for Pharm investigations: water, ethanol, and 1-octanol using a dynamic method and spectroscopic UV-vis method. The experimental solubility data have been correlated by means of the commonly known G(E) equation: the NRTL, with the assumption that the systems studied here have revealed simple eutectic mixtures. pK(a) precise measurement values have been investigated with the Bates-Schwarzenbach spectrophotometric method. © 2011 American Chemical Society

  6. Prediction of the solubility in lipidic solvent mixture: Investigation of the modeling approach and thermodynamic analysis of solubility.

    Science.gov (United States)

    Patel, Shruti V; Patel, Sarsvatkumar

    2015-09-18

    Self-micro emulsifying drug delivery system (SMEDDS) is one of the methods to improve solubility and bioavailability of poorly soluble drug(s). The knowledge of the solubility of pharmaceuticals in pure lipidic solvents and solvent mixtures is crucial for designing the SMEDDS of poorly soluble drug substances. Since, experiments are very time consuming, a model, which allows for solubility predictions in solvent mixtures based on less experimental data is desirable for efficiency. Solvents employed were Labrafil® M1944CS and Labrasol® as lipidic solvents; Capryol-90®, Capryol-PGMC® and Tween®-80 as surfactants; Transcutol® and PEG-400 as co-solvents. Solubilities of both drugs were determined in single solvent systems at temperature (T) range of 283-333K. In present study, we investigated the applicability of the thermodynamic model to understand the solubility behavior of drugs in the lipiodic solvents. By using the Van't Hoff and general solubility theory, the thermodynamic functions like Gibbs free energy, enthalpy and entropy of solution, mixing and solvation for drug in single and mixed solvents were understood. The thermodynamic parameters were understood in the framework of drug-solvent interaction based on their chemical similarity and dissimilarity. Clotrimazole and Fluconazole were used as active ingredients whose solubility was measured in single solvent as a function of temperature and the data obtained were used to derive mathematical models which can predict solubility in multi-component solvent mixtures. Model dependent parameters for each drug were calculated at each temperature. The experimental solubility data of solute in mixed solvent system were measured experimentally and further correlated with the calculates values obtained from exponent model and log-linear model of Yalkowsky. The good correlation was observed between experimental solubility and predicted solubility. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Structure-activity relationships of benzimidazole-based glutaminyl cyclase inhibitors featuring a heteroaryl scaffold.

    Science.gov (United States)

    Ramsbeck, Daniel; Buchholz, Mirko; Koch, Birgit; Böhme, Livia; Hoffmann, Torsten; Demuth, Hans-Ulrich; Heiser, Ulrich

    2013-09-12

    Glutaminyl cyclase (hQC) has emerged as a new potential target for the treatment of Alzheimer's disease (AD). The inhibition of hQC prevents of the formation of the Aβ3(pE)-40,42 species which were shown to be of elevated neurotoxicity and are likely to act as a seeding core, leading to an accelerated formation of Aβ-oligomers and fibrils. This work presents a new class of inhibitors of hQC, resulting from a pharmacophore-based screen. Hit molecules were identified, containing benzimidazole as the metal binding group connected to 1,3,4-oxadiazole as the central scaffold. The subsequent optimization resulted in benzimidazolyl-1,3,4-thiadiazoles and -1,2,3-triazoles with an inhibitory potency in the nanomolar range. Further investigation into the potential binding mode of the new compound classes combined molecular docking and site directed mutagenesis studies.

  8. Solubility of Carbon in Nanocrystalline -Iron

    OpenAIRE

    Alexander Kirchner; Bernd Kieback

    2012-01-01

    A thermodynamic model for nanocrystalline interstitial alloys is presented. The equilibrium solid solubility of carbon in -iron is calculated for given grain size. Inside the strained nanograins local variation of the carbon content is predicted. Due to the nonlinear relation between strain and solubility, the averaged solubility in the grain interior increases with decreasing grain size. The majority of the global solubility enhancement is due to grain boundary enrichment however. Therefor...

  9. Intrinsic solubility estimation and pH-solubility behavior of cosalane (NSC 658586), an extremely hydrophobic diprotic acid.

    Science.gov (United States)

    Venkatesh, S; Li, J; Xu, Y; Vishnuvajjala, R; Anderson, B D

    1996-10-01

    The selection of cosalane (NSC 658586) by the National Cancer Institute for further development as a potential drug candidate for the treatment of AIDS led to the exploration of the solubility behavior of this extremely hydrophobic drug, which has an intrinsic solubility (S0 approaching 1 ng/ml. This study describes attempts to reliably measure the intrinsic solubility of cosalane and examine its pH-solubility behavior. S0 was estimated by 5 different strategies: (a) direct determination in an aqueous suspension: (b) facilitated dissolution; (c) estimation from the octanol/water partition coefficient and octanol solubility (d) application of an empirical equation based on melting point and partition coefficient; and (e) estimation from the hydrocarbon solubility and functional group contributions for transfer from hydrocarbon to water. S0 estimates using these five methods varied over a 5 x 107-fold range Method (a) yielded the highest values, two-orders of magnitude greater than those obtained by method (b) (facilitated dissolution. 1.4 +/- 0.5 ng/ml). Method (c) gave a value 20-fold higher while that from method (d) was in fair agreement with that from facilitated dissolution. Method (e) yielded a value several orders-of-magnitude lower than other methods. A molecular dynamics simulation suggests that folded conformations not accounted for by group contributions may reduce cosalane's effective hydrophobicity. Ionic equilibria calculations for this weak diprotic acid suggested a 100-fold increase in solubility per pH unit increase. The pH-solubility profile of cosalane at 25 degrees C agreed closely with theory. These studies highlight the difficulty in determining solubility of very poorly soluble compounds and the possible advantage of the facilitated dissolution method. The diprotic nature of cosalane enabled a solubility enhancement of > 107-fold by simple pH adjustment.

  10. On the americium oxalate solubility

    International Nuclear Information System (INIS)

    Zakolupin, S.A.; Korablin, Eh.V.

    1977-01-01

    The americium oxalate solubility at different nitric (0.0-1 M) and oxalic (0.0-0.4 M) acid concentrations was investigated in the temperature range from 14 to 60 deg C. The dependence of americium oxalate solubility on the oxalic acid concentration was determined. Increasing oxalic acid concentration was found to reduce the americium oxalate solubility. The dependence of americium oxalate solubility on the oxalic acid concentration was noted to be a minimum at low acidity (0.1-0.3 M nitric acid). This is most likely due to Am(C 2 O 4 ) + , Am(C 2 O 4 ) 2 - and Am(C 2 O 4 ) 3 3- complex ion formation which have different unstability constants. On the basis of the data obtained, a preliminary estimate was carried out for the product of americium oxalate solubility in nitric acid medium (10 -29 -10 -31 ) and of the one in water (6.4x10 -20 )

  11. Distribution and protective function of pituitary adenylate cyclase-activating polypeptide (PACAP in the retina

    Directory of Open Access Journals (Sweden)

    Tomoya eNakamachi

    2012-11-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP, which is found in 27- or 38-amino acid forms, belongs to the VIP/glucagon/secretin family. PACAP and its three receptor subtypes are expressed in neural tissues, with PACAP known to exert a protective effect against several types of neural damage. The retina is considered to be part of the central nervous system, and retinopathy is a common cause of profound and intractable loss of vision. This review will examine the expression and morphological distribution of PACAP and its receptors in the retina, and will summarize the current state of knowledge regarding the protective effect of PACAP against different kinds of retinal damage, such as that identified in association with diabetes, ultraviolet light, hypoxia, optic nerve transection, and toxins. This article will also address PACAP-mediated protective pathways involving retinal glial cells.

  12. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells.

    Science.gov (United States)

    Martín, César; Etxaniz, Asier; Uribe, Kepa B; Etxebarria, Aitor; González-Bullón, David; Arlucea, Jon; Goñi, Félix M; Aréchaga, Juan; Ostolaza, Helena

    2015-09-08

    Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca(2+)-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system.

  13. Solubilities of uranium for TILA-99

    International Nuclear Information System (INIS)

    Ollila, K.; Ahonen, L.

    1998-11-01

    This report presents the evaluation of the uranium solubilities in the reference waters of TILA-99. The behaviour of uranium has been discussed separately in the near-field and far-field conditions. The bentonite/groundwater interactions have been considered in the compositions of the fresh and saline near-field reference waters. The far-field groundwaters' compositions include fresh, brackish, saline and very saline, almost brine-type compositions. The pH and redox conditions, as the main parameters affecting the solubilities, are considered. A literature study was made in order to obtain information on the recent dissolution and leaching experiments of UO 2 and spent fuel. The latest literature includes studies on UO 2 solubility under anoxic conditions, in which the methods for simulating the reducing conditions of deep groundwater have been improved. Studies on natural uraninite and its alteration products give a valuable insight into the long-term behaviour of spent fuel. Also the solubility equilibria for some relevant poorly known uranium minerals have been determined. The solubilities of the selected solubility-limiting phases were calculated using the geochemical code, EQ3/6. The NEA database for uranium was the basis for the modelling. The recently extended and updated SR '97 database was used for comparison. The solubility products for uranophane were taken from the latest literature. The recommended values for solubilities were given after a comparison between the calculated solubilities, experimental information and measured concentrations in natural groundwaters. The experiments include several UO 2 dissolution studies in synthetic groundwaters with compositions close to the reference groundwaters. (author)

  14. Solubilities of uranium for TILA-99

    Energy Technology Data Exchange (ETDEWEB)

    Ollila, K. [VTT Chemical Technology, Espoo (Finland); Ahonen, L. [Geological Survey of Finland, Espoo (Finland)

    1998-11-01

    This report presents the evaluation of the uranium solubilities in the reference waters of TILA-99. The behaviour of uranium has been discussed separately in the near-field and far-field conditions. The bentonite/groundwater interactions have been considered in the compositions of the fresh and saline near-field reference waters. The far-field groundwaters` compositions include fresh, brackish, saline and very saline, almost brine-type compositions. The pH and redox conditions, as the main parameters affecting the solubilities, are considered. A literature study was made in order to obtain information on the recent dissolution and leaching experiments of UO{sub 2} and spent fuel. The latest literature includes studies on UO{sub 2} solubility under anoxic conditions, in which the methods for simulating the reducing conditions of deep groundwater have been improved. Studies on natural uraninite and its alteration products give a valuable insight into the long-term behaviour of spent fuel. Also the solubility equilibria for some relevant poorly known uranium minerals have been determined. The solubilities of the selected solubility-limiting phases were calculated using the geochemical code, EQ3/6. The NEA database for uranium was the basis for the modelling. The recently extended and updated SR `97 database was used for comparison. The solubility products for uranophane were taken from the latest literature. The recommended values for solubilities were given after a comparison between the calculated solubilities, experimental information and measured concentrations in natural groundwaters. The experiments include several UO{sub 2} dissolution studies in synthetic groundwaters with compositions close to the reference groundwaters. (author) 81 refs.

  15. The effects of disordered structure on the solubility and dissolution rates of some hydrophilic, sparingly soluble drugs.

    Science.gov (United States)

    Mosharraf, M; Sebhatu, T; Nyström, C

    1999-01-15

    The effects of experimental design on the apparent solubility of two sparingly soluble hydrophilic compounds (barium sulphate and calcium carbonate) were studied in this paper. The apparent solubility appeared to be primarily dependent on the amount of solute added to the solvent in each experiment, increasing with increased amounts. This effect seems to be due to the existence of a peripheral disordered layer. However physico-chemical methods used in the present study were not able to unambiguously verify the existence of any disorder in the solid state structure of the drugs. At higher proportions of solute to solvent, the solubility reached a plateau corresponding to the solubility of the disordered or amorphous molecular form of the material. Milling the powders caused the plateau to be reached at lower proportions of solute to solvent, since this further disordered the surface of the drug particles. It was also found that the apparent solubility of the drugs tested decreased after storage at high relative humidities. A model for describing the effects of a disordered surface layer of varying thickness and continuity on the solubility of a substance is presented. This model may be used as a method for detection of minute amount of disorder, where no other technique is capable of detecting the disordered structure. It is suggested that recrystallisation of the material occurs via slow solid-state transition at the surface of the drug particle; this would slowly reduce the apparent solubility of the substance at the plateau level to the thermodynamically stable value. A biphasic dissolution rate profile was obtained. The solubility of the disordered surface of the particles appeared to be the rate-determining factor during the initial dissolution phase, while the solubility of the crystalline core was the rate-determining factor during the final slower phase.

  16. Determination of radionuclide solubility limits to be used in SR 97. Uncertainties associated to calculated solubilities

    Energy Technology Data Exchange (ETDEWEB)

    Bruno, J.; Cera, E.; Duro, L.; Jordana, S. [QuantiSci S.L., Barcelona (Spain); Pablo, J. de [DEQ-UPC, Barcelona (Spain); Savage, D. [QuantiSci Ltd., Henley-on-Thames (United Kingdom)

    1997-12-01

    The thermochemical behaviour of 24 critical radionuclides for the forthcoming SR97 PA exercise is discussed. The available databases are reviewed and updated with new data and an extended database for aqueous and solid species of the radionuclides of interest is proposed. We have calculated solubility limits for the radionuclides of interest under different groundwater compositions. A sensitivity analysis of the calculated solubilities with the composition of the groundwater is presented. Besides selecting the most likely solubility limiting phases, in this work we have used coprecipitation approaches in order to calculate more realistic solubility limits for minor radionuclides, such as Ra, Am and Cm. The comparison between the calculated solubilities and the concentrations measured in relevant natural systems (NA) and in spent fuel leaching experiments helps to assess the validity of the methodology used and to derive source term concentrations for the radionuclides studied. The uncertainties associated to the solubilities of the main radionuclides involved in the spent nuclear fuel have also been discussed in this work. The variability of the groundwater chemistry; redox conditions and temperature of the system have been considered the main factors affecting the solubilities. In this case, a sensitivity analysis has been performed in order to study solubility changes as a function of these parameters. The uncertainties have been calculated by including the values found in a major extent in typical granitic groundwaters. The results obtained from this analysis indicate that there are some radionuclides which are not affected by these parameters, i.e. Ag, Cm, Ho, Nb, Ni, Np, Pu, Se, Sm, Sn, Sr, Tc and U

  17. Novel electrosprayed nanospherules for enhanced aqueous solubility and oral bioavailability of poorly water-soluble fenofibrate.

    Science.gov (United States)

    Yousaf, Abid Mehmood; Mustapha, Omer; Kim, Dong Wuk; Kim, Dong Shik; Kim, Kyeong Soo; Jin, Sung Giu; Yong, Chul Soon; Youn, Yu Seok; Oh, Yu-Kyoung; Kim, Jong Oh; Choi, Han-Gon

    2016-01-01

    The purpose of the present research was to develop a novel electrosprayed nanospherule providing the most optimized aqueous solubility and oral bioavailability for poorly water-soluble fenofibrate. Numerous fenofibrate-loaded electrosprayed nanospherules were prepared with polyvinylpyrrolidone (PVP) and Labrafil(®) M 2125 as carriers using the electrospray technique, and the effect of the carriers on drug solubility and solvation was assessed. The solid state characterization of an optimized formulation was conducted by scanning electron microscopy, powder X-ray diffraction, differential scanning calorimetry, and Fourier transform infrared spectroscopic analyses. Oral bioavailability in rats was also evaluated for the formulation of an optimized nanospherule in comparison with free drug and a conventional fenofibrate-loaded solid dispersion. All of the electrosprayed nanospherule formulations had remarkably enhanced aqueous solubility and dissolution compared with free drug. Moreover, Labrafil M 2125, a surfactant, had a positive influence on the solubility and dissolution of the drug in the electrosprayed nanospherule. Increases were observed as the PVP/drug ratio increased to 4:1, but higher ratios gave no significant increases. In particular, an electrosprayed nanospherule composed of fenofibrate, PVP, and Labrafil M 2125 at the weight ratio of 1:4:0.5 resulted in a particle size of water-soluble fenofibrate.

  18. Retrograde curves of solidus and solubility

    International Nuclear Information System (INIS)

    Vasil'ev, M.V.

    1979-01-01

    The investigation was concerned with the constitutional diagrams of the eutectic type with ''retrograde solidus'' and ''retrograde solubility curve'' which must be considered as diagrams with degenerate monotectic transformation. The solidus and the solubility curves form a retrograde curve with a common retrograde point representing the solubility maximum. The two branches of the Aetrograde curve can be described with the aid of two similar equations. Presented are corresponding equations for the Cd-Zn system and shown is the possibility of predicting the run of the solubility curve

  19. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    International Nuclear Information System (INIS)

    Jones, S.B.; Halenda, S.P.; Bylund, D.B.

    1991-01-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism

  20. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    Energy Technology Data Exchange (ETDEWEB)

    Jones, S.B.; Halenda, S.P.; Bylund, D.B. (Univ. of Missouri-Columbia (USA))

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

  1. Interlaboratory validation of small-scale solubility and dissolution measurements of poorly water-soluble drugs

    DEFF Research Database (Denmark)

    Andersson, Sara B. E.; Alvebratt, Caroline; Bevernage, Jan

    2016-01-01

    The purpose of this study was to investigate the interlaboratory variability in determination of apparent solubility (Sapp) and intrinsic dissolution rate (IDR) using a miniaturized dissolution instrument. Three poorly water-soluble compounds were selected as reference compounds and measured at m...

  2. Solubility limits of importance to leaching

    International Nuclear Information System (INIS)

    Ogard, A.; Bentley, G.; Bryant, E.; Duffy, C.; Grisham, J.; Norris, E.; Orth, C.; Thomas, K.

    1981-01-01

    The solubilities of some radionuclides, especially rare earths and actinides, may be an important and controlling factor in leaching of waste forms. These solubilities should be measured accurately as a function of pH and not as a part of a multicomponent system. Individual solubilities should be measured as a function of temperature to determine if a kinetic effect is being observed in the data. A negative temperature coefficient of solubility for actinides and rare earths in water would have important consequences for nuclear reactor safety and for the management of nuclear wastes

  3. Chronic treatment with escitalopram but not R-citalopram translocates Galpha(s) from lipid raft domains and potentiates adenylyl cyclase: a 5-hydroxytryptamine transporter-independent action of this antidepressant compound.

    Science.gov (United States)

    Zhang, Lanqiu; Rasenick, Mark M

    2010-03-01

    Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Galpha(s) from lipid rafts to a nonraft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants, and for serotonin uptake inhibitors, it occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Galpha(s) in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Galpha(s) in lipid rafts, whereas there was no change in overall Galpha(s) content. These effects were drug dose- and exposure time-dependent. Although R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Galpha(s) localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this seemed due to increased coupling between Galpha(s) and adenylyl cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Galpha(s) from lipid rafts, and this effect seems to occur independently of 5-hydroxytryptamine transporters. Our results suggest that, although antidepressants display distinct affinities for well identified targets (e.g., monoamine transporters), several presynaptic and postsynaptic molecules are probably modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs.

  4. Chronic Treatment with Escitalopram but Not R-Citalopram Translocates Gαs from Lipid Raft Domains and Potentiates Adenylyl Cyclase: A 5-Hydroxytryptamine Transporter-Independent Action of This Antidepressant Compound

    Science.gov (United States)

    Zhang, Lanqiu

    2010-01-01

    Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Gαs from lipid rafts to a nonraft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants, and for serotonin uptake inhibitors, it occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Gαs in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Gαs in lipid rafts, whereas there was no change in overall Gαs content. These effects were drug dose- and exposure time-dependent. Although R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Gαs localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this seemed due to increased coupling between Gαs and adenylyl cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Gαs from lipid rafts, and this effect seems to occur independently of 5-hydroxytryptamine transporters. Our results suggest that, although antidepressants display distinct affinities for well identified targets (e.g., monoamine transporters), several presynaptic and postsynaptic molecules are probably modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs. PMID:19996298

  5. Synechocystis sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll a for activity.

    Science.gov (United States)

    Xiong, Wei; Shen, Gaozhong; Bryant, Donald A

    2017-03-01

    The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA 6803 ) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA 6803 gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA 6803 was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA 6803 was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA 6803 is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.

  6. Adenylyl Cyclase Signaling in the Developing Chick Heart: The Deranging Effect of Antiarrhythmic Drugs

    Directory of Open Access Journals (Sweden)

    Lucie Hejnova

    2014-01-01

    Full Text Available The adenylyl cyclase (AC signaling system plays a crucial role in the regulation of cardiac contractility. Here we analyzed the key components of myocardial AC signaling in the developing chick embryo and assessed the impact of selected β-blocking agents on this system. Application of metoprolol and carvedilol, two commonly used β-blockers, at embryonic day (ED 8 significantly downregulated (by about 40% expression levels of AC5, the dominant cardiac AC isoform, and the amount of Gsα protein at ED9. Activity of AC stimulated by forskolin was also significantly reduced under these conditions. Interestingly, when administered at ED4, these drugs did not produce such profound changes in the myocardial AC signaling system, except for markedly increased expression of Giα protein. These data indicate that β-blocking agents can strongly derange AC signaling during the first half of embryonic heart development.

  7. Uranyl Oxalate Solubility

    Energy Technology Data Exchange (ETDEWEB)

    Leturcq, G.; Costenoble, S.; Grandjean, S. [CEA Marcoule DEN/DRCP/SCPS/LCA - BP17171 - 30207 Bagnols sur Ceze cedex (France)

    2008-07-01

    The solubility of uranyl oxalate was determined at ambient temperature by precipitation in oxalic-nitric solutions, using an initial uranyl concentration of 0.1 mol/L. Oxalic concentration varied from 0.075 to 0.3 mol/L while nitric concentration ranged between 0.75 and 3 mol/L. Dissolution tests, using complementary oxalic-nitric media, were carried out for 550 hours in order to study the kinetic to reach thermodynamic equilibrium. Similar solubility values were reached by dissolution and precipitation. Using the results, it was possible to draw the solubility surface versus oxalic and nitric concentrations and to determine both the apparent solubility constant of UO{sub 2}C{sub 2}O{sub 4}, 3H{sub 2}O (Ks) and the apparent formation constant of the first uranyl-oxalate complex UO{sub 2}C{sub 2}O{sub 4} (log {beta}1), for ionic strengths varying between 1 and 3 mol/L. Ks and log {beta}1 values were found to vary from 1.9 10{sup -8} to 9.2 10{sup -9} and from 5.95 to 6.06, respectively, when ionic strength varied from 1 to 3 mol/L. A second model may fit our data obtained at an ionic strength of 3 mol/L suggesting as reported by Moskvin et al. (1959) that no complexes are formed for [H{sup +}] at 3 M. The Ks value would then be 1.3 10{sup -8}. (authors)

  8. Adenylate cyclase regulates elongation of mammalian primary cilia

    International Nuclear Information System (INIS)

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

    2009-01-01

    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-3β 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.

  9. Adenylate cyclase regulates elongation of mammalian primary cilia

    Energy Technology Data Exchange (ETDEWEB)

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J. [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada); Rattner, Jerome B. [Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada); Hoorn, Frans A. van der, E-mail: fvdhoorn@ucalgary.ca [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada)

    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.

  10. Solubility behavior of narcotic analgesics in aqueous media: solubilities and dissociation constants of morphine, fentanyl, and sufentanil.

    Science.gov (United States)

    Roy, S D; Flynn, G L

    1989-02-01

    The pH dependence of the aqueous solubility of morphine, fentanyl, and sufentanil was investigated at 35 degrees C. Dissociation constants and corresponding pKa' values of the drugs were obtained from measured free-base solubilities (determined at high pH's) and the concentrations of saturated solutions at intermediate pH's. Morphine, fentanyl, and sufentanil exhibited pKa' values of 8.08, 8.99, and 8.51, respectively. Over the pH range of 5 to 12.5 the apparent solubilities are determined by the intrinsic solubility of the free base plus the concentration of ionized drug necessary to satisfy the dissociation equilibrium at a given pH. Consequently, the drug concentrations of saturated aqueous solutions fall off precipitously as the pH is raised and ionization is suppressed. Further, at low pH's the aqueous solubility of morphine increased in a linear fashion with increases in the molar strength of citric acid which was added to acidify the medium, suggesting the formation of a soluble morphine-citrate complex.

  11. Solubility studies of Np(IV)

    International Nuclear Information System (INIS)

    Zhang Yingjie; Yao Jun; Jiao Haiyang; Ren Lihong; Zhou Duo; Fan Xianhua

    2001-01-01

    The solubility of Np(IV) in simulated underground water and redistilled water has been measured with the variations of pH(6-12) and storage time (0-100 d) in the presence of reductant (Na 2 S 2 O 4 , metallic Fe). All experiments are performed in a low oxygen concentration glove box containing high purity Ar(99.99%), with an oxygen content of less than 5 x 10 -6 mol/mol. Experimental results show that the variation of pH in solution has little effect on the solubility of Np(IV) in the two kinds of water; the measured solubility of Np(IV) is affected by the composition of solution; with Na 2 S 2 O 4 as a reductant, the solubility of Np(IV) in simulated underground water is (9.23 +- 0.48) x 10 -10 mol/L, and that in redistilled water is (8.31 +- 0.35) x 10 -10 mol/L; with metallic Fe as a reductant, the solubility of Np(IV) in simulated underground water is (1.85 +- 0.56) x 10 -9 mol/L, and that in redistilled water is (1.48 +- 0.66) x 10 -9 mol/L

  12. Solubility of Nd in brine

    International Nuclear Information System (INIS)

    Khalili, F.I.; Symeopoulos, V.; Chen, J.F.; Choppin, G.R.

    1994-01-01

    The solubility of Nd(III) has been measured at 23±3 C in a synthetic brine at pcH 6.4, 8.4, 10.4 and 12.4. The brine consisted predominantly of (Na+K)Cl and MgCl 2 with an ionic strength of 7.8 M (9.4 m) a solid compound of Nd(III) at each pcH was assigned from X-ray diffraction patterns. The log values of the experimental solubilities decrease fomr -3 at pcH 6.4 to -5.8 at pcH 8.4; at pcH 10.4 and 12.4 the solubility was below the detection limit of -7.5. The experimental solubility does not follow closely the variation with pcH estimated from modeling of the species in solution in equilibrium with the Nd solid using S.I.T. (orig.)

  13. Thermodynamic approach to improving solubility prediction of co-crystals in comparison with individual poorly soluble components

    International Nuclear Information System (INIS)

    Perlovich, German L.

    2014-01-01

    Highlights: • Thermodynamic approach for solubility improvement of co-crystal was developed. • The graphical technique for estimation of co-crystal solubility was elaborated. • Hydration enthalpies of some drugs and amino acids were calculated. • Applicability/operability of the approach was exemplified by some drugs and amino acids. - Abstract: A novel thermodynamic approach to compare poorly soluble components (active pharmaceutical ingredient (API)) both in co-crystals and individual compounds was developed. An algorithm of choosing potential co-crystals with improved solubility characteristics on the basis of the known solvation/hydration API and co-former enthalpies is described. The applicability and operability of the algorithm were tested exemplified by some drugs and amino acids

  14. Heme-based sensors in biological systems.

    Science.gov (United States)

    Rodgers, K R

    1999-04-01

    The past several years have been witness to a staggering rate of advancement in the understanding of how organisms respond to changes in the availability of diatomic molecules that are toxic and/or crucial to survival. Heme-based sensors presently constitute the majority of the proteins known to sense NO, O2 and CO and to initiate the chemistry required to adapt to changes in their availabilities. Knowledge of the three characterized members of this class, soluble guanylate cyclase, FixL and CooA, has grown substantially during the past year. The major advances have resulted from a broad range of approaches to elucidation of both function and mechanism. They include growth in the understanding of the interplay between the heme and protein in soluble guanylate cyclase, as well as alternate means for its stimulation. Insight into the O2-induced structural changes in FixL has been supplied by the single crystal structure of the heme domain of Bradyrhizobium japonicum. Finally, the ligation environment and ligand interchange that facilitates CO sensing by CooA has been established by spectroscopic and mutagenesis techniques.

  15. Kaempferol stimulates large conductance Ca2+-activated K+ (BKCa) channels in human umbilical vein endothelial cells via a cAMP/PKA-dependent pathway

    Science.gov (United States)

    Xu, Y C; Leung, G P H; Wong, P Y D; Vanhoutte, P M; Man, R Y K

    2008-01-01

    Background and purpose: Kaempferol has been shown to possess a vasodilator effect but its mechanism of action remains unclear. In this study, experiments were carried out to study the effect of kaempferol on K+ channels in endothelial cells. Experimental approach: K+ channel activities in human umbilical vein endothelial cells (HUVECs) were studied by conventional whole cell and cell-attached patch-clamp electrophysiology. Key results: Kaempferol stimulated an outward-rectifying current in HUVECs in a dose-dependent manner with an EC50 value of 2.5±0.02 μM. This kaempferol-induced current was abolished by large conductance Ca2+-activated K+ (BKCa) channel blockers, such as iberiotoxin (IbTX) and charybdotoxin (ChTX), whereas the small conductance Ca2+-activated K+ (SKCa) channel blocker, apamin, and the voltage-dependent K+ (KV) channel blocker, 4-aminopyridine, had no effect. Cell-attached patches demonstrated that kaempferol increased the open probability of BkCa channels in HUVECs. Clamping intracellular Ca2+ did not prevent kaempferol-induced increases in outward current. In addition, the kaempferol-induced current was diminished by the adenylyl cyclase inhibitor SQ22536, the cAMP antagonist Rp-8-Br-cAMP and the PKA inhibitor KT5720, but was not affected by the guanylyl cyclase inhibitor ODQ, the cGMP antagonist Rp-8-Br-cGMP and the PKG inhibitor KT5823. The activation of BKCa channels by kaempferol caused membrane hyperpolarization of HUVECs. Conclusion and implications: These results demonstrate that kaempferol activates the opening of BKCa channels in HUVECs via a cAMP/PKA-dependent pathway, resulting in membrane hyperpolarization. This mechanism may partly account for the vasodilator effects of kaempferol. PMID:18493242

  16. Solubility database for TILA-99

    Energy Technology Data Exchange (ETDEWEB)

    Vuorinen, U.; Carlsson, T. [VTT Chemical Technology, Espoo (Finland); Kulmala, S.; Hakanen, M. [Helsinki Univ. (Finland). Lab. of Radiochemistry; Ahonen, L. [Geological Survey of Finland, Espoo (Finland)

    1998-11-01

    The safety assessment of spent fuel disposal requires solubility values for several elements estimated in Finnish disposal conditions. In Finland four sites (Haestholmen, Kivetty, Olkiluoto and Romuvaara) are investigated for the disposal of spent fuel. Haestholmen and OLkiluoto are onshore sites, while Kivetty and Romuvaara are inland sites. Based on groundwater analysis and classification according to salinity at the planned disposal depth mainly fresh groundwater is encountered at Kivetty and Romuvaara, while brackish and saline water-types are met at Haestholmen and Olkiluoto. Very saline, almost brine-type water ({approx}70 g/l) has been found in the deepest parts of the investigated bedrock at one of the sites (Olkiluoto). The reference waters and conditions were chosen according to the water-types. The considered reference conditions incorporated both the near- and far-field, and both oxidizing and reducing conditions were considered. In the reference conditions, the changes in solubilities were also estimated as caused by possible variations in the pH, carbonate content and redox conditions. Uranium, which is the main component of spent fuel is dealt with in a separate report presenting the solubility of uranium and spent fuel dissolution. In this work the solubilities of all the other elements of concern (Am, Cu, Nb, Np, Pa, Pd, Pu, Ra, Se, Sn, Tc, Zr, Cm, Ni, Sr, Th, C, Cl, Cs, Fe, Ho, I, and Sm) in the safety assessment are considered. Some discussion on the corrosion of the spent fuel canister is also presented. For the estimation of solubilities of the elements in question, literature data was collected that mainly comprised experimentally measured concentrations. The sources used were spent fuel experiments, concentrations measured in solubility measurements, natural concentrations and concentrations from natural analogue sites (especially Palmottu and Hyrkkoelae in Finland) as well as the concentrations measured at the Finnish investigation sites

  17. Solubility database for TILA-99

    International Nuclear Information System (INIS)

    Vuorinen, U.; Carlsson, T.; Kulmala, S.; Hakanen, M.

    1998-11-01

    The safety assessment of spent fuel disposal requires solubility values for several elements estimated in Finnish disposal conditions. In Finland four sites (Haestholmen, Kivetty, Olkiluoto and Romuvaara) are investigated for the disposal of spent fuel. Haestholmen and OLkiluoto are onshore sites, while Kivetty and Romuvaara are inland sites. Based on groundwater analysis and classification according to salinity at the planned disposal depth mainly fresh groundwater is encountered at Kivetty and Romuvaara, while brackish and saline water-types are met at Haestholmen and Olkiluoto. Very saline, almost brine-type water (∼70 g/l) has been found in the deepest parts of the investigated bedrock at one of the sites (Olkiluoto). The reference waters and conditions were chosen according to the water-types. The considered reference conditions incorporated both the near- and far-field, and both oxidizing and reducing conditions were considered. In the reference conditions, the changes in solubilities were also estimated as caused by possible variations in the pH, carbonate content and redox conditions. Uranium, which is the main component of spent fuel is dealt with in a separate report presenting the solubility of uranium and spent fuel dissolution. In this work the solubilities of all the other elements of concern (Am, Cu, Nb, Np, Pa, Pd, Pu, Ra, Se, Sn, Tc, Zr, Cm, Ni, Sr, Th, C, Cl, Cs, Fe, Ho, I, and Sm) in the safety assessment are considered. Some discussion on the corrosion of the spent fuel canister is also presented. For the estimation of solubilities of the elements in question, literature data was collected that mainly comprised experimentally measured concentrations. The sources used were spent fuel experiments, concentrations measured in solubility measurements, natural concentrations and concentrations from natural analogue sites (especially Palmottu and Hyrkkoelae in Finland) as well as the concentrations measured at the Finnish investigation sites. The

  18. Transmembrane segments of complement receptor 3 do not participate in cytotoxic activities but determine receptor structure required for action of Bordetella adenylate cyclase toxin

    Czech Academy of Sciences Publication Activity Database

    Wald, Tomáš; Osičková, Adriana; Mašín, Jiří; Matyska Lišková, Petra; Petry-Podgorska, Inga; Matoušek, Tomáš; Šebo, Peter; Osička, Radim

    2016-01-01

    Roč. 74, č. 3 (2016), flw008 ISSN 2049-632X R&D Projects: GA ČR(CZ) GAP302/11/0580; GA ČR GAP302/12/0460; GA ČR GA13-14547S Institutional support: RVO:61388971 ; RVO:68081715 Keywords : adenylate cyclase toxin * ICP-MS * CD11b/CD18 Subject RIV: EE - Microbiology, Virology; CB - Analytical Chemistry, Separation (UIACH-O) Impact factor: 2.335, year: 2016

  19. Overview of milling techniques for improving the solubility of poorly water-soluble drugs

    Directory of Open Access Journals (Sweden)

    Zhi Hui Loh

    2015-07-01

    Full Text Available Milling involves the application of mechanical energy to physically break down coarse particles to finer ones and is regarded as a “top–down” approach in the production of fine particles. Fine drug particulates are especially desired in formulations designed for parenteral, respiratory and transdermal use. Most drugs after crystallization may have to be comminuted and this physical transformation is required to various extents, often to enhance processability or solubility especially for drugs with limited aqueous solubility. The mechanisms by which milling enhances drug dissolution and solubility include alterations in the size, specific surface area and shape of the drug particles as well as milling-induced amorphization and/or structural disordering of the drug crystal (mechanochemical activation. Technology advancements in milling now enable the production of drug micro- and nano-particles on a commercial scale with relative ease. This review will provide a background on milling followed by the introduction of common milling techniques employed for the micronization and nanonization of drugs. Salient information contained in the cited examples are further extracted and summarized for ease of reference by researchers keen on employing these techniques for drug solubility and bioavailability enhancement.

  20. Noble gases solubility in water

    International Nuclear Information System (INIS)

    Crovetto, Rosa; Fernandez Prini, Roberto.

    1980-07-01

    The available experimental data of solubility of noble gases in water for temperatures smaller than 330 0 C have been critically surveyed. Due to the unique structure of the solvent, the solubility of noble gases in water decreases with temperature passing through a temperature of minimum solubility which is different for each gas, and then increases at higher temperatures. As aresult of the analysis of the experimental data and of the features of the solute-solvent interaction, a generalized equation is proposed which enables thecalculation of Henry's coefficient at different temperatures for all noble gases. (author) [es

  1. Solubility of xenon in amino-acid solutions. II. Nine less-soluble amino acids

    Science.gov (United States)

    Kennan, Richard P.; Himm, Jeffrey F.; Pollack, Gerald L.

    1988-05-01

    Ostwald solubility (L) of xenon gas, as the radioisotope 133Xe, has been measured as a function of solute concentration, at 25.0 °C, in aqueous solutions of nine amino acids. The amino-acid concentrations investigated covered much of their solubility ranges in water, viz., asparagine monohydrate (0-0.19 M), cysteine (0-1.16 M), glutamine (0-0.22 M), histidine (0-0.26 M), isoleucine (0-0.19 M), methionine (0-0.22 M), serine (0-0.38 M), threonine (0-1.4 M), and valine (0-0.34 M). We have previously reported solubility results for aqueous solutions of six other, generally more soluble, amino acids (alanine, arginine, glycine, hydroxyproline, lysine, and proline), of sucrose and sodium chloride. In general, L decreases approximately linearly with increasing solute concentration in these solutions. If we postulate that the observed decreases in gas solubility are due to hydration, the results under some assumptions can be used to calculate hydration numbers (H), i.e., the number of H2O molecules associated with each amino-acid solute molecule. The average values of hydration number (H¯) obtained at 25.0 °C are 15.3±1.5 for asparagine, 6.8±0.3 for cysteine, 11.5±1.1 for glutamine, 7.3±0.7 for histidine, 5.9±0.4 for isoleucine, 10.6±0.8 for methionine, 11.2±1.3 for serine, 7.7± 1.0 for threonine, and 6.6±0.6 for valine. We have also measured the temperature dependence of solubility L(T) from 5-40 °C for arginine, glycine, and proline, and obtained hydration numbers H¯(T) in this range. Between 25-40 °C, arginine has an H¯ near zero. This may be evidence for an attractive interaction between xenon and arginine molecules in aqueous solution.

  2. The Emerging Role of Soluble Adenylyl Cyclase in Primary Biliary Cholangitis

    NARCIS (Netherlands)

    Chang, Jung-Chin; Beuers, Ulrich; Oude Elferink, Ronald P. J.

    2017-01-01

    Primary biliary cholangitis (PBC; previously referred to as primary biliary cirrhosis) is a chronic fibrosing cholangiopathy with the signature of an autoimmune disease and features of intrahepatic cholestasis. Immunosuppressing treatments are largely unsuccessful. Responsiveness to ursodeoxycholic

  3. Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro

    KAUST Repository

    Mulaudzi, Takalani

    2011-09-01

    While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5′-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O 2 and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  4. Pituitary adenylate cyclase activating peptide (PACAP participates in adipogenesis by activating ERK signaling pathway.

    Directory of Open Access Journals (Sweden)

    Tatjana Arsenijevic

    Full Text Available Pituitary adenylate cyclase activating peptide (PACAP belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP family. Its action can be mediated by three different receptor subtypes: PAC1, which has exclusive affinity for PACAP, and VPAC1 and VPAC2 which have equal affinity for PACAP and VIP. We showed that all three receptors are expressed in 3T3-L1 cells throughout their differentiation into adipocytes. We established the activity of these receptors by cAMP accumulation upon induction by PACAP. Together with insulin and dexamethasone, PACAP induced adipogenesis in 3T3-L1 cell line. PACAP increased cAMP production within 15 min upon stimulation and targeted the expression and phosphorylation of MAPK (ERK1/2, strengthened by the ERK1/2 phosphorylation being partially or completely abolished by different combinations of PACAP receptors antagonists. We therefore speculate that ERK1/2 activation is crucial for the activation of CCAAT/enhancer- binding protein β (C/EBPβ.

  5. Determination of soluble protein contents from RVNRL

    International Nuclear Information System (INIS)

    Wan Manshol Wan Zin; Nurulhuda Othman

    1996-01-01

    This project was carried out to determine the soluble protein contents on RVNRL film vulcanisates, with respect to the RVNRL storage time, gamma irradiation dose absorbed by the latex and the effect of different leaching time and leaching conditions. These three factors are important in the hope to determine the best possible mean of minimizing the soluble protein contents in products made from RVNRL. Within the nine months storage period employed in the study, the results show that, the longer the storage period the less the soluble protein extracted from the film samples. Gamma irradiation dose absorbed by the samples, between 5.3 kGy to 25.2 kGy seems to influence the soluble protein contents of the RVNRL films vulcanisates. The higher the dose the more was the soluble protein extracted from the film samples. At an absorbed dose of 5.3 kGy and 25.2 kGy, the soluble contents were 0. 198 mg/ml and 0.247 mg/ml respectively. At a fixed leaching temperature, the soluble proteins increases with leaching time and at a fixed leaching time, the soluble proteins increases with leaching temperature. ne highest extractable protein contents was determined at a leaching time of 10 minutes and leaching temperature of 90'C The protein analysis were done by using Modified Lowry Method

  6. Overcoming the solubility limit with solubility-enhancement tags: successful applications in biomolecular NMR studies

    International Nuclear Information System (INIS)

    Zhou Pei; Wagner, Gerhard

    2010-01-01

    Although the rapid progress of NMR technology has significantly expanded the range of NMR-trackable systems, preparation of NMR-suitable samples that are highly soluble and stable remains a bottleneck for studies of many biological systems. The application of solubility-enhancement tags (SETs) has been highly effective in overcoming solubility and sample stability issues and has enabled structural studies of important biological systems previously deemed unapproachable by solution NMR techniques. In this review, we provide a brief survey of the development and successful applications of the SET strategy in biomolecular NMR. We also comment on the criteria for choosing optimal SETs, such as for differently charged target proteins, and recent new developments on NMR-invisible SETs.

  7. Solubility of Tc(IV) oxides

    International Nuclear Information System (INIS)

    Liu, D.J.; Fan, X.H.

    2005-01-01

    Full text of publication follows: The deep geological disposal of the high level radioactive wastes is expected to be a safer disposal method in most countries. The long-lived fission product 99 Tc is present in large quantities in nuclear wastes and its chemical behavior in aqueous solution is of considerable interest. Under the reducing conditions, expected to exist in a deep geological repository, it is generally predicted that technetium will be present as TcO 2 .nH 2 O. The solubility of Tc(IV) is used as a source term in performance assessment of radioactive waste repository. Technetium oxide was prepared by reduction of a technetate solution with Sn 2+ . The solubility of Tc(IV) oxide has been determined in simulated groundwater and re-distilled water under aerobic and anaerobic conditions. The effects of pH and CO 3 2- concentration of solution on solubility of Tc(IV) oxide were studied. The concentration of total technetium and Tc(IV) species in the solutions were periodically determined by separating the oxidized and reduced technetium species using a solvent extraction procedure and counting the beta activity of the 99 Tc with a liquid scintillation counter. The experimental results show that the rate of oxidation of Tc(IV) in simulated groundwater and re-distilled water is about (1.49∼1.86) x 10 -9 mol/(L.d) under aerobic conditions, but Tc(IV) in simulated groundwater and re-distilled water is not oxidized under anaerobic conditions. Under aerobic or anaerobic conditions the solubility of Tc(IV) oxide in simulated groundwater and re-distilled water is equal on the whole after centrifugation or ultrafiltration. The solubility of Tc(IV) oxide decreases with the increase of pH at pH 10 and is pH independent in the range 2 -8 to 10 -9 mol/L at 2 3 2- concentration. These data could be used to estimate the Tc(IV) solubility for cases where solubility limits transport of technetium in reducing environments of high-level waste repositories. (authors)

  8. Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

    Science.gov (United States)

    Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H-J

    2011-04-08

    Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

  9. Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

    Science.gov (United States)

    Banta, Amy B.; Wei, Jeremy H.; Gill, Clare C. C.; Giner, José-Luis; Welander, Paula V.

    2017-01-01

    Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

  10. Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana

    Science.gov (United States)

    Wang, Zhonghua; Yeats, Trevor; Han, Hong; Jetter, Reinhard

    2010-01-01

    The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C30H50O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761–779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores. PMID:20610397

  11. Effect of composition of simulated intestinal media on the solubility of poorly soluble compounds investigated by design of experiments

    DEFF Research Database (Denmark)

    Madsen, Cecilie Maria; Feng, Kung-I; Leithead, Andrew

    2018-01-01

    The composition of the human intestinal fluids varies both intra- and inter-individually. This will influence the solubility of orally administered drug compounds, and hence, the absorption and efficacy of compounds displaying solubility limited absorption. The purpose of this study was to assess...... studies feasible compared to single SIF solubility studies. Applying this DoE approach will lead to a better understanding of the impact of intestinal fluid composition on the solubility of a given drug compound....

  12. pH-metric solubility. 3. Dissolution titration template method for solubility determination.

    Science.gov (United States)

    Avdeef, A; Berger, C M

    2001-12-01

    The main objective of this study was to develop an effective potentiometric saturation titration protocol for determining the aqueous intrinsic solubility and the solubility-pH profile of ionizable molecules, with the specific aim of overcoming incomplete dissolution conditions, while attempting to shorten the data collection time. A modern theory of dissolution kinetics (an extension of the Noyes-Whitney approach) was applied to acid-base titration experiments. A thermodynamic method was developed, based on a three-component model, to calculate interfacial, diffusion-layer, and bulk-water reactant concentrations in saturated solutions of ionizable compounds perturbed by additions of acid/base titrant, leading to partial dissolution of the solid material. Ten commercial drugs (cimetidine, diltiazem hydrochloride, enalapril maleate, metoprolol tartrate, nadolol, propoxyphene hydrochloride, quinine hydrochloride, terfenadine, trovafloxacin mesylate, and benzoic acid) were chosen to illustrate the new titration methodology. It was shown that the new method is about 10 times faster in determining equilibrium solubility constants, compared to the traditional saturation shake-flask methods.

  13. Sibutramine characterization and solubility, a theoretical study

    Science.gov (United States)

    Aceves-Hernández, Juan M.; Nicolás Vázquez, Inés; Hinojosa-Torres, Jaime; Penieres Carrillo, Guillermo; Arroyo Razo, Gabriel; Miranda Ruvalcaba, René

    2013-04-01

    Solubility data from sibutramine (SBA) in a family of alcohols were obtained at different temperatures. Sibutramine was characterized by using thermal analysis and X-ray diffraction technique. Solubility data were obtained by the saturation method. The van't Hoff equation was used to obtain the theoretical solubility values and the ideal solvent activity coefficient. No polymorphic phenomena were found from the X-ray diffraction analysis, even though this compound is a racemic mixture of (+) and (-) enantiomers. Theoretical calculations showed that the polarisable continuum model was able to reproduce the solubility and stability of sibutramine molecule in gas phase, water and a family of alcohols at B3LYP/6-311++G (d,p) level of theory. Dielectric constant, dipolar moment and solubility in water values as physical parameters were used in those theoretical calculations for explaining that behavior. Experimental and theoretical results were compared and good agreement was obtained. Sibutramine solubility increased from methanol to 1-octanol in theoretical and experimental results.

  14. Ideal gas solubilities and solubility selectivities in a binary mixture of room-temperature ionic liquids.

    Science.gov (United States)

    Finotello, Alexia; Bara, Jason E; Narayan, Suguna; Camper, Dean; Noble, Richard D

    2008-02-28

    This study focuses on the solubility behaviors of CO2, CH4, and N2 gases in binary mixtures of imidazolium-based room-temperature ionic liquids (RTILs) using 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C2mim][Tf2N]) and 1-ethyl-3-methylimidazolium tetrafluoroborate ([C2mim][BF4]) at 40 degrees C and low pressures (approximately 1 atm). The mixtures tested were 0, 25, 50, 75, 90, 95, and 100 mol % [C2mim][BF4] in [C2mim][Tf2N]. Results show that regular solution theory (RST) can be used to describe the gas solubility and selectivity behaviors in RTIL mixtures using an average mixture solubility parameter or an average measured mixture molar volume. Interestingly, the solubility selectivity, defined as the ratio of gas mole fractions in the RTIL mixture, of CO2 with N2 or CH4 in pure [C2mim][BF4] can be enhanced by adding 5 mol % [C2mim][Tf2N].

  15. Solubility of carbohydrates in heavy water.

    Science.gov (United States)

    Cardoso, Marcus V C; Carvalho, Larissa V C; Sabadini, Edvaldo

    2012-05-15

    The solubility of several mono-(glucose and xylose), di-(sucrose and maltose), tri-(raffinose) and cyclic (α-cyclodextrin) saccharides in H(2)O and in D(2)O were measured over a range of temperatures. The solution enthalpies for the different carbohydrates in the two solvents were determined using the vant' Hoff equation and the values in D(2)O are presented here for the first time. Our findings indicate that the replacement of H(2)O by D(2)O remarkably decreases the solubilities of the less soluble carbohydrates, such as maltose, raffinose and α-cyclodextrin. On the other hand, the more soluble saccharides, glucose, xylose, and sucrose, are practically insensitive to the H/D replacement in water. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Novel electrosprayed nanospherules for enhanced aqueous solubility and oral bioavailability of poorly water-soluble fenofibrate

    Directory of Open Access Journals (Sweden)

    Yousaf AM

    2016-01-01

    Full Text Available Abid Mehmood Yousaf,1,2 Omer Mustapha,1 Dong Wuk Kim,1 Dong Shik Kim,1 Kyeong Soo Kim,1 Sung Giu Jin,1 Chul Soon Yong,3 Yu Seok Youn,4 Yu-Kyoung Oh,5 Jong Oh Kim,3 Han-Gon Choi1 1College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan, Gyeonggi, South Korea; 2Faculty of Pharmacy, University of Central Punjab, Johar, Lahore, Pakistan; 3College of Pharmacy, Yeungnam University, Gyongsan, North Gyeongsang, 4School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi, 5College of Pharmacy, Seoul National University, Seoul, South Korea Purpose: The purpose of the present research was to develop a novel electrosprayed nanospherule providing the most optimized aqueous solubility and oral bioavailability for poorly water-soluble fenofibrate.Methods: Numerous fenofibrate-loaded electrosprayed nanospherules were prepared with polyvinylpyrrolidone (PVP and Labrafil® M 2125 as carriers using the electrospray technique, and the effect of the carriers on drug solubility and solvation was assessed. The solid state characterization of an optimized formulation was conducted by scanning electron microscopy, powder X-ray diffraction, differential scanning calorimetry, and Fourier transform infrared spectroscopic analyses. Oral bioavailability in rats was also evaluated for the formulation of an optimized nanospherule in comparison with free drug and a conventional fenofibrate-loaded solid dispersion.Results: All of the electrosprayed nanospherule formulations had remarkably enhanced aqueous solubility and dissolution compared with free drug. Moreover, Labrafil M 2125, a surfactant, had a positive influence on the solubility and dissolution of the drug in the electrosprayed nanospherule. Increases were observed as the PVP/drug ratio increased to 4:1, but higher ratios gave no significant increases. In particular, an electrosprayed nanospherule composed of fenofibrate, PVP, and Labrafil M 2125 at the weight ratio of 1

  17. Water-soluble dietary fibers and cardiovascular disease.

    Science.gov (United States)

    Theuwissen, Elke; Mensink, Ronald P

    2008-05-23

    One well-established way to reduce the risk of developing cardiovascular disease (CVD) is to lower serum LDL cholesterol levels by reducing saturated fat intake. However, the importance of other dietary approaches, such as increasing the intake of water-soluble dietary fibers is increasingly recognized. Well-controlled intervention studies have now shown that four major water-soluble fiber types-beta-glucan, psyllium, pectin and guar gum-effectively lower serum LDL cholesterol concentrations, without affecting HDL cholesterol or triacylglycerol concentrations. It is estimated that for each additional gram of water-soluble fiber in the diet serum total and LDL cholesterol concentrations decrease by -0.028 mmol/L and -0.029 mmol/L, respectively. Despite large differences in molecular structure, no major differences existed between the different types of water-soluble fiber, suggesting a common underlying mechanism. In this respect, it is most likely that water-soluble fibers lower the (re)absorption of in particular bile acids. As a result hepatic conversion of cholesterol into bile acids increases, which will ultimately lead to increased LDL uptake by the liver. Additionally, epidemiological studies suggest that a diet high in water-soluble fiber is inversely associated with the risk of CVD. These findings underlie current dietary recommendations to increase water-soluble fiber intake.

  18. Transport of soluble species in backfill and rock

    International Nuclear Information System (INIS)

    Chambre, P.L.; Lee, W.W.L.; Light, W.B.; Pigford, T.H.

    1992-03-01

    In this report we study the release and transport of soluble species from spent nuclear fuel. By soluble species we mean a fraction of certain fission product species. Our previously developed methods for calculating release rates of solubility-limited species need to be revised for these soluble species. Here we provide methods of calculating release rates of soluble species directly into rock and into backfill and then into rock. Section 2 gives a brief discussion of the physics of fission products dissolution from U0 2 spent fuel. Section 3 presents the mathematics for calculating release rates of soluble species into backfill and then into rock. The calculation of release rates directly into rock is a special case. Section 4 presents numerical illustrations of the analytic results

  19. 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

    International Nuclear Information System (INIS)

    Liang, B.T.

    1989-01-01

    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)

  20. Solubility limits on radionuclide dissolution

    Energy Technology Data Exchange (ETDEWEB)

    Kerrisk, J.F.

    1984-12-31

    This paper examines the effects of solubility in limiting dissolution rates of a number of important radionuclides from spent fuel and high-level waste. Two simple dissolution models were used for calculations that would be characteristics of a Yucca Mountain repository. A saturation-limited dissolution model, in which the water flowing through the repository is assumed to be saturated with each waste element, is very conservative in that it overestimates dissolution rates. A diffusion-limited dissolution model, in which element-dissolution rates are limited by diffusion of waste elements into water flowing past the waste, is more realistic, but it is subject to some uncertainty at this time. Dissolution rates of some elements (Pu, Am, Sn, Th, Zr, Sm) are always limited by solubility. Dissolution rates of other elements (Cs, Tc, Np, Sr, C, I) are never solubility limited; their release would be limited by dissolution of the bulk waste form. Still other elements (U, Cm, Ni, Ra) show solubility-limited dissolution under some conditions. 9 references, 3 tables.

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

    Science.gov (United States)

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

    1988-10-25

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

  2. Optogenetic long-term manipulation of behavior and animal development.

    Directory of Open Access Journals (Sweden)

    Christian Schultheis

    Full Text Available Channelrhodopsin-2 (ChR2 is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development. We used "slow" ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days with low-intensity light. Photoactivation of ChR2(C128S in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the "dauer" larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.

  3. Cellular signaling with nitric oxide and cyclic GMP

    Directory of Open Access Journals (Sweden)

    F. Murad

    1999-11-01

    Full Text Available During the past two decades, nitric oxide signaling has been one of the most rapidly growing areas in biology. This simple free radical gas can regulate an ever growing list of biological processes. In most instances nitric oxide mediates its biological effects by activating guanylyl cyclase and increasing cyclic GMP synthesis. However, the identification of effects of nitric oxide that are independent of cyclic GMP is also growing at a rapid rate. The effects of nitric oxide can mediate important physiological regulatory events in cell regulation, cell-cell communication and signaling. Nitric oxide can function as an intracellular messenger, neurotransmitter and hormone. However, as with any messenger molecule, there can be too much or too little of the substance and pathological events ensue. Methods to regulate either nitric oxide formation, metabolism or function have been used therapeutically for more than a century as with nitroglycerin therapy. Current and future research should permit the development of an expanded therapeutic armamentarium for the physician to manage effectively a number of important disorders. These expectations have undoubtedly fueled the vast research interests in this simple molecule.

  4. The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies

    Science.gov (United States)

    Iacobucci, Claudio; Piotrowski, Christine; Rehkamp, Anne; Ihling, Christian H.; Sinz, Andrea

    2018-04-01

    Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. [Figure not available: see fulltext.

  5. Hydrogen solubility in austenite of Fe-Ni-Cr alloys

    International Nuclear Information System (INIS)

    Zhirnova, V.V.; Mogutnov, B.M.; Tomilin, I.A.

    1981-01-01

    Hydrogen solubility in Fe-Ni-Cr alloys at 600-1000 deg C is determined. Hydrogen solubility in ternary alloys can not be predicted on the basis of the data on its solubility in binary Fe-Ni, Fe-Cr alloys. Chromium and nickel effect on hydrogen solubility in iron is insignificant in comparison with the effect of these elements on carbon or nitrogen solubility [ru

  6. Carcinogenicity assessment of water-soluble nickel compounds.

    Science.gov (United States)

    Goodman, Julie E; Prueitt, Robyn L; Dodge, David G; Thakali, Sagar

    2009-01-01

    IARC is reassessing the human carcinogenicity of nickel compounds in 2009. To address the inconsistencies among results from studies of water-soluble nickel compounds, we conducted a weight-of-evidence analysis of the relevant epidemiological, toxicological, and carcinogenic mode-of-action data. We found the epidemiological evidence to be limited, in that some, but not all, data suggest that exposure to soluble nickel compounds leads to increased cancer risk in the presence of certain forms of insoluble nickel. Although there is no evidence that soluble nickel acts as a complete carcinogen in animals, there is limited evidence that suggests it may act as a tumor promoter. The mode-of-action data suggest that soluble nickel compounds will not be able to cause genotoxic effects in vivo because they cannot deliver sufficient nickel ions to nuclear sites of target cells. Although the mode-of-action data suggest several possible non-genotoxic effects of the nickel ion, it is unclear whether soluble nickel compounds can elicit these effects in vivo or whether these effects, if elicited, would result in tumor promotion. The mode-of-action data equally support soluble nickel as a promoter or as not being a causal factor in carcinogenesis at all. The weight of evidence does not indicate that soluble nickel compounds are complete carcinogens, and there is only limited evidence that they could act as tumor promoters.

  7. Solubility study of Tc(IV) oxides

    International Nuclear Information System (INIS)

    Liu, D.J.; Fan, X.H.

    2005-01-01

    The deep geological disposal of the high level radioactive wastes is expected to be a safer disposal method in most countries. The long-lived fission product 99 Tc is present in large quantities in nuclear wastes and its chemical behavior in aqueous solution is of considerable interest. Under oxidizing conditions technetium exists as the anionic species TcO 4 - whereas under the reducing conditions, expected to exist in a deep geological repository, it is generally predicted that technetium will be present as TcO 2 ·nH 2 O. Hence, the mobility of Tc(IV) in reducing groundwater may be limited by the solubility of TcO 2 ·nH 2 O under these conditions. Due to this fact it is important to investigate the solubility of TcO 2 ·nH 2 O. The solubility determines the release of radionuclides from waste form and is used as a source term in radionuclide migration analysis in performance assessment of radioactive waste repository. Technetium oxide was prepared by reduction of a technetate solution with Sn 2 + . The solubility of Tc(IV) oxide has been determined in simulated groundwater and redistilled water under aerobic and anaerobic conditions. The effects of pH and CO 3 2- concentration of solution on solubility of Tc(IV) oxide were studied. The concentration of total technetium and Tc(IV) species in the solutions were periodically determined by separating the oxidized and reduced technetium species using a solvent extraction procedure and counting the beta activity of the 99 Tc with a liquid scintillation counter. The experimental results show that the rate of oxidation of Tc(IV) in simulated groundwater and redistilled water is about (1.49-1.86) x 10 -9 mol/(L·d) under aerobic conditions, but Tc(IV) in simulated groundwater and redistilled water is not oxidized under anaerobic conditions. Under aerobic or anaerobic conditions the solubility of Tc(IV) oxide in simulated groundwater and redistilled water is equal on the whole after centrifugation or ultrafiltration. The

  8. A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate

    OpenAIRE

    Pirger, Zsolt; László, Zita; Kemenes, Ildikó; Tóth, Gábor; Reglődi, Dóra; Kemenes, György

    2010-01-01

    Similar to other invertebrate and vertebrate animals, cAMP dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unkno...

  9. Solubility of cefoxitin acid in different solvent systems

    International Nuclear Information System (INIS)

    Yuan, Fuhong; Wang, Yongli; Xiao, Liping; Huang, Qiaoyin; Xu, Jinchao; Jiang, Chen; Hao, Hongxun

    2016-01-01

    Highlights: • The solubility of cefoxitin acid in different solvent systems was measured. • Three models were used to correlate the solubility data. • The dissolution enthalpy of the dissolution process was calculated. - Abstract: Cefoxitin acid is one kind of important pharmaceutical intermediate. Its solubility is crucial for designing and optimizing the crystallization processes. In this work, the solubility of cefoxitin acid in organic solvents (methanol, acetonitrile, ethanol, isopropanol, n-propanol and ethyl acetate), water and water-methanol mixtures was measured spectrophotometrically using a shake-flask method within the temperature range 278.15–303.15 K. PXRD data and the Karl Fischer method were used to verify the crystal form stability of cefoxitin acid in the solubility measuring process. The melting points, the enthalpy and entropy of fusion were estimated. Results showed that the solubility of cefoxitin acid increases with the increasing temperature in all tested solvents in this work, and the solubility of cefoxitin acid increases with the increasing methanol concentration in water-methanol mixtures. The experimental solubility values were well correlated using the modified Apelblat equation, NRTL model and CNIBS/R-K model. An equation proposed by Williamson was adopted to calculate the molar enthalpy during the dissolution process.

  10. Active intestinal drug absorption and the solubility-permeability interplay.

    Science.gov (United States)

    Porat, Daniel; Dahan, Arik

    2018-02-15

    The solubility-permeability interplay deals with the question: what is the concomitant effect on the drug's apparent permeability when increasing the apparent solubility with a solubility-enabling formulation? The solubility and the permeability are closely related, exhibit certain interplay between them, and ongoing research throughout the past decade shows that treating the one irrespectively of the other may be insufficient. The aim of this article is to provide an overview of the current knowledge on the solubility-permeability interplay when using solubility-enabling formulations for oral lipophilic drugs, highlighting active permeability aspects. A solubility-enabling formulation may affect the permeability in opposite directions; the passive permeability may decrease as a result of the apparent solubility increase, according to the solubility-permeability tradeoff, but at the same time, certain components of the formulation may inhibit/saturate efflux transporters (when relevant), resulting in significant apparent permeability increase. In these cases, excipients with both solubilizing and e.g. P-gp inhibitory properties may lead to concomitant increase of both the solubility and the permeability. Intelligent development of such formulation will account for the simultaneous effects of the excipients' nature/concentrations on the two arms composing the overall permeability: the passive and the active arms. Overall, thorough mechanistic understanding of the various factors involved in the solubility-permeability interplay may allow developing better solubility-enabling formulations, thereby exploiting the advantages analyzed in this article, offering oral delivery solution even for BCS class IV drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Study of solubility of some metal cyclohexane carbonates

    International Nuclear Information System (INIS)

    Niyazov, A.N.; Amanov, K.B.; Trapeznikova, V.F.; Kul'maksimov, A.; Kolosova, N.

    1978-01-01

    The solubility of calcium, magnesium, strontium, barium, cabalt, copper and aluminium cyclohexane, carbonates (CHC) in water has been studied at 25 deg C. The salt solubility has been calculated according to the metal ion concentration in saturated solutions. It has been established, that the cobalt and rare earth cyclohexane carbonates are relatively very soluble in water and have solubility products of SP > 1x10 -5 . The solubility of CHC of multivalent metals increases with the decrease of pH values. Each salt has some ''limiting'' pH value of a solution, below which it decomposes completely and can not exist in a solution in the form of solid phase

  12. Hydrothermal solubility of uraninite. Final technical report

    International Nuclear Information System (INIS)

    Parks, G.A.; Pohl, D.C.

    1985-01-01

    Experimental measurements of the solubility of UO 2 from 100 to 300 0 C under 500 bars H 2 , in NaCl solutions at pH from 1 to 8 do not agree with solubilities calculated using existing thermodynamic databases. For pH 2 (hyd) has precipitated and is controlling solubility. For pH > 8, solubilities at all temperatures are much lower than predicted, suggesting that the U(OH)/sub delta/ - complex is much weaker than predicted. Extrapolated to 25 0 C, high pH solubility agrees within experimental error with the upper limit suggested by Ryan and Rai (1983). In the pH range 2 to 6, solubilities are up to three orders of magnitude lower than predicted for temperatures exceeding 200 0 C and up to two orders higher than predicted at lower temperatures. pH dependence in this region is negligible suggesting that U(OH) 4 (aq) predominates, thus the stability of this species is higher than presently estimated at low temperatures, but the enthalpy of solution is smaller. A low maximum observed near pH approx. =3 is presently unexplained. 40 refs., 16 figs., 12 tabs

  13. Comparison between dopamine-stimulated adenylate cyclase and 3H-SCH 23390 binding in rat striatum

    International Nuclear Information System (INIS)

    Andersen, P.H.; Groenvald, F.C.; Jansen, J.A.

    1985-01-01

    Methods for measuring 3 H-SCH 23990 binding and dopamine (DA) stimulated adenylate cyclase (AC) were established in identical tissue preparations and under similar experimental conditions. Pharmacological characterization revealed that both assays involved interaction with the D1 receptor or closely associated sites. In order to investigate whether the binding sites for 3 H-SCH 23390 and DA in fact are identical, the antagonistic effects of a variety of pharmacologically active compounds were examined. Surprisingly, the K/sub i/-values obtained from Schild-plot analysis of the antagonism of DA-stimulated AC, were 80-240 times higher than the K/sub i/-values obtained from competition curves of 3 H-SCH 23390 binding. Since both assays were performed under identical conditions, the differences in K/sub i/-values indicate the possibility of different binding sites for DA and 3 H-SCH 23390 or, that DA and 3 -SCH 23390 label different states of the same receptor. 19 references, 7 figures, 2 tables

  14. Diagnosing solubility limitations – the example of hydrate formation

    Directory of Open Access Journals (Sweden)

    Joerg Berghausen

    2014-07-01

    Full Text Available Solubility is regarded as one of the key challenges in many drug discovery projects. Thus, it’s essential to support the lead finding and optimization efforts by appropriate solubility data. In silico solubility prediction remains challenging and therefore a screening assay is used as a first filter, followed by selected follow-up assays to reveal what causes the low solubility of a specific compound or chemotype. Results from diagnosing the underlying reason for solubility limitation are discussed. As lipophilicity and crystal lattice forces are regarded as main contributors to limiting solubility, changes in solid state are important to be recognized. Solubility limitation by various factors will be presented and the impact of the solid-state is exemplified by compounds that are able to form hydrates.

  15. Serum Soluble Corin is Decreased in Stroke.

    Science.gov (United States)

    Peng, Hao; Zhu, Fangfang; Shi, Jijun; Han, Xiujie; Zhou, Dan; Liu, Yan; Zhi, Zhongwen; Zhang, Fuding; Shen, Yun; Ma, Juanjuan; Song, Yulin; Hu, Weidong

    2015-07-01

    Soluble corin was decreased in coronary heart disease. Given the connections between cardiac dysfunction and stroke, circulating corin might be a candidate marker of stroke risk. However, the association between circulating corin and stroke has not yet been studied in humans. Here, we aimed to examine the association in patients wtith stroke and community-based healthy controls. Four hundred eighty-one patients with ischemic stroke, 116 patients with hemorrhagic stroke, and 2498 healthy controls were studied. Serum soluble corin and some conventional risk factors of stroke were examined. Because circulating corin was reported to be varied between men and women, the association between serum soluble corin and stroke was evaluated in men and women, respectively. Patients with ischemic and hemorrhagic stroke had a significantly lower level of serum soluble corin than healthy controls in men and women (all P values, stroke than men in the highest quartile. Women in the lowest quartile of serum soluble corin were also more likely to have ischemic (OR, 3.10; 95% confidence interval, 1.76-5.44) and hemorrhagic (OR, 8.54; 95% confidence interval, 2.35-31.02) stroke than women in the highest quartile. ORs of ischemic and hemorrhagic stroke were significantly increased with the decreasing levels of serum soluble corin in men and women (all P values for trend, stroke compared with healthy controls. Our findings raise the possibility that serum soluble corin may have a pathogenic role in stroke. © 2015 American Heart Association, Inc.

  16. Solubility and degradation of paracetamol in subcritical water

    Directory of Open Access Journals (Sweden)

    Emire Zuhal

    2017-01-01

    Full Text Available In this study, solubility and degradation of paracetamol were examined using subcritical water. Effect of temperature and static time was investigated during solubility process in subcritical water at constant pressure (50 bar. Experimental results show that temperature and static time have crucial effect on the degradation and solubility rates. Maximum mole fraction for solubility of paracetamol was obtained at 403 K as (14.68 ± 0.74×103. Approximation model for solubility of paracetamol was proposed. O2 and H2O2 were used in degradation process of paracetamol. Maximum degradation rate was found as 68.66 ± 1.05 and 100 ± 0.00 % using O2 and H2O2, respectively.

  17. Solubility of hydrogen in bio-oil compounds

    International Nuclear Information System (INIS)

    Qureshi, Muhammad Saad; Touronen, Jouni; Uusi-Kyyny, Petri; Richon, Dominique; Alopaeus, Ville

    2016-01-01

    Highlights: • Solubility of Hydrogen was measured in bio-oil compounds in the at temperatures from 342 to 473 K and pressures up to 16 MPa. • Phase equilibrium data were acquired using a visualization enabled continuous flow synthetic apparatus. • The measured solubility is modeled with Peng-Robinson EoS. - Abstract: The knowledge of accurate hydrogen solubility values in bio-oil compounds is essential for the design and optimization of hydroprocesses relevant to biofuel industry. This work reports the solubility of hydrogen in three industrially relevant bio-oil compounds (allyl alcohol, furan, and eugenol) at temperatures from 342 to 473 K and pressures up to 16 MPa. Phase equilibrium data were acquired using a continuous flow synthetic method. The method is based on the visual observation of the bubble point using a high resolution camera. The measured solubility is modeled with Peng-Robinson EoS with classical van der Waals one fluid mixing rules.

  18. Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction.

    Science.gov (United States)

    Brownfoot, Fiona C; Hastie, Roxanne; Hannan, Natalie J; Cannon, Ping; Tuohey, Laura; Parry, Laura J; Senadheera, Sevvandi; Illanes, Sebastian E; Kaitu'u-Lino, Tu'uhevaha J; Tong, Stephen

    2016-03-01

    Preeclampsia is associated with placental ischemia/hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble endoglin into the maternal circulation. This causes widespread endothelial dysfunction that manifests clinically as hypertension and multisystem organ injury. Recently, small molecule inhibitors of hypoxic inducible factor 1α have been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin secretion. However, their safety profile in pregnancy is unknown. Metformin is safe in pregnancy and is also reported to inhibit hypoxic inducible factor 1α by reducing mitochondrial electron transport chain activity. The purposes of this study were to determine (1) the effects of metformin on placental soluble fms-like tyrosine kinase 1 and soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion are regulated through the mitochondrial electron transport chain, and (3) to examine its effects on endothelial dysfunction, maternal blood vessel vasodilation, and angiogenesis. We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from placenta, endothelial cells, and placental villous explants. We used succinate, mitochondrial complex II substrate, to examine whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion were mediated through the mitochondria. We also isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron transport chain activity using kinetic spectrophotometric assays. Endothelial cells or whole maternal vessels were incubated with metformin to determine whether it rescued endothelial dysfunction induced by either tumor necrosis factor-α (to endothelial cells) or placenta villous

  19. The solubility of palladium(II) bis-dimethylglyoximate

    International Nuclear Information System (INIS)

    Maghzian, R.

    1978-01-01

    The solubility of palladium(II) bis-dimethylglyoximate in different solutions has been determined. Values obtained for the solubility of the palladium complex are tabulated. The solubility is the lowest in water, ammonium acetate and a 25% acetone-water mixture. It is highest in dilute HCl and acetone but precipitation from aqueous acetone should be satisfactory for most purposes if the acetone content of the solvent is roughly less than 50% by volume. The solubility in dilute HCl reflects the concern by previous workers for losses in precipitation from mineral acid. In general, however, the losses are unlikely to be significant unless the quantity of palladium to be precipitated and weighed is small. (T.G.)

  20. Solubility and physical properties of sugars in pressurized water

    International Nuclear Information System (INIS)

    Saldaña, Marleny D.A.; Alvarez, Víctor H.; Haldar, Anupam

    2012-01-01

    Highlights: ► Sugar solubility in pressurized water and density at high pressures were measured. ► Glucose solubility was higher than that of lactose as predicted by their σ-profiles. ► Sugar aqueous solubility decreased with an increase in pressure from 15 to 120 bar. ► Aqueous glucose molecular packing shows high sensitivity to pressure. ► The COSMO-SAC model qualitatively predicted the sugar solubility data. - Abstract: In this study, the solubility, density, and refractive index of glucose and lactose in water as a function of temperature were measured. For solubility of sugars in pressurized water, experimental data were obtained at pressures of (15 to 120) bar and temperatures of (373 to 433) K using a dynamic flow high pressure system. Density data for aqueous sugar solutions were obtained at pressures of (1 to 300) bar and temperatures of (298 to 343) K. The refractive index of aqueous sugar solutions was obtained at 293 K and atmospheric pressure. Activity coefficient models, Van Laar and the Conductor-like Screening Model-Segment Activity Coefficient (COSMO-SAC), were used to fit and predict the experimental solubility data, respectively. The results obtained showed that the solubility of both sugars in pressurized water increase with an increase in temperature. However, with the increase of pressure from 15 bar to 120 bar, the solubility of both sugars in pressurized water decreased. The Van Laar model fit the experimental aqueous solubility data with deviations lower than 13 and 53% for glucose and lactose, respectively. The COSMO-SAC model predicted qualitatively the aqueous solubility of these sugars.

  1. Dual Activity of Hydroxypropyl-β-Cyclodextrin and Water-Soluble Carriers on the Solubility of Carvedilol.

    Science.gov (United States)

    Zoghbi, Abdelmoumin; Geng, Tianjiao; Wang, Bo

    2017-11-01

    Carvedilol (CAR) is a non-selective α and β blocker categorized as class II drug with low water solubility. Several recent studies have investigated ways to overcome this problem. The aim of the present study was to combine two of these methods: the inclusion complex using hydroxypropyl-β-cyclodextrin (HPβCD) with solid dispersion using two carriers: Poloxamer 188 (PLX) and Polyvinylpyrrolidone K-30 (PVP) to enhance the solubility, bioavailability, and the stability of CAR. Kneading method was used to prepare CAR-HPβCD inclusion complex (KD). The action of different carriers separately and in combination on Carvedilol solubility was investigated in three series. CAR-carrier and KD-carrier solid dispersions were prepared by solvent evaporation method. In vitro dissolution test was conducted in three different media: double-distilled water (DDW), simulative gastric fluid (SGF), and PBS pH 6.8 (PBS). The interactions between CAR, HPβCD, and different carriers were explored by Fourier transform infrared spectroscopy (FTIR), powder X-ray diffractometry (XRD), and differential scanning colorimetry (DSC). The results showed higher solubility of CAR in KD-PVP solid dispersions up to 70, 25, and 22 fold compared to pure CAR in DDW, SGF, and PBS, respectively. DSC and XRD analyses indicated an improved degree of transformation of CAR in KD-PVP solid dispersion from crystalline to amorphous state. This study provides a new successful combination of two polymers with the dual action of HPβCD and PLX/PVP on water solubility and bioavailability of CAR.

  2. Effect of Cyclodextrin Complexation on the Aqueous Solubility and Solubility/Dose Ratio of Praziquantel

    OpenAIRE

    Maragos, Stratos; Archontaki, Helen; Macheras, Panos; Valsami, Georgia

    2009-01-01

    Praziquantel (PZQ), the primary drug of choice in the treatment of schistosomiasis, is a highly lipophilic drug that possesses high permeability and low aqueous solubility and is, therefore, classified as a Class II drug according to the Biopharmaceutics Classification System (BCS). In this work, β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) were used in order to determine whether increasing the aqueous solubility of a drug by complexation with CDs, a BCS-Class II compound ...

  3. Research on solubility characteristics of gaseous methyl iodide

    International Nuclear Information System (INIS)

    Zhou Yanmin; Sun Zhongning; Gu Haifeng; Wang Junlong

    2014-01-01

    With the deionized water as the absorbent, the solubility characteristics of the gaseous methyl iodide were studied under different temperature and pressure conditions, using a dynamic measuring method. The results show that within the range of experiment parameters, namely temperature is below 80℃ and pressure is lower than 0.3 MPa, the physical dissolution process of gaseous methyl iodide in water obeys Henry's law. The solubility coefficient under different temperature and pressure conditions was calculated based on the measurement results. Further research indicates that at atmospheric pressure, the solubility coefficient of methyl iodide in water decreases exponentially with the increase of temperature. While the pressure changes from 0.1 MPa to 0.3 MPa with equal interval, the solubility coefficient also increases linearly. The variation of the solubility coefficient with temperature under different pressure conditions all decreases exponentially. An equation is given to calculate the solubility coefficient of methyl iodide under different pressure and temperature conditions. (authors)

  4. Uranium solubility and speciation in ground water

    International Nuclear Information System (INIS)

    Ollila, K.

    1985-04-01

    The purpose of this study has been to assess the solubility and possible species of uranium in groundwater at the disposal conditions of spent fuel. The effects of radiolysis and bentonite are considered. The assessment is based on the theoretical calculations found in the literature. The Finnish experimental results are included. The conservative estimate for uranium solubility under the oxidizing conditions caused by alpha radiolysis is based on the oxidation of uranium to the U(VI) state and formation of carbonate complex. For the groundwater with the typical carbonate content of 275 mg/l and the high carbonate content of 485 mg/l due to bentonite, the solubility values of 360 mg u/l and 950 mg U/l, are obtained, respectively. The experimental results predict considerably lower values, 0.5-20 mg U/l. The solubility of uranium under the undisturbed reducing conditions may be calculated based on the hydrolysis, carbonate complexation and redox reactions. The results vary considerably depending on the thermodynamic data used. The wide ranges of the most important groundwater parameters are seen in the solubility values. The experimental results show the same trends. As a conservative value for the solubility in reducing groundwater 50-500 μg U/l is estimated. (author)

  5. Pituitary adenylate cyclase activating polypeptide in stress-related disorders: data convergence from animal and human studies.

    Science.gov (United States)

    Hammack, Sayamwong E; May, Victor

    2015-08-01

    The maladaptive expression and function of several stress-associated hormones have been implicated in pathological stress and anxiety-related disorders. Among these, recent evidence has suggested that pituitary adenylate cyclase activating polypeptide (PACAP) has critical roles in central neurocircuits mediating stress-related emotional behaviors. We describe the PACAPergic systems, the data implicating PACAP in stress biology, and how altered PACAP expression and signaling may result in psychopathologies. We include our work implicating PACAP signaling within the bed nucleus of the stria terminalis in mediating the consequences of stressor exposure and relatedly, describe more recent studies suggesting that PACAP in the central nucleus of the amygdala may impact the emotional aspects of chronic pain states. In aggregate, these results are consistent with data suggesting that PACAP dysregulation is associated with posttraumatic stress disorder in humans. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  6. Sildenafil, a selective phosphodiesterase type 5 inhibitor, enhances memory reconsolidation of an inhibitory avoidance task in mice.

    Science.gov (United States)

    Boccia, M M; Blake, M G; Krawczyk, M C; Baratti, C M

    2011-07-07

    Intracellular levels of the second messengers cAMP and cGMP are maintained through a balance between production, carried out by adenyl cyclase (AC) and guanylyl cyclase (GC), and degradation, carried out by phosphodiesterases (PDEs). Recently, PDEs have gained increased attention as potential new targets for cognition enhancement, with particular reference to phosphodiesterase type 5 (PDE5A). It is accepted that once consolidation is completed memory becomes permanent, but it has also been suggested that reactivation (memory retrieval) of the original memory makes it sensitive to the same treatments that affect memory consolidation when given after training. This new period of sensitivity coined the term reconsolidation. Sildenafil (1, 3, and 10mg/kg, ip), a cGMP-PDE5 inhibitor, facilitated retention performance of a one-trial step-through inhibitory avoidance task, when administered to CF-1 male mice immediately after retrieval. The effects of sildenafil (1mg/kg, ip) were time-dependent, long-lasting and inversely correlated with memory age. The administration of sildenafil (1mg/kg, ip) 30 min prior to the 2nd retention test did not affect retention of mice given post-retrieval injections of either vehicle or sildenafil (1mg/kg, ip). Finally, an enhancement of retention was also observed in CF-1 female mice receiving sildenafil (1mg/kg, ip) immediately, but not 180 min after retrieval. In the present paper we reported for the first time that systemic administration of sildenafil after memory reactivation enhances retention performance of the original learning. Our results indirectly point out cGMP, a component of the NO/cGMP/PKG pathway, as a necessary factor for memory reconsolidation. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Molecular properties of mammalian proteins that interact with cGMP: protein kinases, cation channels, phosphodiesterases, and multi-drug anion transporters.

    Science.gov (United States)

    Francis, Sharron H; Blount, Mitsi A; Zoraghi, Roya; Corbin, Jackie D

    2005-09-01

    Cyclic GMP is a critical second messenger signaling molecule in many mammalian cell types. It is synthesized by a family of guanylyl cyclases that is activated in response to stimuli from hormones such as natriuretic peptides, members of the guanylin family, and chemical stimuli including nitric oxide and carbon monoxide. The resulting elevation of cGMP modulates myriad physiological processes. Three major groups of cellular proteins bind cGMP specifically at allosteric sites; interaction of cGMP with these sites modulates the activities and functions of other domains within these protein groups to bring about physiological effects. These proteins include the cyclic nucleotide (cN)-dependent protein kinases, cN-gated cation channels, and cGMP-binding phosphodiesterases (PDE). Cyclic GMP also interacts with the catalytic sites of many cN PDEs and with some members of the multi-drug anion transporter family (MRPs) which can extrude nucleotides from cells. The allosteric cN-binding sites in the kinases and the cN-gated channels are evolutionarily and biochemically related, whereas the allosteric cGMP-binding sites in PDEs (also known as GAF domains), the catalytic sites of PDEs , and the ligand-binding sites in the MRPs are evolutionarily and biochemically distinct from each other and from those in the kinase and channel families. The sites that interact with cGMP within each of these groups of proteins have unique properties that provide for cGMP binding. Within a given cell, cGMP can potentially interact with members of all these groups of proteins if they are present. The relative abundance and affinities of these various cGMP-binding sites in conjunction with their subcellular compartmentation, proximity to cyclases and PDEs, and post-translational modification contribute importantly in determining the impact of these respective proteins to cGMP signaling within a particular cell.

  8. Dry season aerosol iron solubility in tropical northern Australia

    Directory of Open Access Journals (Sweden)

    V. H. L. Winton

    2016-10-01

    Full Text Available Marine nitrogen fixation is co-limited by the supply of iron (Fe and phosphorus in large regions of the global ocean. The deposition of soluble aerosol Fe can initiate nitrogen fixation and trigger toxic algal blooms in nitrate-poor tropical waters. We present dry season soluble Fe data from the Savannah Fires in the Early Dry Season (SAFIRED campaign in northern Australia that reflects coincident dust and biomass burning sources of soluble aerosol Fe. The mean soluble and total aerosol Fe concentrations were 40 and 500 ng m−3 respectively. Our results show that while biomass burning species may not be a direct source of soluble Fe, biomass burning may substantially enhance the solubility of mineral dust. We observed fractional Fe solubility up to 12 % in mixed aerosols. Thus, Fe in dust may be more soluble in the tropics compared to higher latitudes due to higher concentrations of biomass-burning-derived reactive organic species in the atmosphere. In addition, biomass-burning-derived particles can act as a surface for aerosol Fe to bind during atmospheric transport and subsequently be released to the ocean upon deposition. As the aerosol loading is dominated by biomass burning emissions over the tropical waters in the dry season, additions of biomass-burning-derived soluble Fe could have harmful consequences for initiating nitrogen-fixing toxic algal blooms. Future research is required to quantify biomass-burning-derived particle sources of soluble Fe over tropical waters.

  9. A systematic evaluation of solubility enhancing excipients to enable the generation of permeability data for poorly soluble compounds in Caco-2 model.

    Science.gov (United States)

    Shah, Devang; Paruchury, Sundeep; Matta, Muralikrishna; Chowan, Gajendra; Subramanian, Murali; Saxena, Ajay; Soars, Matthew G; Herbst, John; Haskell, Roy; Marathe, Punit; Mandlekar, Sandhya

    2014-01-01

    The study presented here identified and utilized a panel of solubility enhancing excipients to enable the generation of flux data in the Human colon carcinoma (Caco-2) system for compounds with poor solubility. Solubility enhancing excipients Dimethyl acetamide (DMA) 1 % v/v, polyethylene glycol (PEG) 400 1% v/v, povidone 1% w/v, poloxamer 188 2.5% w/v and bovine serum albumin (BSA) 4% w/v did not compromise Caco-2 monolayer integrity as assessed by trans-epithelial resistance measurement (TEER) and Lucifer yellow (LY) permeation. Further, these excipients did not affect P-glycoprotein (P-gp) mediated bidirectional transport of digoxin, permeabilities of high (propranolol) or low permeability (atenolol) compounds, and were found to be inert to Breast cancer resistant protein (BCRP) mediated transport of cladribine. This approach was validated further using poorly soluble tool compounds, atazanavir (poloxamer 188 2.5% w/v) and cyclosporine A (BSA 4% w/v) and also applied to new chemical entity (NCE) BMS-A in BSA 4% w/v, for which Caco-2 data could not be generated using the traditional methodology due to poor solubility (solubility of atazanavir by >8 fold whereas BSA 4% w/v increased the solubility of cyclosporine A and BMS-A by >2-4 fold thereby enabling permeability as well as efflux liability estimation in the Caco-2 model with reasonable recovery values. To conclude, addition of excipients such as poloxamer 188 2.5% w/v and BSA 4% w/v to HBSS leads to a significant improvement in the solubility of the poorly soluble compounds resulting in enhanced recoveries without modulating transporter-mediated efflux, expanding the applicability of Caco-2 assays to poorly soluble compounds.

  10. Nanosuspension Technology for Solubilizing Poorly Soluble Drugs

    OpenAIRE

    Deoli Mukesh

    2012-01-01

    Poor water solubility for many drugs and drug candidates remains a major obstacle to their development and clinical application. It is estimated that around 40% of drugs in the pipeline cannot be delivered through the preferred route or in some cases, at all owing to poor water solubility. Conventional formulations to improve solubility suffer from low bioavailability and poor pharmacokinetics, with some carriers rendering systemic toxicities (e.g. Cremophor1 EL). To date, nanoscale systems f...

  11. Phenylephrine Decreases Vascular Tension in Goat Arteries in Specific Circumstances.

    Directory of Open Access Journals (Sweden)

    Renu R Raj

    Full Text Available Phenylephrine (PE causes vasoconstriction through alpha adrenergic receptors. PE-induced vasodilatation has also been reported earlier in pre-constricted vessels. Here we demonstrate in spiral strips of goat arteries that addition of PE can decrease tone even from base-line levels (i.e. not pre-constricted and show that this process requires nitric oxide (NO and alpha adrenergic stimulation, but is cGMP-independent. Under control conditions, PE caused vasoconstriction, but under conditions where NO levels are higher, as with L-Arginine or sodium nitroprusside, PE decreased vessel tension. L-Arginine/PE combination was not able to decrease tension when alpha adrenoceptors were blocked with Phentolamine or endothelial nitric oxide synthase (eNOS was blocked with Nω-Nitro-L-arginine (L-NNA. Propranolol, a beta blocker, was unable to prevent the reduction in tension by the L-Arginine/PE combination. Adrenaline and noradrenaline (and not isoproterenol also reduced vessel tension in the presence of L-Arginine. Even when NO levels were not enhanced, relieving NO from having to stimulate the enzyme soluble guanylyl cyclase (sGC (either by using sGC blockers, namely ODQ or methylene blue, or by enhancing cGMP levels (with sildenafil which by negative feedback probably inhibits sGC led to PE-induced reduction of vascular tension. PMA-phorbol myristate acetate-an agonist which stimulates Protein Kinase C was able to prevent the ability of PE to reduce vascular tension in a high NO environment. Our conclusion is that PE reduces vascular tension through alpha adrenoceptors if there is excess NO availability to activate a putative pathway. Though the reduction of vessel tone by PE is dependent on NO, it is independent of cGMP. Prior treatment with PMA or PE itself can prevent further PE-induced reduction of tension in a high NO environment. The results here suggest, counter-intuitively, that alpha blockers may be of help in the treatment of septic shock

  12. Identification of mechanisms involved in the relaxation of rabbit cavernous smooth muscle by a new nitric oxide donor ruthenium compound

    Directory of Open Access Journals (Sweden)

    João Batista Gadelha de Cerqueira

    2012-10-01

    Full Text Available PURPOSE: The aim of this study was to evaluate the relaxation in vitro of cavernous smooth muscle induced by a new NO donor of the complex nitrosil-ruthenium, named trans-[Ru(NH34(caffeine(NO]C13 (Rut-Caf and sodium nitroprusside (SNP. MATERIALS AND METHODS: The tissues, immersed in isolated bath systems, were pre-contracted with phenilephrine (PE (1 µM and then concentration-response curves (10-12 - 10-4 M were obtained. To clarify the mechanism of action involved, it was added to the baths ODQ (10 µM, 30 µM, oxyhemoglobin (10 µM, L-cysteine (100 µM, hydroxicobalamine (100 µM, glibenclamide, iberotoxin and apamine. Tissue samples were frozen in liquid nitrogen to measure the amount of cGMP and cAMP produced. RESULTS: The substances provoked significant relaxation of the cavernous smooth muscle. Both Rut-Caf and SNP determined dose-dependent relaxation with similar potency (pEC50 and maximum effect (Emax. The substances showed activity through activation of the soluble guanylyl cyclase (sGC, because the relaxations were inhibited by ODQ. Oxyhemoglobin significantly diminished the relaxation effect of the substances. L-cysteine failed to modify the relaxations caused by the agents. Hydroxicobalamine significantly diminished the relaxation effect of Rut-Caf. Glibenclamide significantly increased the efficacy of Rut-Caf (pEC50 4.09 x 7.09. There were no alterations of potency or maximum effect of the substances with the addition of the other ion channel blockers. Rut-Caf induced production of significant amounts of cGMP and cAMP during the relaxation process. CONCLUSIONS: In conclusion, Rut-Caf causes relaxation of smooth muscle of corpus cavernosum by means of activation of sGC with intracellular production of cGMP and cAMP; and also by release of NO in the intracellular environment. Rut-Caf releases the NO free radical and it does not act directly on the potassium ion channels.

  13. Antidepressant effect of pramipexole in mice forced swimming test: A cross talk between dopamine receptor and NMDA/nitric oxide/cGMP pathway.

    Science.gov (United States)

    Ostadhadi, Sattar; Imran Khan, Muhammad; Norouzi-Javidan, Abbas; Dehpour, Ahmad-Reza

    2016-07-01

    Pramipexole is a dopamine D2 receptor agonist indicated for treating Parkinson disorder. This study was aimed to investigate the effect of pramipexole in forced swimming test (FST) in mice and the possible involvement of activation of D2 receptors and inhibition of N-methyl-d-aspartate (NMDA) receptors and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) on this effect. Intraperitoneal administration of pramipexole (1-3mg/kg) reduced the immobility time in the FST similar to fluoxetine (20mg/kg, i.p.). This effect of pramipexole (1mg/kg, i.p.) was ceased when mice were pretreated with haloperidol (0.15mg/kg, i.p,) and sulpiride (5mg/kg, i.p) as D2 receptor antagonists, NMDA (75mg/kg,i.p.), l-arginine (750mg/kg, i.p., a substrate for nitric oxide synthase) or sildenafil (5mg/kg, i.p., a phosphodiesterase 5 inhibitor). The administration of MK-801 (0.05mg/kg, i.p., a NMDA receptor antagonist) l-NG-Nitro arginine methyl ester (l-NAME, 10mg/kg, i.p., a non-specific nitric oxide synthase (NOS) inhibitor), 7-nitroindazole (30mg/kg, i.p., a neuronal NOS inhibitor) and methylene blue (10mg/kg, i.p.), an inhibitor of both NOS and soluble guanylyl cyclase (sGC) in combination with the sub-effective dose of pramipexole (0.3mg/kg, i.p.) reduced the immobility. Altogether, our data suggest that the antidepressant-like effect of pramipexole is dependent on the activation of D2 receptor and inhibition of either NMDA receptors and/or NO-cGMP synthesis. These results contribute to the understanding of the mechanisms underlying the antidepressant-like effect of pramipexole and reinforce the role of D2 receptors, NMDA receptors and l-arginine-NO-GMP pathway in the antidepressant mechanism of this agent. Copyright © 2016. Published by Elsevier Masson SAS.

  14. Cyclic GMP-mediated memory enhancement in the object recognition test by inhibitors of phosphodiesterase-2 in mice.

    Science.gov (United States)

    Lueptow, Lindsay M; Zhan, Chang-Guo; O'Donnell, James M

    2016-02-01

    Cyclic nucleotide phosphodiesterase-2 (PDE2) is a potential therapeutic target for the treatment of cognitive dysfunction. Using the object recognition test (ORT), this study assessed the effects of two PDE2 inhibitors, Bay 60-7550 and ND7001, on learning and memory, and examined underlying mechanisms. To assess the role of PDE2 inhibition on phases of memory, Bay 60-7550 (3 mg/kg) was administered: 30 min prior to training; 0, 1, or 3 h after training; or 30 min prior to recall testing. To assess cyclic nucleotide involvement in PDE2 inhibitor-enhanced memory consolidation, either the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 20 mg/kg; intraperitoneal (IP)), soluble guanylyl cyclase inhibitor 1H-[-1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; 20 mg/kg; IP), protein kinase G inhibitor KT5823 (2.5 μg; intracerebroventricular (ICV)), or protein kinase A inhibitor H89 (1 μg; ICV) was administered 30 min prior to the PDE2 inhibitor Bay 60-7550 (3 mg/kg) or ND7001 (3 mg/kg). Changes in the phosphorylation of 3'5'-cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) at Ser-133 and vasodilator-stimulated phosphoprotein (VASP) at Ser-239 were determined to confirm activation of cAMP and 3'5'-cyclic guanosine monophosphate (cGMP) signaling. Bay 60-7550 (3 mg/kg) enhanced memory of mice in the ORT when given 30 min prior to training, immediately after training, or 30 min prior to recall. Inhibitors of the cGMP pathway blocked the memory-enhancing effects of both Bay 60-7550 (3 mg/kg) and ND7001 (3 mg/kg) on early consolidation processes. Bay 60-7550 (3 mg/kg) enhanced phosphorylation of CREB and VASP, both targets of cGMP-dependent protein kinase (PKG). These results confirm a potential of PDE2, or components of its signaling pathway, as a therapeutic target for drug discovery focused on restoring memory function.

  15. Stimulation of the cardiac myocyte Na+-K+ pump due to reversal of its constitutive oxidative inhibition.

    Science.gov (United States)

    Chia, Karin K M; Liu, Chia-Chi; Hamilton, Elisha J; Garcia, Alvaro; Fry, Natasha A; Hannam, William; Figtree, Gemma A; Rasmussen, Helge H

    2015-08-15

    Protein kinase C can activate NADPH oxidase and induce glutathionylation of the β1-Na(+)-K(+) pump subunit, inhibiting activity of the catalytic α-subunit. To examine if signaling of nitric oxide-induced soluble guanylyl cyclase (sGC)/cGMP/protein kinase G can cause Na(+)-K(+) pump stimulation by counteracting PKC/NADPH oxidase-dependent inhibition, cardiac myocytes were exposed to ANG II to activate NADPH oxidase and inhibit Na(+)-K(+) pump current (Ip). Coexposure to 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) to stimulate sGC prevented the decrease of Ip. Prevention of the decrease was abolished by inhibition of protein phosphatases (PP) 2A but not by inhibition of PP1, and it was reproduced by an activator of PP2A. Consistent with a reciprocal relationship between β1-Na(+)-K(+) pump subunit glutathionylation and pump activity, YC-1 decreased ANG II-induced β1-subunit glutathionylation. The decrease induced by YC-1 was abolished by a PP2A inhibitor. YC-1 decreased phosphorylation of the cytosolic p47(phox) NADPH oxidase subunit and its coimmunoprecipitation with the membranous p22(phox) subunit, and it decreased O2 (·-)-sensitive dihydroethidium fluorescence of myocytes. Addition of recombinant PP2A to myocyte lysate decreased phosphorylation of p47(phox) indicating the subunit could be a substrate for PP2A. The effects of YC-1 to decrease coimmunoprecipitation of p22(phox) and p47(phox) NADPH oxidase subunits and decrease β1-Na(+)-K(+) pump subunit glutathionylation were reproduced by activation of nitric oxide-dependent receptor signaling. We conclude that sGC activation in cardiac myocytes causes a PP2A-dependent decrease in NADPH oxidase activity and a decrease in β1 pump subunit glutathionylation. This could account for pump stimulation with neurohormonal oxidative stress expected in vivo. Copyright © 2015 the American Physiological Society.

  16. Cardiac hyporesponsiveness in severe sepsis is associated with nitric oxide-dependent activation of G protein receptor kinase.

    Science.gov (United States)

    Dal-Secco, Daniela; DalBó, Silvia; Lautherbach, Natalia E S; Gava, Fábio N; Celes, Mara R N; Benedet, Patricia O; Souza, Adriana H; Akinaga, Juliana; Lima, Vanessa; Silva, Katiussia P; Kiguti, Luiz Ricardo A; Rossi, Marcos A; Kettelhut, Isis C; Pupo, André S; Cunha, Fernando Q; Assreuy, Jamil

    2017-07-01

    G protein-coupled receptor kinase isoform 2 (GRK2) has a critical role in physiological and pharmacological responses to endogenous and exogenous substances. Sepsis causes an important cardiovascular dysfunction in which nitric oxide (NO) has a relevant role. The present study aimed to assess the putative effect of inducible NO synthase (NOS2)-derived NO on the activity of GRK2 in the context of septic cardiac dysfunction. C57BL/6 mice were submitted to severe septic injury by cecal ligation and puncture (CLP). Heart function was assessed by isolated and perfused heart, echocardiography, and β-adrenergic receptor binding. GRK2 was determined by immunofluorescence and Western blot analysis in the heart and isolated cardiac myocytes. Sepsis increased NOS2 expression in the heart, increased plasma nitrite + nitrate levels, and reduced isoproterenol-induced isolated ventricle contraction, whole heart tension development, and β-adrenergic receptor density. Treatment with 1400W or with GRK2 inhibitor prevented CLP-induced cardiac hyporesponsiveness 12 and 24 h after CLP. Increased labeling of total and phosphorylated GRK2 was detected in hearts after CLP. With treatment of 1400W or in hearts taken from septic NOS2 knockout mice, the activation of GRK2 was reduced. 1400W or GRK2 inhibitor reduced mortality, improved echocardiographic cardiac parameters, and prevented organ damage. Therefore, during sepsis, NOS2-derived NO increases GRK2, which leads to a reduction in β-adrenergic receptor density, contributing to the heart dysfunction. Isolated cardiac myocyte data indicate that NO acts through the soluble guanylyl cyclase/cGMP/PKG pathway. GRK2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction. NEW & NOTEWORTHY The main novelty presented here is to show that septic shock induces cardiac hyporesponsiveness to isoproterenol by a mechanism dependent on nitric oxide and mediated by G protein-coupled receptor kinase isoform 2. Therefore

  17. Investigation and characterization of receptors for pituitary adenylate cyclase-activating polypeptide in human brain by radioligand binding and chemical cross-linking

    International Nuclear Information System (INIS)

    Suda, K.; Smith, D.M.; Ghatei, M.A.; Murphy, J.K.; Bloom, S.R.

    1991-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel peptide of hypothalamic origin which increases adenylate cyclase activity in rat anterior pituitary cell cultures. The 38-amino acid peptide shows a close sequence homology to vasoactive intestinal peptide (VIP). Binding sites for PACAP in membranes from postmortem human brain tissue were studied using [ 125 I]PACAP27 as the radioligand. High specific binding sites (amount of specific binding measured at 0.25 nM [ 125 I]PACAP27 in femtomoles per mg protein +/- SEM; n = 4) were present in hypothalamus (344.5 +/- 13.0), brain stem (343.0 +/- 29.3), cerebellum (292.0 +/- 21.1), cortex (259.6 +/- 19.8), and basal ganglia (259.2 +/- 50.3). Specific binding sites in pituitary, although present, were less abundant (35.0 +/- 8.9). Binding of [ 125 I]PACAP27 was reversible and time, pH, and temperature dependent. Despite the homology with VIP, VIP was a poor inhibitor of [ 125 I]PACAP27 binding (IC50, greater than 1 microM) compared with PACAP27 (IC50, 0.5-1.3 nM) and PACAP38 (IC50, 0.2-1.3 nM). Scatchard plots of [ 125 I]PACAP27 binding showed the presence of both high and lower affinity sites. Chemical cross-linking of PACAP-binding sites revealed that [ 125 I]PACAP27 was bound to polypeptide chains of 67,000 and 48,000 mol wt. Thus, we have demonstrated the presence of PACAP-specific receptors in human brain which are not VIP receptors. This opens the possibility of PACAP functioning as a novel neurotransmitter/neuromodulator in human brain

  18. Solubility of pllutonium in alkaline salt solutions

    International Nuclear Information System (INIS)

    Hobbs, D.T.; Edwards, T.B.

    1993-01-01

    Plutonium solubility data from several studies have been evaluated. For each data set, a predictive model has been developed where appropriate. In addition, a statistical model and corresponding prediction intervals for plutonium solubility as a quadratic function of the hydroxide concentration have been developed. Because of the wide range of solution compositions, the solubility of plutonium can vary by as much as three orders of magnitude for any given hydroxide concentration and still remain within the prediction interval. Any nuclear safety assessments that depend on the maximum amount of plutonium dissolved in alkaline salt solutions should use concentrations at least as great as the upper prediction limits developed in this study. To increase the confidence in the prediction model, it is recommended that additional solubility tests be conducted at low hydroxide concentrations and with all of the other solution components involved. To validate the model for application to actual waste solutions, it is recommended that the plutonium solubilities in actual waste solutions be determined and compared to the values predicted by the quadratic model

  19. Enhancing the solubility and bioavailability of poorly water-soluble drugs using supercritical antisolvent (SAS) process.

    Science.gov (United States)

    Abuzar, Sharif Md; Hyun, Sang-Min; Kim, Jun-Hee; Park, Hee Jun; Kim, Min-Soo; Park, Jeong-Sook; Hwang, Sung-Joo

    2018-03-01

    Poor water solubility and poor bioavailability are problems with many pharmaceuticals. Increasing surface area by micronization is an effective strategy to overcome these problems, but conventional techniques often utilize solvents and harsh processing, which restricts their use. Newer, green technologies, such as supercritical fluid (SCF)-assisted particle formation, can produce solvent-free products under relatively mild conditions, offering many advantages over conventional methods. The antisolvent properties of the SCFs used for microparticle and nanoparticle formation have generated great interest in recent years, because the kinetics of the precipitation process and morphologies of the particles can be accurately controlled. The characteristics of the supercritical antisolvent (SAS) technique make it an ideal tool for enhancing the solubility and bioavailability of poorly water-soluble drugs. This review article focuses on SCFs and their properties, as well as the fundamentals of overcoming poorly water-soluble drug properties by micronization, crystal morphology control, and formation of composite solid dispersion nanoparticles with polymers and/or surfactants. This article also presents an overview of the main aspects of the SAS-assisted particle precipitation process, its mechanism, and parameters, as well as our own experiences, recent advances, and trends in development. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Impact of Dendrimers on Solubility of Hydrophobic Drug Molecules

    Directory of Open Access Journals (Sweden)

    Sonam Choudhary

    2017-05-01

    Full Text Available Adequate aqueous solubility has been one of the desired properties while selecting drug molecules and other bio-actives for product development. Often solubility of a drug determines its pharmaceutical and therapeutic performance. Majority of newly synthesized drug molecules fail or are rejected during the early phases of drug discovery and development due to their limited solubility. Sufficient permeability, aqueous solubility and physicochemical stability of the drug are important for achieving adequate bioavailability and therapeutic outcome. A number of different approaches including co-solvency, micellar solubilization, micronization, pH adjustment, chemical modification, and solid dispersion have been explored toward improving the solubility of various poorly aqueous-soluble drugs. Dendrimers, a new class of polymers, possess great potential for drug solubility improvement, by virtue of their unique properties. These hyper-branched, mono-dispersed molecules have the distinct ability to bind the drug molecules on periphery as well as to encapsulate these molecules within the dendritic structure. There are numerous reported studies which have successfully used dendrimers to enhance the solubilization of poorly soluble drugs. These promising outcomes have encouraged the researchers to design, synthesize, and evaluate various dendritic polymers for their use in drug delivery and product development. This review will discuss the aspects and role of dendrimers in the solubility enhancement of poorly soluble drugs. The review will also highlight the important and relevant properties of dendrimers which contribute toward drug solubilization. Finally, hydrophobic drugs which have been explored for dendrimer assisted solubilization, and the current marketing status of dendrimers will be discussed.

  1. Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.

    Science.gov (United States)

    Merriam, Laura A; Baran, Caitlin N; Girard, Beatrice M; Hardwick, Jean C; May, Victor; Parsons, Rodney L

    2013-03-06

    After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

  2. pH-metric solubility. 2: correlation between the acid-base titration and the saturation shake-flask solubility-pH methods.

    Science.gov (United States)

    Avdeef, A; Berger, C M; Brownell, C

    2000-01-01

    The objective of this study was to compare the results of a normal saturation shake-flask method to a new potentiometric acid-base titration method for determining the intrinsic solubility and the solubility-pH profiles of ionizable molecules, and to report the solubility constants determined by the latter technique. The solubility-pH profiles of twelve generic drugs (atenolol, diclofenac.Na, famotidine, flurbiprofen, furosemide, hydrochlorothiazide, ibuprofen, ketoprofen, labetolol.HCl, naproxen, phenytoin, and propranolol.HCl), with solubilities spanning over six orders of magnitude, were determined both by the new pH-metric method and by a traditional approach (24 hr shaking of saturated solutions, followed by filtration, then HPLC assaying with UV detection). The 212 separate saturation shake-flask solubility measurements and those derived from 65 potentiometric titrations agreed well. The analysis produced the correlation equation: log(1/S)titration = -0.063(+/- 0.032) + 1.025(+/- 0.011) log(1/S)shake-flask, s = 0.20, r2 = 0.978. The potentiometrically-derived intrinsic solubilities of the drugs were: atenolol 13.5 mg/mL, diclofenac.Na 0.82 microg/mL, famotidine 1.1 mg/ mL, flurbiprofen 10.6 microg/mL, furosemide 5.9 microg/mL, hydrochlorothiazide 0.70 mg/mL, ibuprofen 49 microg/mL, ketoprofen 118 microg/mL, labetolol.HCl 128 microg/mL, naproxen 14 microg/mL, phenytoin 19 microg/mL, and propranolol.HCl 70 microg/mL. The new potentiometric method was shown to be reliable for determining the solubility-pH profiles of uncharged ionizable drug substances. Its speed compared to conventional equilibrium measurements, its sound theoretical basis, its ability to generate the full solubility-pH profile from a single titration, and its dynamic range (currently estimated to be seven orders of magnitude) make the new pH-metric method an attractive addition to traditional approaches used by preformulation and development scientists. It may be useful even to discovery

  3. Water Soluble Polymers for Pharmaceutical Applications

    Directory of Open Access Journals (Sweden)

    Veeran Gowda Kadajji

    2011-11-01

    Full Text Available Advances in polymer science have led to the development of novel drug delivery systems. Some polymers are obtained from natural resources and then chemically modified for various applications, while others are chemically synthesized and used. A large number of natural and synthetic polymers are available. In the present paper, only water soluble polymers are described. They have been explained in two categories (1 synthetic and (2 natural. Drug polymer conjugates, block copolymers, hydrogels and other water soluble drug polymer complexes have also been explained. The general properties and applications of different water soluble polymers in the formulation of different dosage forms, novel delivery systems and biomedical applications will be discussed.

  4. Solubility of actinides and surrogates in nuclear glasses

    International Nuclear Information System (INIS)

    Lopez, Ch.

    2003-01-01

    The nuclear wastes are currently incorporated in borosilicate glass matrices. The resulting glass must be perfectly homogeneous. The work discussed here is a study of actinide (thorium and plutonium) solubility in borosilicate glass, undertaken to assess the extent of actinide solubility in the glass and to understand the mechanisms controlling actinide solubilization. Glass specimens containing; actinide surrogates were used to prepare and optimize the fabrication of radioactive glass samples. These preliminary studies revealed that actinide Surrogates solubility in the glass was enhanced by controlling the processing temperature, the dissolution kinetic of the surrogate precursors, the glass composition and the oxidizing versus reducing conditions. The actinide solubility was investigated in the borosilicate glass. The evolution of thorium solubility in borosilicate glass was determined for temperatures ranging from 1200 deg C to 1400 deg C.Borosilicate glass specimens containing plutonium were fabricated. The experimental result showed that the plutonium solubility limit ranged from 1 to 2.5 wt% PuO 2 at 1200 deg C. A structural approach based on the determination of the local structure around actinides and their surrogates by EXAFS spectroscopy was used to determine their structural role in the glass and the nature of their bonding with the vitreous network. This approach revealed a correlation between the length of these bonds and the solubility of the actinides and their surrogates. (author)

  5. Solubility limited radionuclide transport through geologic media

    International Nuclear Information System (INIS)

    Muraoka, Susumu; Iwamoto, Fumio; Pigford, T.H.

    1980-11-01

    Prior analyses for the migration of radionuclides neglect solubility limits of resolved radionuclide in geologic media. But actually some of the actinides may appear in chemical forms of very low solubility. In the present report we have proposed the migration model with no decay parents in which concentration of radionuclide is limited in concentration of solubility in ground water. In addition, the analytical solutions of the space-time-dependent concentration are presented in the case of step release, band release and exponential release. (author)

  6. Molecular Thermodynamic Modeling of Mixed Solvent Solubility

    DEFF Research Database (Denmark)

    Ellegaard, Martin Dela; Abildskov, Jens; O’Connell, John P.

    2010-01-01

    A method based on statistical mechanical fluctuation solution theory for composition derivatives of activity coefficients is employed for estimating dilute solubilities of 11 solid pharmaceutical solutes in nearly 70 mixed aqueous and nonaqueous solvent systems. The solvent mixtures range from...... nearly ideal to strongly nonideal. The database covers a temperature range from 293 to 323 K. Comparisons with available data and other existing solubility methods show that the method successfully describes a variety of observed mixed solvent solubility behaviors using solute−solvent parameters from...

  7. Solubility Products of M(II) - Carbonates

    International Nuclear Information System (INIS)

    Grauer, Rolf; Berner, Urs

    1999-01-01

    Many solubility data for M(II) carbonates commonly compiled in tables are contradictory and sometimes obviously wrong. The quality of such data has been evaluated based on the original publications and reliable solubility constants have been selected for the carbonates of Mn, Fe, Co, Ni, Cu, Zn, Cd and Pb with the help of cross-comparisons. (author)

  8. Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

    Science.gov (United States)

    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.

  9. Micelles from lipid derivatives of water-soluble polymers as delivery systems for poorly soluble drugs.

    Science.gov (United States)

    Lukyanov, Anatoly N; Torchilin, Vladimir P

    2004-05-07

    Polymeric micelles have a whole set of unique characteristics, which make them very promising drug carriers, in particular, for poorly soluble drugs. Our review article focuses on micelles prepared from conjugates of water-soluble polymers, such as polyethylene glycol (PEG) or polyvinyl pyrrolidone (PVP), with phospholipids or long-chain fatty acids. The preparation of micelles from certain polymer-lipid conjugates and the loading of these micelles with various poorly soluble anticancer agents are discussed. The data on the characterization of micellar preparations in terms of their morphology, stability, longevity in circulation, and ability to spontaneously accumulate in experimental tumors via the enhanced permeability and retention (EPR) effect are presented. The review also considers the preparation of targeted immunomicelles with specific antibodies attached to their surface. Available in vivo results on the efficiency of anticancer drugs incorporated into plain micelles and immunomicelles in animal models are also discussed.

  10. Solubility of lithium deuteride in liquid lithium

    International Nuclear Information System (INIS)

    Veleckis, E.; Yonco, R.M.; Maroni, V.A.

    1977-01-01

    The solubility of LiD in liquid lithium between the eutectic and monotectic temperatures was measured using a direct sampling method. Solubilities were found to range from 0.0154 mol.% LiD at 199 0 C to 3.32 mol.% LiD at 498 0 C. The data were used in the derivation of an expression for the activity coefficient of LiD as a function of temperature and composition and an equation relating deuteride solubility and temperature, thus defining the liquidus curve. Similar equations were also derived for the Li-LiH system using the existing solubility data. Extrapolation of the liquidus curves yielded the eutectic concentrations (0.040 mol.% LiH and 0.035 mol.% LiD) and the freezing point depressions (0.23 0 C for Li-LiH and 0.20 0 C for Li-LiD) at the eutectic point. The results are compared with the literature data for hydrogen and deuterium. The implications of the relatively high solubility of hydrogen isotopes in lithium just above the melting point are discussed with respect to the cold trapping of tritium in fusion reactor blankets. (Auth.)

  11. Thorium oxalate solubility and morphology

    International Nuclear Information System (INIS)

    Monson, P.R. Jr.; Hall, R.

    1981-10-01

    Thorium was used as a stand-in for studying the solubility and precipitation of neptunium and plutonium oxalates. Thorium oxalate solubility was determined over a range of 0.001 to 10.0 in the concentration parameter [H 2 C 2 O 4 ]/[HNO 3 ] 2 . Morphology of thorium oxide made from the oxalate precipitates was characterized by scanning electron microscopy. The different morphologies found for oxalate-lean and oxalate-rich precipitations were in agreement with predictions based on precipitation theory

  12. The amorphous solid dispersion of the poorly soluble ABT-102 forms nano/microparticulate structures in aqueous medium: impact on solubility

    Directory of Open Access Journals (Sweden)

    Frank KJ

    2012-11-01

    Full Text Available Kerstin J Frank,1,3 Ulrich Westedt,2 Karin M Rosenblatt,2 Peter Hölig,2 Jörg Rosenberg,2 Markus Mägerlein,2 Gert Fricker,3 Martin Brandl11Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark; 2Abbott GmbH and Co. KG, Ludwigshafen, Germany; 3Department of Pharmaceutical Technology, University of Heidelberg, Heidelberg, GermanyAbstract: Amorphous solid dispersions (ASDs are a promising formulation approach for poorly soluble active pharmaceutical ingredients (APIs, because they ideally enhance both dissolution rate and solubility. However, the mechanism behind this is not understood in detail. In the present study, we investigated the supramolecular and the nano/microparticulate structures that emerge spontaneously upon dispersion of an ASD in aqueous medium and elucidated their influence on solubility. The ASD, prepared by hot melt extrusion, contained the poorly soluble ABT-102 (solubility in buffer, 0.05 µg/mL, a hydrophilic polymer, and three surfactants. The apparent solubility of ABT-102 from the ASD-formulation was enhanced up to 200 times in comparison to crystalline ABT-102. At the same time, the molecular solubility, as assessed by inverse equilibrium dialysis, was enhanced two times. Asymmetrical flow field-flow fractionation in combination with a multiangle light-scattering detector, an ultraviolet detector, and a refractometer enabled us to separate and identify the various supramolecular assemblies that were present in the aqueous dispersions of the API-free ASD (placebo and of binary/ternary blends of the ingredients. Thus, the supramolecular assemblies with a molar mass between 20,000 and 90,000 could be assigned to the polyvinylpyrrolidone/vinyl acetate 64, while two other kinds of assemblies were assigned to different surfactant assemblies (micelles. The amount of ABT-102 remaining associated with each of the assemblies upon fractionation was quantified offline with high

  13. Characterization of CYP264B1 and a terpene cyclase of a terpene biosynthesis gene cluster from the myxobacterium Sorangium cellulosum So ce56

    OpenAIRE

    Ly, Thuy Thi Bich

    2011-01-01

    In the work presented here, CYP264B1 and the terpene cyclase GeoA of Sorangium cellulosum So ce56 have been characterized. CYP264B1 is able to convert norisoprenoids (a-ionone and b-ionone) and diverse sesquiterpene compounds, including nootkatone. Three products, 3-hydroxy-a-ionone, 3-hydroxy-b-ionone and 13-hydroxy-nootkatone were characterized using HPLC and 1H and 13C NMR. CYP264B1 is the first enzyme reported to be capable to hydroxylate regioselectively both norisoprenoids at the positi...

  14. Tetraphenylborate Solubility in High Ionic Strength Salt Solutions

    International Nuclear Information System (INIS)

    Serkiz, S.M.; Ginn, J.D.; Jurgensen, A.R.

    1998-04-01

    Solubility of sodium and potassium salts of the tetraphenylborate ion (TPB) in simulated Savannah River Site High Level Waste was investigated. Data generated from this study allow more accurate predictions of TPB solubility at the In-Tank Precipitation (ITP) facility. Because previous research showed large deviations in the observed solubility of TPB salts when compared with model predictions, additional data were generated to better understand the solubility of TPB in more complex systems of high ionic strength and those containing both potassium and sodium. These data allow evaluation of the ability of current models to accurately predict equilibrium TPB concentrations over the range of experimental conditions investigated in this study

  15. Analysis of chemical concepts as the basic of virtual laboratory development and process science skills in solubility and solubility product subject

    Science.gov (United States)

    Syafrina, R.; Rohman, I.; Yuliani, G.

    2018-05-01

    This study aims to analyze the concept characteristics of solubility and solubility products that will serve as the basis for the development of virtual laboratory and students' science process skills. Characteristics of the analyzed concepts include concept definitions, concept attributes, and types of concepts. The concept analysis method uses concept analysis according to Herron. The results of the concept analysis show that there are twelve chemical concepts that become the prerequisite concept before studying the solubility and solubility and five core concepts that students must understand in the solubility and Solubility product. As many as 58.3% of the definitions of the concepts contained in high school textbooks support students' science process skills, the rest of the definition of the concept is memorized. Concept attributes that meet three levels of chemical representation and can be poured into a virtual laboratory have a percentage of 66.6%. Type of concept, 83.3% is a concept based on principle; and 16.6% concepts that state the process. Meanwhile, the science process skills that can be developed based on concept analysis are the ability to observe, calculate, measure, predict, interpret, hypothesize, apply, classify, and inference.

  16. Solubility and stability of inorganic carbonates

    International Nuclear Information System (INIS)

    Taylor, P.

    1987-01-01

    The chemistry of inorganic carbonates is reviewed, with emphasis on solubility and hydrolytic stability, in order to identify candidate waste forms for immobilization and disposal of 14 C. At present, CaCO 3 and BaCO 3 are the two most widely favoured wasted forms, primarily because they are the products of proven CO 2 -scrubbing technology. However, they have relatively high solubilities in non-alkaline solutions, necessitating care in selecting and assessing an appropriate disposal environment. Three compounds with better solubility characteristics in near-neutral waters are identified: bismutite, (BiO) 2 CO 3 ; hydrocerussite, Pb 3 (OH) 2 (CO 3 ) 2 ; and rhodochrosite, MnCO 3 . Some of the limitations of each of these alternative waste forms are discussed

  17. Water-soluble resorcin[4]arene based cavitands

    NARCIS (Netherlands)

    Grote gansey, M.H.B.; Grote Gansey, Marcel H.B.; Bakker, Frank K.G.; Feiters, Martinus C.; Geurts, Hubertus P.M.; Verboom, Willem; Reinhoudt, David

    1998-01-01

    Water-soluble resorcin[4]arene based cavitands were obtained in good yields by reaction of bromomethylcavitands with pyridine. Their solubility was determined by conductometry. The behaviour in water depends on the alkyl chain length; the methylcavitand does not aggregate, whereas the pentyl- and

  18. Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

    Directory of Open Access Journals (Sweden)

    Mounira Tlili

    2015-01-01

    Full Text Available 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.

  19. Expression of adenylyl cyclase types III and VI in human hyperfunctioning thyroid nodules.

    Science.gov (United States)

    Celano, M; Arturi, F; Presta, I; Bruno, R; Scarpelli, D; Calvagno, M G; Cristofaro, C; Bulotta, S; Giannasio, P; Sacco, R; Filetti, S; Russo, D

    2003-05-30

    Hyperfunctioning thyroid nodules are characterized by the presence of spontaneous somatic mutations responsible for constitutive activation of the cAMP pathway. However, alterations affecting other elements of the cAMP signaling system may counteract the effects of the mutations. In this study, the expression of the adenylyl cyclase (AC) types III and VI was investigated by Western blot in 18 hyperfunctioning thyroid nodules; in 12 samples, we also assessed the presence of TSH receptor (TSHR) or gsp mutations and levels of AC VI and III mRNA. We found that the expression of nodular AC VI (but not AC III) was significantly lower (85.1% of normal, P=0.014) than the expression of both adenylyl cycles types of perinodular tissue from the same patients. Slightly, but not significant differences were detected in nodules with or without mutations and AC protein levels generally showed correlation with the levels of the transcripts detected by RT-PCR. In addition, AC III and AC VI expression levels within a given nodule were characterized by a significant positive correlation. These findings indicate that a diminished expression of AC type VI may be part of the mechanisms occurring in the hyperfunctioning nodules, independently of the presence of TSHR or gsp mutations, which influence the resulting phenotype.

  20. Determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts.

    Science.gov (United States)

    Tsukatani, Tadayuki; Suenaga, Hikaru; Ishiyama, Munetaka; Ezoe, Takatoshi; Matsumoto, Kiyoshi

    2011-07-15

    A method for the determination of water-soluble vitamins using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8)} via 2-methyl-1,4-napthoquinone (NQ) was developed. Measurement conditions were optimized for the microbiological determination of water-soluble vitamins, such as vitamin B(6), biotin, folic acid, niacin, and pantothenic acid, using microorganisms that have a water-soluble vitamin requirement. A linear relationship between absorbance and water-soluble vitamin concentration was obtained. The proposed method was applied to determine the concentration of vitamin B(6) in various foodstuffs. There was good agreement between vitamin B(6) concentrations determined after 24h using the WST-8 colorimetric method and those obtained after 48h using a conventional method. The results suggest that the WST-8 colorimetric assay is a useful method for the rapid determination of water-soluble vitamins in a 96-well microtiter plate. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. Respiratory carcinogenicity assessment of soluble nickel compounds.

    Science.gov (United States)

    Oller, Adriana R

    2002-10-01

    The many chemical forms of nickel differ in physicochemical properties and biological effects. Health assessments for each main category of nickel species are needed. The carcinogenicity assessment of water-soluble nickel compounds has proven particularly difficult. Epidemiologic evidence indicates an association between inhalation exposures to nickel refinery dust containing soluble nickel compounds and increased risk of respiratory cancers. However, the nature of this association is unclear because of limitations of the exposure data, inconsistent results across cohorts, and the presence of mixed exposures to water-insoluble nickel compounds and other confounders that are known or suspected carcinogens. Moreover, well-conducted animal inhalation studies, where exposures were solely to soluble nickel, failed to demonstrate a carcinogenic potential. Similar negative results were seen in animal oral studies. A model exists that relates respiratory carcinogenic potential to the bioavailability of nickel ion at nuclear sites within respiratory target cells. This model helps reconcile human, animal, and mechanistic data for soluble nickel compounds. For inhalation exposures, the predicted lack of bioavailability of nickel ion at target sites suggests that water-soluble nickel compounds, by themselves, will not be complete human carcinogens. However, if inhaled at concentrations high enough to induce chronic lung inflammation, these compounds may enhance carcinogenic risks associated with inhalation exposure to other substances. Overall, the weight of evidence indicates that inhalation exposure to soluble nickel alone will not cause cancer; moreover, if exposures are kept below levels that cause chronic respiratory toxicity, any possible tumor-enhancing effects (particularly in smokers) would be avoided.

  2. 40 CFR Table 7 to Subpart Vvvvvv... - Partially Soluble HAP

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 14 2010-07-01 2010-07-01 false Partially Soluble HAP 7 Table 7 to... Pt. 63, Subpt. VVVVVV, Table 7 Table 7 to Subpart VVVVVV of Part 63—Partially Soluble HAP As required... partially soluble HAP listed in the following table. Partially soluble HAP name CAS No. 1. 1,1,1...

  3. Enhanced Iron Solubility at Low pH in Global Aerosols

    Directory of Open Access Journals (Sweden)

    Ellery D. Ingall

    2018-05-01

    Full Text Available The composition and oxidation state of aerosol iron were examined using synchrotron-based iron near-edge X-ray absorption spectroscopy. By combining synchrotron-based techniques with water leachate analysis, impacts of oxidation state and mineralogy on aerosol iron solubility were assessed for samples taken from multiple locations in the Southern and the Atlantic Oceans; and also from Noida (India, Bermuda, and the Eastern Mediterranean (Crete. These sampling locations capture iron-containing aerosols from different source regions with varying marine, mineral dust, and anthropogenic influences. Across all locations, pH had the dominating influence on aerosol iron solubility. When aerosol samples were approximately neutral pH, iron solubility was on average 3.4%; when samples were below pH 4, the iron solubility increased to 35%. This observed aerosol iron solubility profile is consistent with thermodynamic predictions for the solubility of Fe(III oxides, the major iron containing phase in the aerosol samples. Source regions and transport paths were also important factors affecting iron solubility, as samples originating from or passing over populated regions tended to contain more soluble iron. Although the acidity appears to affect aerosol iron solubility globally, a direct relationship for all samples is confounded by factors such as anthropogenic influence, aerosol buffer capacity, mineralogy and physical processes.

  4. Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

    Directory of Open Access Journals (Sweden)

    Elena Elizabeth Bagley

    2014-06-01

    Full Text Available Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1 currents in periaqueductal gray (PAG neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1

  5. Solubility Part 1

    NARCIS (Netherlands)

    Tantra, Ratna; Bolea, Eduardo; Bouwmeester, H.; Rey-Castro, Carlos; David, C.A.A.; Dogné, Jean Michel; Laborda, Francisco; Laloy, Julie; Robinson, Kenneth N.; Undas, A.K.; Zande, van der M.

    2016-01-01

    This chapter gives an overview of different methods that can potentially be used to determine the solubility of nanomaterials. In general, the methods presented can be broadly divided into four categories: separation methods, methods to quantify free ions, methods to quantify total dissolved

  6. Molybdenum solubility in aluminium nitrate solutions

    Energy Technology Data Exchange (ETDEWEB)

    Heres, X.; Sans, D.; Bertrand, M.; Eysseric, C. [CEA, Centre de Marcoule, Nuclear Energy Division, DRCP, BP 17171, 30207 Bagnols-sur-Ceze Cedex (France); Brackx, E.; Domenger, R.; Excoffier, E. [CEA, Centre de Marcoule, Nuclear Energy Division, DTEC, BP 17171, 30207 Bagnols-sur-Ceze Cedex (France); Valery, J.F. [AREVA-NC, DOR/RDP, Paris - La Defense (France)

    2016-07-01

    For over 60 years, research reactors (RR or RTR for research testing reactors) have been used as neutron sources for research, radioisotope production ({sup 99}Mo/{sup 99m}Tc), nuclear medicine, materials characterization, etc... Currently, over 240 of these reactors are in operation in 56 countries. They are simpler than power reactors and operate at lower temperature (cooled to below 100 C. degrees). The fuel assemblies are typically plates or cylinders of uranium alloy and aluminium (U-Al) coated with pure aluminium. These fuels can be processed in AREVA La Hague plant after batch dissolution in concentrated nitric acid and mixing with UOX fuel streams. The aim of this study is to accurately measure the solubility of molybdenum in nitric acid solution containing high concentrations of aluminium. The higher the molybdenum solubility is, the more flexible reprocessing operations are, especially when the spent fuels contain high amounts of molybdenum. To be most representative of the dissolution process, uranium-molybdenum alloy and molybdenum metal powder were dissolved in solutions of aluminium nitrate at the nominal dissolution temperature. The experiments showed complete dissolution of metallic elements after 30 minutes long stirring, even if molybdenum metal was added in excess. After an induction period, a slow precipitation of molybdic acid occurs for about 15 hours. The data obtained show the molybdenum solubility decreases with increasing aluminium concentration. The solubility law follows an exponential relation around 40 g/L of aluminium with a high determination coefficient. Molybdenum solubility is not impacted by the presence of gadolinium, or by an increasing concentration of uranium. (authors)

  7. Residual nilpotence and residual solubility of groups

    International Nuclear Information System (INIS)

    Mikhailov, R V

    2005-01-01

    The properties of the residual nilpotence and the residual solubility of groups are studied. The main objects under investigation are the class of residually nilpotent groups such that each central extension of these groups is also residually nilpotent and the class of residually soluble groups such that each Abelian extension of these groups is residually soluble. Various examples of groups not belonging to these classes are constructed by homological methods and methods of the theory of modules over group rings. Several applications of the theory under consideration are presented and problems concerning the residual nilpotence of one-relator groups are considered.

  8. Radionuclide solubilities at elevated temperatures. A literature study

    International Nuclear Information System (INIS)

    Carlsson, T.; Vuorinen, U.

    1997-07-01

    This literature study contains experimental data and modelling data collected in order to illustrate how temperature affects radionuclide solubilities under conditions similar to those expected in the vicinity of a planned repository for spent nuclear fuel. The elements considered were Ni, Se, Zr, Tc, Pd, Sn, Ra, Th, Pa, U, Np, Pu and Am. The temperatures of main interest are restricted to the interval between room temperature and 100 deg C. The study showed that the literature on radionuclide solubility at temperatures above room temperature is scarce. Therefore, also work that refers to conditions slightly varying from the expected repository conditions has been considered. A minor modelling exercise was done in this study in order to show the effect of temperature on the solubilities of Ni, Np and U under various conditions. The results from the literature survey and our modelling demonstrate the complexity of groundwater systems and the difficulty in finding simple and general relationships between temperature and radionuclide solubilities. Often an increase in temperature (below 100 deg C) leads to a reduction of the radionuclide solubility or leaves it roughly unchanged. However, examples are also found where the rise in temperature increases the radionuclide solubility by several orders of magnitude. (orig.)

  9. Radionuclide solubilities at elevated temperatures. A literature study

    Energy Technology Data Exchange (ETDEWEB)

    Carlsson, T.; Vuorinen, U. [Technical Research Centre of Finland, Espoo (Finland)

    1997-07-01

    This literature study contains experimental data and modelling data collected in order to illustrate how temperature affects radionuclide solubilities under conditions similar to those expected in the vicinity of a planned repository for spent nuclear fuel. The elements considered were Ni, Se, Zr, Tc, Pd, Sn, Ra, Th, Pa, U, Np, Pu and Am. The temperatures of main interest are restricted to the interval between room temperature and 100 deg C. The study showed that the literature on radionuclide solubility at temperatures above room temperature is scarce. Therefore, also work that refers to conditions slightly varying from the expected repository conditions has been considered. A minor modelling exercise was done in this study in order to show the effect of temperature on the solubilities of Ni, Np and U under various conditions. The results from the literature survey and our modelling demonstrate the complexity of groundwater systems and the difficulty in finding simple and general relationships between temperature and radionuclide solubilities. Often an increase in temperature (below 100 deg C) leads to a reduction of the radionuclide solubility or leaves it roughly unchanged. However, examples are also found where the rise in temperature increases the radionuclide solubility by several orders of magnitude. (orig.). 54 refs.

  10. Feasibility study of SMART core with soluble boron

    International Nuclear Information System (INIS)

    Kim, Kang Seog; Lee, Chung Chan; Zee, Sung Quun

    2000-11-01

    The excess reactivity of SMART core without soluble boron is effectively controlled by 49 CEDM. We suggest another method to control the core excess reactivity using both the checkerboard type of 25 CEDM and soluble boron and perform a feasibility calculation. The soluble boron operation is categorized into the on-line and the off-line mechanisms. The former is to successively control the boron concentration according to the excess reactivity during operation and the latter is to add and change some soluble boron during refueling and repairing. Since the on-line soluble boron control system of SMART is conceptually identical to that of the commercial pressurized water reactor, we did not perform the analysis. Since the soluble boron in the complete off-line system increases the moderator temperature coefficient, the reactivity defect between hot and cold moderator temperature is decreased. However, the decrease of the reactivity is not big to satisfy the core reactivity limits. When using 25 CEDM, the possible mechanism is to control the excess reactivity by both control rod and on-line boron control mechanism between cold and hot zero power and by only control rod at hot full power. We selected the loading pattern satisfying the requirement in the view of nuclear design

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

  12. In vitro dynamic solubility test: influence of various parameters.

    Science.gov (United States)

    Thélohan, S; de Meringo, A

    1994-10-01

    This article discusses the dissolution of mineral fibers in simulated physiological fluids (SPF), and the parameters that affect the solubility measurement in a dynamic test where an SPF runs through a cell containing fibers (Scholze and Conradt test). Solutions simulate either the extracellular fluid (pH 7.6) or the intracellular fluid (pH 4.5). The fibers have various chemical compositions and are either continuously drawn or processed as wool. The fiber solubility is determined by the amount of SiO2 (and occasionally other ions) released in the solution. Results are stated as percentage of the initial silica content released or as dissolution rate v in nm/day. The reproducibility of the test is higher with the less soluble fibers (10% solubility), than with highly soluble fibers (20% solubility). The influence of test parameters, including SPF, test duration, and surface area/volume (SA/V), has been studied. The pH and the inorganic buffer salts have a major influence: industrial glasswool composition is soluble at pH 7.6 but not at pH 4.5. The opposite is true for rock- (basalt) wool composition. For slightly soluble fibers, the dissolution rate v remains constant with time, whereas for highly soluble fibers, the dissolution rate decreases rapidly. The dissolution rates believed to occur are v1, initial dissolution rate, and v2, dissolution rate of the residual fibers. The SA of fibers varies with the mass of the fibers tested, or with the fiber diameter at equal mass. Volume, V, is the chosen flow rate. An increase in the SA/V ratio leads to a decrease in the dissolution rate.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Estimation of aqueous solubility of TODGA using group contribution method

    International Nuclear Information System (INIS)

    Balasubramonian, S.; Kumar, Shekhar; Sampath, M.; Sivakumar, D.; Kamachi Mudali, U.

    2017-01-01

    The aqueous solubility of N, N, N', N'-tetraoctyl-3-oxapentanediamide normally referred as TODGA is experimentally measured. The aqueous solubility was also predicted using Marrero and Gani group contribution method. The modification of original Marrero and Gani method was proposed to accurately predict TODGA solubility. The predicted solubility of TODGA using original Marrero and Gani method, Modified Marrero and Gani method and UNIFAC Model was compared. The predicted solubility of TODGA using modified Marrero and Gani method is 0.0237 g/l against the experimentally measured value of 0.0226 g/l. (author)

  14. On nitrogen solubility in water

    International Nuclear Information System (INIS)

    Kalajda, Yu.A.; Katkov, Yu.D.; Kuznetsov, V.A.; Lastovtsev, A.Yu.; Lastochkin, A.P.; Susoev, V.S.

    1980-01-01

    Presented are the results of experimental investigations on nitrogen solubility in water under 0-15 MPa pressure, at the temperature of 100-340 deg C and nitrogen concentration of 0-5000 n.ml. N 2 /kg H 2 O. Empiric equations are derived and a diagram of nitrogen solubility in water is developed on the basis of the experimental data, as well as critically evaluated published data. The investigation results can be used in analyzing water-gas regime of a primary heat carrier in stream-generating plants with water-water reactors

  15. (S)Pot on Mitochondria: Cannabinoids Disrupt Cellular Respiration to Limit Neuronal Activity.

    Science.gov (United States)

    Harkany, Tibor; Horvath, Tamas L

    2017-01-10

    Classical views posit G protein-coupled cannabinoid receptor 1s (CB1Rs) at the cell surface with cytosolic Giα-mediated signal transduction. Hebert-Chatelain et al. (2016) instead place CB 1 Rs at mitochondria limiting neuronal respiration by soluble adenylyl cyclase-dependent modulation of complex I activity. Thus, neuronal bioenergetics link to synaptic plasticity and, globally, learning and memory. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. A framework for API solubility modelling

    DEFF Research Database (Denmark)

    Conte, Elisa; Gani, Rafiqul; Crafts, Peter

    . In addition, most of the models are not predictive and requires experimental data for the calculation of the needed parameters. This work aims at developing an efficient framework for the solubility modelling of Active Pharmaceutical Ingredients (API) in water and organic solvents. With this framework......-SAFT) are used for solubility calculations when the needed interaction parameters or experimental data are available. The CI-UNIFAC is instead used when the previous models lack interaction parameters or when solubility data are not available. A new GC+ model for APIs solvent selection based...... on the hydrophobicity, hydrophilicity and polarity information of the API and solvent is also developed, for performing fast solvent selection and screening. Eventually, all the previous developments are integrated in a framework for their efficient and integrated use. Two case studies are presented: the first...

  17. The solubility of metals in Pb-17Li liquid alloy

    International Nuclear Information System (INIS)

    Borgstedt, H.U.; Feuerstein, H.

    1992-01-01

    The solubility data of iron in the eutectic alloy Pb-17Li which were evaluated from corrosion tests in a turbulent flow of the molten alloy are discussed in the frame of solubilities of the transition metals in liquid lead. It is shown that the solubility of iron in the alloy is close to that in lead. This is also the fact for several other alloying elements of steels. A comparison of all known data shows that they are in agreement with generally shown trends for the solubility of the transition metals in low melting metals. These trends indicate comparably high solubilities of nickel and manganese in the liquid metals, lower saturation concentration of vanadium, chromium, iron, and cobalt, and extremely low solubility of molybdenum. (orig.)

  18. Solubility Study of Curatives in Various Rubbers

    NARCIS (Netherlands)

    Guo, R.; Talma, Auke; Datta, Rabin; Dierkes, Wilma K.; Noordermeer, Jacobus W.M.

    2008-01-01

    The previous works on solubility of curatives in rubbers were mainly carried out in natural rubber. Not too much information available on dissimilar rubbers and this is important because most of the compounds today are blends of dissimilar rubbers. Although solubility can be expected to certain

  19. Characterization of soluble protein BCP 11/24 from bovine corneal epithelium, different from the principal soluble protein BCP 54

    NARCIS (Netherlands)

    Bakker, C.; Pasmans, S.; Verhagen, C.; van Haren, M.; van der Gaag, R.; Hoekzema, R.

    1992-01-01

    The water-soluble fraction of bovine corneal epithelium was analysed by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Next to the principal soluble protein BCP 54, which has recently been identified as a corneal aldehyde dehydrogenase (ALDH), another abundant protein was

  20. Progress in the research of neptunium solubility

    International Nuclear Information System (INIS)

    Jiang Tao; Liu Yongye; Yao Jun

    2012-01-01

    237 Np is considered a possible long-term potential threat for environment, because of its long half-life, high toxicity and its mobile nature under aerobic conditions due to the high chemical stability of its pentavalent state. Therefore 237 Np is considered as one of high-level radioactive waste and need to be disposed in deep geologic disposal repository. The dissolution behavior is an important aspect of migration research. The solubility is considered very important for high level waste geological disposal safety and environmental evaluation. The solubility determines the maximum concentration of the discharge, and then it is initial concentration of the radionuclides migration to the environment. The solubility impact directly on radionuclides migration in host rock, and can be used to predict the concentration and speciation of radionuclides in groundwater around disposal sites many years later. This paper focused on research results of the solubility, some proposals for Np dissolution chemistry research were also been suggested. (authors)

  1. On barium oxide solubility in barium-containing chloride melts

    International Nuclear Information System (INIS)

    Nikolaeva, Elena V.; Zakiryanova, Irina D.; Bovet, Andrey L.; Korzun, Iraida V.

    2016-01-01

    Oxide solubility in chloride melts depends on temperature and composition of molten solvent. The solubility of barium oxide in the solvents with barium chloride content is essentially higher than that in molten alkali chlorides. Spectral data demonstrate the existence of oxychloride ionic groupings in such melts. This work presents the results of the BaO solubility in two molten BaCl 2 -NaCl systems with different barium chloride content. The received data together with earlier published results revealed the main regularities of BaO solubility in molten BaO-BaCl 2 -MCl systems.

  2. The solubilities and solubility products of zirconium hydroxide and oxide after aging at 278, 313, and 333 K

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Taishi; Uemura, Takuya; Sasaki, Takayuki; Takagi, Ikuji [Kyoto Univ. (Japan). Dept. of Nuclear Engineering; Moriyama, Hirotake [Kyoto Univ. (Japan). Research Reactor Inst.

    2016-07-01

    The solubilities of zirconium hydroxide and oxide after aging at 278, 313, and 333 K were measured at 278, 298, 313, and 333 K in the pH{sub c} range of 0.3-7 in a 0.5 M ionic strength solution of NaClO{sub 4} and HClO{sub 4}. Size distributions of the colloidal species were investigated by ultrafiltration using membranes with different pore sizes, and the solid phases were examined by X-ray diffraction. The apparent solubility of zirconium amorphous hydroxide (Zr(OH){sub 4}(am)), prepared by the oversaturation method, decreased with increasing aging temperature (T{sub a}), and the size distributions obtained after aging at elevated temperatures indicated the growth of the colloidal species. We, therefore, suggested that agglomeration of the colloidal species and dehydration and crystallization of Zr(OH){sub 4}(am) as the solubility-limiting solid phase occurred over the course of aging at elevated temperatures. For sample solutions of the crystalline oxide (ZrO{sub 2}(cr)), the aging temperature had no significant effect on the solubility, but the solubility data at lower temperatures were found to be slightly higher than those at higher temperatures, implying a small fraction of the amorphous components. In the analysis of different solid phases (Zr(OH){sub 4}(s,T{sub a}), T{sub a} = 278, 313, and 333 K) depending on the aging temperatures, the solubility products (K{sub sp}, T{sub a}) were determined at different measurement temperatures, and the enthalpy change (Δ{sub r}H {sup circle}) for Zr{sup 4+} 4OH{sup -} <=> Zr(OH){sub 4}(s,T{sub a}) was calculated using the van't Hoff equation. The solid-phase-transformation process at elevated temperatures was also analyzed based on the obtained K{sub sp}, T{sub a} and Δ{sub r}H {sup circle} values.

  3. Three new hydrochlorothiazide cocrystals: Structural analyses and solubility studies

    Science.gov (United States)

    Ranjan, Subham; Devarapalli, Ramesh; Kundu, Sudeshna; Vangala, Venu R.; Ghosh, Animesh; Reddy, C. Malla

    2017-04-01

    Hydrochlorothiazide (HCT) is a diuretic BCS class IV drug with poor aqueous solubility and low permeability leading to poor oral absorption. The present work explores the cocrystallization technique to enhance the aqueous solubility of HCT. Three new cocrystals of HCT with water soluble coformers phenazine (PHEN), 4-dimethylaminopyridine (DMAP) and picolinamide (PICA) were prepared successfully by solution crystallization method and characterized by single crystal X-ray diffraction (SCXRD), powder X-ray diffraction (PXRD), fourier transform -infraredspectroscopy (FT-IR), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). Structural characterization revealed that the cocrystals with PHEN, DMAP and PICA exists in P21/n, P21/c and P21/n space groups, respectively. The improved solubility of HCT-DMAP (4 fold) and HCT-PHEN (1.4 fold) cocrystals whereas decreased solubility of HCT-PICA (0.5 fold) as compared to the free drug were determined after 4 h in phosphate buffer, pH 7.4, at 25 °C by using shaking flask method. HCT-DMAP showed a significant increase in solubility than all previously reported cocrystals of HCT suggest the role of a coformer. The study demonstrates that the selection of coformer could have pronounced impact on the physicochemical properties of HCT and cocrystallization can be a promising approach to improve aqueous solubility of drugs.

  4. Solubility of Meloxicam in Mixed Solvent Systems | Babu | Ethiopian ...

    African Journals Online (AJOL)

    The solubility of meloxicam is higher in phosphate buffer (pH 7.4) compared to water, probably due to ionization of the drug. The solubility of meloxicam is marginally enhanced in surfactant systems (Tween 80 and Brij 35) at concentrations higher than cmc, proving the micellar solubilization. Meloxicam solubility studies in ...

  5. Solubility of inorganic salts in pure ionic liquids

    International Nuclear Information System (INIS)

    Pereiro, A.B.; Araújo, J.M.M.; Oliveira, F.S.; Esperança, J.M.S.S.; Canongia Lopes, J.N.; Marrucho, I.M.; Rebelo, L.P.N.

    2012-01-01

    Highlights: ► We report the solubility of different conventional salts in several ionic liquids. ► The solubility was initially screened using a visual detection method. ► The most promising mixtures were quantitatively re-measured using an ATR–FTIR. - Abstract: The solubility of different conventional salts in several room-temperature ionic liquids – containing ammonium, phosphonium or imidazolium cations combined with acetate, sulfate, sulfonate, thiocyanate, chloride, tetracyano-borate, tris(pentafluoroethyl)trifluoro-phosphate, L-lactate, bis(trifluoromethylsulfonyl)imide or trifluoromethylsulfonate anions – were screened using a visual detection method. The most promising mixtures were then re-measured using an ATR–FTIR (Attenuated Total Reflection Fourier Transform Infra Red) spectroscopy technique in order to accurately and quantitatively determine the corresponding solubility at 298.15 K.

  6. Measurement and correlation of solubility of carbon dioxide in triglycerides

    International Nuclear Information System (INIS)

    Howlader, Md Shamim; French, William Todd; Toghiani, Hossein; Hartenbower, Ben; Pearson, Larry; DuBien, Janice; Rai, Neeraj

    2017-01-01

    Graphical abstract: Comparison of experimental results with correlation for solubility of CO 2 in triglycerides as a function pressure at two different temperatures of 289.15 and 303.15 K, respectively. - Highlights: • New pressure drop gas apparatus was developed to determine the solubility of gases in liquids. • Solubility of CO 2 in triglycerides was measured at different temperatures and pressures. • Experimental solubility data were correlated using three thermodynamic models. • Enthalpy, entropy and Gibbs energy of dissolution for CO 2 -triglyceride were determined. - Abstract: A new pressure drop solubility gas apparatus was developed to determine the solubility of carbon dioxide in canola oil, a triglyceride consisting primarily of oleic, linoleic, and alpha linoleic acid radicals. Solubility of CO 2 in triglycerides was determined at different temperatures (283.2–303.2 K) and pressures (600–2450 kPa). It was found that the solubility of CO 2 in triglycerides is higher than that of pure water because triglycerides lack strong hydrogen bond networks that exist in liquid water at the ambient conditions. The experimental solubility was correlated using Krichevsky–Kasarnovsky (KK), Mather-Jou (MJ), and Carvalho-Coutinho (CC) correlations. We find that KK and MJ equations can predict the solubility with higher accuracy. The enthalpy and entropy of absorption of CO 2 were calculated using the van’t Hoff plot and were found to be −7.165 kJ.mol −1 , and −28.791 J.mol −1 .K −1 , respectively.

  7. Thermodynamic data development using the solubility method (Joint research)

    International Nuclear Information System (INIS)

    Rai, Dhanpat; Yui, Mikazu

    2013-05-01

    The solubility method is one of the most powerful tools to obtain reliable thermodynamic data for 1) solubility products of discrete solids and double salts, 2) complexation constants for various ligands, 3) development of data in a wide range of pH values, 4) evaluation of data for metals that form very insoluble solids (e.g. tetravalent actinides), 5) determining solubility-controlling solids in different types of wastes and 6) elevated temperatures for redox sensitive systems. This document is focused on describing various aspects of obtaining thermodynamic data using the solubility method. This manuscript deals with various aspects of conducting solubility studies, including selecting the study topic, modeling to define important variables, selecting the range of variables and experimental parameters, anticipating results, general equipment requirements, conducting experiments, and interpreting experimental data. (author)

  8. Solubility of corrosion products in high temperature water

    International Nuclear Information System (INIS)

    Srinivasan, M.P.; Narasimhan, S.V.

    1995-01-01

    A short review of solubility of corrosion products at high temperature in either neutral or alkaline water as encountered in BWR, PHWR and PWR primary coolant reactor circuits is presented in this report. Based on the available literature, various experimental techniques involved in the study of the solubility, theory for fitting the solubility data to the thermodynamic model and discussion of the published results with a scope for future work have been brought out. (author). 17 refs., 7 figs

  9. The Hildebrand solubility parameters of ionic liquids-part 2.

    Science.gov (United States)

    Marciniak, Andrzej

    2011-01-01

    The Hildebrand solubility parameters have been calculated for eight ionic liquids. Retention data from the inverse gas chromatography measurements of the activity coefficients at infinite dilution were used for the calculation. From the solubility parameters, the enthalpies of vaporization of ionic liquids were estimated. Results are compared with solubility parameters estimated by different methods.

  10. Sensitivity analysis of uranium solubility under strongly oxidizing conditions

    International Nuclear Information System (INIS)

    Liu, L.; Neretnieks, I.

    1999-01-01

    To evaluate the effect of geochemical conditions in the repository on the solubility of uranium under strongly oxidizing conditions, a mathematical model has been developed to determine the solubility, by utilizing a set of nonlinear algebraic equations to describe the chemical equilibria in the groundwater environment. The model takes into account the predominant precipitation-dissolution reactions, hydrolysis reactions and complexation reactions that may occur under strongly oxidizing conditions. The model also includes the solubility-limiting solids induced by the presence of carbonate, phosphate, silicate, calcium, and sodium in the groundwater. The thermodynamic equilibrium constants used in the solubility calculations are essentially taken from the NEA Thermochemical Data Base of Uranium, with some modification and some uranium minerals added, such as soddyite, rutherfordite, uranophane, uranyl orthophosphate, and becquerelite. By applying this model, the sensitivities of uranium solubility to variations in the concentrations of various groundwater component species are systematically investigated. The results show that the total analytical concentrations of carbonate, phosphate, silicate, and calcium in deep groundwater play the most important role in determining the solubility of uranium under strongly oxidizing conditions

  11. Soluble L-selectin levels predict survival in sepsis

    DEFF Research Database (Denmark)

    Seidelin, Jakob B; Nielsen, Ole H; Strøm, Jens

    2002-01-01

    To evaluate serum soluble L-selectin as a prognostic factor for survival in patients with sepsis.......To evaluate serum soluble L-selectin as a prognostic factor for survival in patients with sepsis....

  12. Cocoa procyanidins modulate transcriptional pathways linked to inflammation and metabolism in human dendritic cells

    DEFF Research Database (Denmark)

    Midttun, Helene L E; Ramsay, Aina; Mueller-Harvey, Irene

    2018-01-01

    the mechanistic basis of this inhibition, here we conducted transcriptomic analysis on DCs cultured with either LPS or LPS combined with oligomeric cocoa PC. Procyanidins suppressed a number of genes encoding cytokines and chemokines such as CXCL1, but also genes involved in the cGMP pathway such as GUCY1A3...... (encoding guanylate cyclase soluble subunit alpha-3). Upregulated genes were involved in diverse metabolic pathways, but notably two of the four most upregulated genes (NMB, encoding neuromedin B and ADCY3, encoding adenyl cyclase type 3) were involved in the cAMP signalling pathway. Gene-set enrichment...... analysis demonstrated that upregulated gene pathways were primarily involved in nutrient transport, carbohydrate metabolism and lysosome function, whereas down-regulated gene pathways involved cell cycle, signal transduction and gene transcription, as well as immune function. qPCR analysis verified...

  13. Scoring function to predict solubility mutagenesis

    Directory of Open Access Journals (Sweden)

    Deutsch Christopher

    2010-10-01

    Full Text Available Abstract Background Mutagenesis is commonly used to engineer proteins with desirable properties not present in the wild type (WT protein, such as increased or decreased stability, reactivity, or solubility. Experimentalists often have to choose a small subset of mutations from a large number of candidates to obtain the desired change, and computational techniques are invaluable to make the choices. While several such methods have been proposed to predict stability and reactivity mutagenesis, solubility has not received much attention. Results We use concepts from computational geometry to define a three body scoring function that predicts the change in protein solubility due to mutations. The scoring function captures both sequence and structure information. By exploring the literature, we have assembled a substantial database of 137 single- and multiple-point solubility mutations. Our database is the largest such collection with structural information known so far. We optimize the scoring function using linear programming (LP methods to derive its weights based on training. Starting with default values of 1, we find weights in the range [0,2] so that predictions of increase or decrease in solubility are optimized. We compare the LP method to the standard machine learning techniques of support vector machines (SVM and the Lasso. Using statistics for leave-one-out (LOO, 10-fold, and 3-fold cross validations (CV for training and prediction, we demonstrate that the LP method performs the best overall. For the LOOCV, the LP method has an overall accuracy of 81%. Availability Executables of programs, tables of weights, and datasets of mutants are available from the following web page: http://www.wsu.edu/~kbala/OptSolMut.html.

  14. Hansen Solubility Parameters for Octahedral Oligomeric Silsesquioxanes

    Science.gov (United States)

    2012-08-28

    1997, 80, 386-&. 5. Hansen, C. M. The three-dimensional solubility parameter -- key to paint component affinities I. J. Paint Technol. 1967, 39, 104...Chai, J.; Zhang, Q. X.; Han, D. X.; Niu, L. Synthesis and Application of Widely Soluble Graphene Sheets. Langmuir 2010, 26, 12314-12320. 12. Hansen, C

  15. A Colorful Solubility Exercise for Organic Chemistry

    Science.gov (United States)

    Shugrue, Christopher R.; Mentzen, Hans H., II; Linton, Brian R.

    2015-01-01

    A discovery chemistry laboratory has been developed for the introductory organic chemistry student to investigate the concepts of polarity, miscibility, solubility, and density. The simple procedure takes advantage of the solubility of two colored dyes in a series of solvents or solvent mixtures, and the diffusion of colors can be easily…

  16. The Hildebrand Solubility Parameters of Ionic Liquids—Part 2

    Science.gov (United States)

    Marciniak, Andrzej

    2011-01-01

    The Hildebrand solubility parameters have been calculated for eight ionic liquids. Retention data from the inverse gas chromatography measurements of the activity coefficients at infinite dilution were used for the calculation. From the solubility parameters, the enthalpies of vaporization of ionic liquids were estimated. Results are compared with solubility parameters estimated by different methods. PMID:21747694

  17. The Hildebrand Solubility Parameters of Ionic Liquids—Part 2

    Directory of Open Access Journals (Sweden)

    Andrzej Marciniak

    2011-06-01

    Full Text Available The Hildebrand solubility parameters have been calculated for eight ionic liquids. Retention data from the inverse gas chromatography measurements of the activity coefficients at infinite dilution were used for the calculation. From the solubility parameters, the enthalpies of vaporization of ionic liquids were estimated. Results are compared with solubility parameters estimated by different methods.

  18. Phorbol esters alter adenylate cyclase responses to vasoactive intestinal peptide and forskolin in the GH cell line

    Energy Technology Data Exchange (ETDEWEB)

    Summers, S.; Florio, T.; Cronin, M.

    1986-05-01

    Activation of protein kinase C with phorbol ester modifies cyclic AMP production in several anterior pituitary cell systems. In the GH cell line from a rat pituitary tumor, exposure to phorbol 12-myristate 13-acetate (PMA: 100 nM) for 30 minutes significantly reduces vasoactive intestinal peptide (VIP: 100 nM) stimulated adenylate cyclase (AC) activity in subsequent membrane preparations to 62 + 4% of control (n = 6 independent studies). In contrast, these same membrane preparations respond to forskolin (1 ..mu..M) with significantly more activity, 130 +/- 6% of controls (n = 6 independent studies). Finally, phorbol ester does not block an inhibitory hormone input into the AC system; somatostatin (100 nM) reduction of VIP-stimulated AC activity is not significantly different in membrane preparations from PMA treated and control cells (n = 3 independent studies). These other findings lead the authors to propose that protein kinase C can modify several sites in the AC complex in anterior pituitary cells.

  19. X-radiation effect on soluble proteins of gastric mucosa

    International Nuclear Information System (INIS)

    Sukhomlinov, B.F.; Chajka, Ya.P.; Fedorovich, A.N.

    1979-01-01

    Using the method of electrophoresis in agar gel soluble proteins of gastric mucosa of rats were separated into 11 fractions. Proteins posessing a proteolytic (pH 1.8) and lipase (pH 7.4) activity were localized within the second and third prealbumin fractions. Soluble proteins of gastric mucosa contain glyco- and lipoproteid complexes. Exposure of rats to 1000 R of X-rays induces quantitative redistribution within the electrophoretic spectrum of soluble proteins and a considerable disturbance of the proteolytic activity of total soluble proteins throughout the entire period of observation (from 10 min to 72h)

  20. Soluble Molecularly Imprinted Nanorods for Homogeneous Molecular Recognition

    Directory of Open Access Journals (Sweden)

    Rongning Liang

    2018-03-01

    Full Text Available Nowadays, it is still difficult for molecularly imprinted polymers (MIPs to achieve homogeneous recognition since they cannot be easily dissolved in organic or aqueous phase. To address this issue, soluble molecularly imprinted nanorods have been synthesized by using soluble polyaniline doped with a functionalized organic protonic acid as the polymer matrix. By employing 1-naphthoic acid as a model, the proposed imprinted nanorods exhibit an excellent solubility and good homogeneous recognition ability. The imprinting factor for the soluble imprinted nanoroads is 6.8. The equilibrium dissociation constant and the apparent maximum number of the proposed imprinted nanorods are 248.5 μM and 22.1 μmol/g, respectively. We believe that such imprinted nanorods may provide an appealing substitute for natural receptors in homogeneous recognition related fields.

  1. Soluble Molecularly Imprinted Nanorods for Homogeneous Molecular Recognition

    Science.gov (United States)

    Liang, Rongning; Wang, Tiantian; Zhang, Huan; Yao, Ruiqing; Qin, Wei

    2018-03-01

    Nowadays, it is still difficult for molecularly imprinted polymer (MIPs) to achieve homogeneous recognition since they cannot be easily dissolved in organic or aqueous phase. To address this issue, soluble molecularly imprinted nanorods have been synthesized by using soluble polyaniline doped with a functionalized organic protonic acid as the polymer matrix. By employing 1-naphthoic acid as a model, the proposed imprinted nanorods exhibit an excellent solubility and good homogeneous recognition ability. The imprinting factor for the soluble imprinted nanoroads is 6.8. The equilibrium dissociation constant and the apparent maximum number of the proposed imprinted nanorods are 248.5 μM and 22.1 μmol/g, respectively. We believe that such imprinted nanorods may provide an appealing substitute for natural receptors in homogeneous recognition related fields.

  2. Review on theoretical calculation of the magnetite solubility

    International Nuclear Information System (INIS)

    Kim, Myongjin; Kim, Hongpyo

    2013-01-01

    FAC is influenced by many factors such as water chemistry (temperature, pH, dissolved oxygen (D. O.) in a solution, and etc.), chemical composition of carbon steel, and fluid dynamics. Magnetite is formed at the inner surface of carbon steel, and protects the integrity of pipes from the damage. The magnetite has a stable state at each equilibrium condition, so that it can be dissolved into the fluid under conditions that satisfy the equilibrium state. The iron solubility can be calculated by considering the reaction equilibrium constants for prediction of a change in the magnetite layer. In the present work, studies on the magnetite solubility were reviewed for the theoretical calculation of magnetite, and iron solubility data were compared to find the proper solubility values of each study

  3. Soluble Non-ammonia Nitrogen in Ruminal and Omasal Digesta of Korean Native Steers Supplemented with Soluble Proteins

    Directory of Open Access Journals (Sweden)

    C. W. Choi

    2012-09-01

    Full Text Available An experiment was conducted to study the effect of soluble protein supplements on concentration of soluble non-ammonia nitrogen (SNAN in the liquid phase of ruminal (RD and omasal digesta (OD of Korean native steers, and to investigate diurnal pattern in SNAN concentration in RD and OD. Three ruminally cannulated Korean native steers in a 3×3 Latin square design consumed a basal diet of rice straw and corn-based concentrate (control, and that supplemented (kg/d DM basis with intact casein (0.24; IC or acid hydrolyzed casein (0.46; AHC. Ruminal digesta was sampled using a vacuum pump, whereas OD was collected using an omasal sampling system at 2.0 h intervals after a morning feeding. The SNAN fractions (free amino acid (AA, peptide and soluble protein in RD and OD were assessed using the ninhydrin assay. Concentrations of free AA and total SNAN in RD were significantly (p<0.05 lower than those in OD. Although free AA concentration was relatively high, mean peptide was quantitatively the most important fraction of total SNAN in both RD and OD, indicating that degradation of peptide to AA rather than hydrolysis of soluble protein to peptide or deamination may be the most limiting step in rumen proteolysis of Korean native steers. Diurnal variation in peptide concentration in OD for the soluble protein supplemented diets during the feeding cycle peaked 2 h post-feeding and decreased thereafter whereas that for the control was relatively constant during the entire feeding cycle. Diurnal variation in peptide concentration was rather similar between RD and OD.

  4. A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.

    Directory of Open Access Journals (Sweden)

    Robert W Moon

    2009-09-01

    Full Text Available The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3', 5'-cyclic monophosphate (cGMP as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDEdelta, unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GCbeta, showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.

  5. An olfactory subsystem that detects carbon disulfide and mediates food-related social learning.

    Science.gov (United States)

    Munger, Steven D; Leinders-Zufall, Trese; McDougall, Lisa M; Cockerham, Renee E; Schmid, Andreas; Wandernoth, Petra; Wennemuth, Gunther; Biel, Martin; Zufall, Frank; Kelliher, Kevin R

    2010-08-24

    Olfactory signals influence social interactions in a variety of species. In mammals, pheromones and other social cues can promote mating or aggression behaviors; can communicate information about social hierarchies, genetic identity and health status; and can contribute to associative learning. However, the molecular, cellular, and neural mechanisms underlying many olfactory-mediated social interactions remain poorly understood. Here, we report that a specialized olfactory subsystem that includes olfactory sensory neurons (OSNs) expressing the receptor guanylyl cyclase GC-D, the cyclic nucleotide-gated channel subunit CNGA3, and the carbonic anhydrase isoform CAII (GC-D(+) OSNs) is required for the acquisition of socially transmitted food preferences (STFPs) in mice. Using electrophysiological recordings from gene-targeted mice, we show that GC-D(+) OSNs are highly sensitive to the volatile semiochemical carbon disulfide (CS(2)), a component of rodent breath and a known social signal mediating the acquisition of STFPs. Olfactory responses to CS(2) are drastically reduced in mice lacking GC-D, CNGA3, or CAII. Disruption of this sensory transduction cascade also results in a failure to acquire STFPs from either live or surrogate demonstrator mice or to exhibit hippocampal correlates of STFP retrieval. Our findings indicate that GC-D(+) OSNs detect chemosignals that facilitate food-related social interactions. Copyright 2010 Elsevier Ltd. All rights reserved.

  6. Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

    International Nuclear Information System (INIS)

    Sakamoto, C.; Matozaki, T.; Nagao, M.; Baba, S.

    1987-01-01

    Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125 I-[Tyr 1 ]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p]>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg 2+ . When pancreatic acini were treated with 1 μg/ml pertussis toxin for 4 h, subsequent 125 I-[Tyr 1 ]somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor

  7. SITE-94. Radionuclide solubilities for SITE-94

    Energy Technology Data Exchange (ETDEWEB)

    Arthur, R.; Apted, M. [QuantiSci, Denver, CO (United States)

    1996-12-01

    In this report, solubility constraints are evaluated on radioelement source-term concentrations supporting the SITE-94 performance assessment. Solubility models are based on heterogeneous-equilibrium, mass- and charge-balance constraints incorporated into the EQ3/6 geochemical software package, which is used to calculate the aqueous speciation behavior and solubilities of U, Th, Pu, Np, Am, Ni, Ra, Se, Sn, Sr, Tc and Zr in site groundwaters and near-field solutions. The chemical evolution of the near field is approximated using EQ3/6 in terms of limiting conditions at equilibrium, or steady state, in three closed systems representing fully saturated bentonite, Fe{sup o} corrosion products of the canister, and spent fuel. The calculations consider both low-temperature (15 deg C) and high-temperature (80 deg C) conditions in the near field, and the existence of either reducing or strongly oxidizing conditions in each of the bentonite, canister, and spent-fuel barriers. Heterogeneities in site characteristics are evaluated through consideration of a range of initial groundwaters and their interactions with engineered barriers. Aqueous speciation models for many radioelements are constrained by thermodynamic data that are estimated with varying degrees of accuracy. An important question, however, is how accurate do these models need to be for purposes of estimating source-term concentrations? For example, it is unrealistic to expect a high degree of accuracy in speciation models if such models predict solubilities that are below the analytical detection limit for a given radioelement. From a practical standpoint, such models are irrelevant if calculated solubilities cannot be tested by direct comparison to experimental data. In the absence of models that are both accurate and relevant for conditions of interest, the detection limit could define a pragmatic upper limit on radioelement solubility 56 refs, 25 tabs, 10 figs

  8. SITE-94. Radionuclide solubilities for SITE-94

    International Nuclear Information System (INIS)

    Arthur, R.; Apted, M.

    1996-12-01

    In this report, solubility constraints are evaluated on radioelement source-term concentrations supporting the SITE-94 performance assessment. Solubility models are based on heterogeneous-equilibrium, mass- and charge-balance constraints incorporated into the EQ3/6 geochemical software package, which is used to calculate the aqueous speciation behavior and solubilities of U, Th, Pu, Np, Am, Ni, Ra, Se, Sn, Sr, Tc and Zr in site groundwaters and near-field solutions. The chemical evolution of the near field is approximated using EQ3/6 in terms of limiting conditions at equilibrium, or steady state, in three closed systems representing fully saturated bentonite, Fe o corrosion products of the canister, and spent fuel. The calculations consider both low-temperature (15 deg C) and high-temperature (80 deg C) conditions in the near field, and the existence of either reducing or strongly oxidizing conditions in each of the bentonite, canister, and spent-fuel barriers. Heterogeneities in site characteristics are evaluated through consideration of a range of initial groundwaters and their interactions with engineered barriers. Aqueous speciation models for many radioelements are constrained by thermodynamic data that are estimated with varying degrees of accuracy. An important question, however, is how accurate do these models need to be for purposes of estimating source-term concentrations? For example, it is unrealistic to expect a high degree of accuracy in speciation models if such models predict solubilities that are below the analytical detection limit for a given radioelement. From a practical standpoint, such models are irrelevant if calculated solubilities cannot be tested by direct comparison to experimental data. In the absence of models that are both accurate and relevant for conditions of interest, the detection limit could define a pragmatic upper limit on radioelement solubility

  9. Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.

    Science.gov (United States)

    Mori, Takaaki; Kamiya, Koki; Tomita, Masahiro; Yoshimura, Tetsuro; Tsumoto, Kanta

    2014-06-01

    Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

  10. Iron solubility in highly boron-doped silicon

    International Nuclear Information System (INIS)

    McHugo, S.A.; McDonald, R.J.; Smith, A.R.; Hurley, D.L.; Weber, E.R.

    1998-01-01

    We have directly measured the solubility of iron in high and low boron-doped silicon using instrumental neutron activation analysis. Iron solubilities were measured at 800, 900, 1000, and 1100thinsp degree C in silicon doped with either 1.5x10 19 or 6.5x10 14 thinspboronthinspatoms/cm 3 . We have measured a greater iron solubility in high boron-doped silicon as compared to low boron-doped silicon, however, the degree of enhancement is lower than anticipated at temperatures >800thinsp degree C. The decreased enhancement is explained by a shift in the iron donor energy level towards the valence band at elevated temperatures. Based on this data, we have calculated the position of the iron donor level in the silicon band gap at elevated temperatures. We incorporate the iron energy level shift in calculations of iron solubility in silicon over a wide range of temperatures and boron-doping levels, providing a means to accurately predict iron segregation between high and low boron-doped silicon. copyright 1998 American Institute of Physics

  11. Soluble CD163

    DEFF Research Database (Denmark)

    Møller, Holger J

    2012-01-01

    CD163 is an endocytic receptor for haptoglobin-hemoglobin complexes and is expressed solely on macrophages and monocytes. As a result of ectodomain shedding, the extracellular portion of CD163 circulates in blood as a soluble protein (sCD163) at 0.7-3.9 mg/l in healthy individuals. The function o...

  12. Solubilities of boric acid in heavy water

    International Nuclear Information System (INIS)

    Nakai, Shigetsugu; Aoi, Hideki; Hayashi, Ken-ichi; Katoh, Taizo; Watanabe, Takashi.

    1988-01-01

    A gravimetric analysis using meta-boric acid (HBO 2 or DBO 2 ) as a weighing form has been developed for solubility measurement. The method gave satisfactory results in preliminary measurement of solubilities of boric acid in light water. By using this method, the solubilities of 10 B enriched D 3 BO 3 in heavy water were measured. The results are as follows; 2.67 (7deg C), 3.52 (15deg C), 5.70 (30deg C), 8.87 (50deg C) and 12.92 (70deg C) w/o, respectively. These values are about 10% lower than those in light water. Thermodynamical consideration based on the data shows that boric acid is the water structure breaker. (author)

  13. Solubility Products of M(II) - Carbonates

    Energy Technology Data Exchange (ETDEWEB)

    Grauer, Rolf; Berner, Urs [ed.

    1999-01-01

    Many solubility data for M(II) carbonates commonly compiled in tables are contradictory and sometimes obviously wrong. The quality of such data has been evaluated based on the original publications and reliable solubility constants have been selected for the carbonates of Mn, Fe, Co, Ni, Cu, Zn, Cd and Pb with the help of cross-comparisons. (author) translated from a PSI internal report written in German in 1994 (TM-44-94-05). 5 figs., 1 tab., 68 refs.

  14. Enhancing the Solubility and Oral Bioavailability of Poorly Water-Soluble Drugs Using Monoolein Cubosomes.

    Science.gov (United States)

    Ali, Md Ashraf; Kataoka, Noriko; Ranneh, Abdul-Hackam; Iwao, Yasunori; Noguchi, Shuji; Oka, Toshihiko; Itai, Shigeru

    2017-01-01

    Monoolein cubosomes containing either spironolactone (SPI) or nifedipine (NI) were prepared using a high-pressure homogenization technique and characterized in terms of their solubility and oral bioavailability. The mean particle size, polydispersity index (PDI), zeta potential, solubility and encapsulation efficiency (EE) values of the SPI- and NI-loaded cubosomes were determined to be 90.4 nm, 0.187, -13.4 mV, 163 µg/mL and 90.2%, and 91.3 nm, 0.168, -12.8 mV, 189 µg/mL and 93.0%, respectively, which were almost identical to those of the blank cubosome. Small-angle X-ray scattering analyses confirmed that the SPI-loaded, NI-loaded and blank cubosomes existed in the cubic space group Im3̄m. The lattice parameters of the SPI- and NI-loaded cubosomes were 147.6 and 151.6 Å, respectively, making them almost identical to that of blank cubosome (151.0 Å). The in vitro release profiles of the SPI- and NI-loaded cubosomes showed that they released less than 5% of the drugs into various media over 12-48 h, indicating that most of the drug remained encapsulated within the cubic phase of their lipid bilayer. Furthermore, the in vivo pharmacokinetic results suggested that these cubosomes led to a considerable increase in the systemic oral bioavailability of the drugs compared with pure dispersions of the same materials. Notably, the stability results indicated that the mean particle size and PDI values of these cubosomes were stable for at least 4 weeks. Taken together, these results demonstrate that monoolein cubosomes represent promising drug carriers for enhancing the solubility and oral bioavailability of poorly water-soluble drugs.

  15. Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon.

    Directory of Open Access Journals (Sweden)

    Natalia López-del Hoyo

    Full Text Available 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

  16. Solubility of iron in liquid lead

    International Nuclear Information System (INIS)

    Ali-Khan, I.

    1981-01-01

    The use of liquid lead in high temperature chemical and metallurgical processes is well known. The structural materials applied for the containment of these processes are either iron base alloys or possess iron as an alloying element. Besides that, lead itself is alloyed in some steels to achieve some very useful properties. For understanding the effect of liquid lead in such structural materials, it is important to determine the solubility of iron in liquid lead which would also be indicative of the stability of these alloys. At the institute of reactor materials of KFA Juelich, investigations have been conducted to determine the solubility of iron in liquid lead up to a temperature of about 1000 0 C. In this presentation the data concerning the solubility of iron in liquid lead are brought up to date and discussed including the results of our previous investigations. (orig.)

  17. pH-potentiometric determination of solubility of barely soluble organic extracting agents in water and aqueous solutions of neutral salts

    International Nuclear Information System (INIS)

    Pavlovskaya, E.M.; Charykov, A.K.; Tikhomirov, V.I.

    1977-01-01

    A pH-potentiometric method has been used to estimate the solubility of chloroform, benzene and nitrobenzene in water. The desalting effect is studied of alkali metal chlorides on chloroform solubility to establish the following phenomenological series of alkali metal cations by their desalting action: Li + + + + + . The non-conformity of chloroform solubility values in water-isoactive solutions of different salts is indicative of the high specificity of desalting processes with respect to the chemical nature of the desalting cation. Salt effects also essentially depend on the chemical nature of the desalted substance, particularly on its acid-base properties

  18. Enhancement of carvedilol solubility by solid dispersion technique using cyclodextrins, water soluble polymers and hydroxyl acid.

    Science.gov (United States)

    Yuvaraja, K; Khanam, Jasmina

    2014-08-05

    Aim of the present work is to enhance aqueous solubility of carvedilol (CV) by solid dispersion technique using wide variety of carriers such as: β-cyclodextrin (βCD), hydroxypropyl-β-cyclodextrin (HPβCD), tartaric acid (TA), polyvinyl pyrrolidone K-30 (PVP K-30) and poloxamer-407 (PLX-407). Various products of 'CV-solid dispersion' had been studied extensively in various pH conditions to check enhancement of solubility and dissolution characteristics of carvedilol. Any physical change upon interaction between CV and carriers was confirmed by instrumental analysis: XRD, DSC, FTIR and SEM. Negative change of Gibb's free energy and complexation constants (Kc, 75-240M(-1), for cyclodextrins and 1111-20,365M(-1), for PVP K-30 and PLX-407) were the evidence of stable nature of the binding between CV and carriers. 'Solubility enhancement factor' of ionized-CV was found high enough (340 times) with HPβCD in presence of TA. TA increases the binding efficiency of cyclodextrin and changing the pH of microenvironment in dissolution medium. In addition, ionization process was used to increase the apparent intrinsic solubility of drug. In vitro, dissolution time of CV was remarkably reduced in the solid dispersion system compared to that of pure drug. This may be attributed to increased wettability, dispersing ability and transformation of crystalline state of drug to amorphous one. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Influence of milling process on efavirenz solubility

    Directory of Open Access Journals (Sweden)

    Erizal Zaini

    2017-01-01

    Full Text Available Introduction: The aim of this study was to investigate the influence of the milling process on the solubility of efavirenz. Materials and Methods: Milling process was done using Nanomilling for 30, 60, and 180 min. Intact and milled efavirenz were characterized by powder X-ray diffraction, scanning electron microscopy (SEM, spectroscopy infrared (IR, differential scanning calorimetry (DSC, and solubility test. Results: The X-ray diffractogram showed a decline on peak intensity of milled efavirenz compared to intact efavirenz. The SEM graph depicted the change from crystalline to amorphous habit after milling process. The IR spectrum showed there was no difference between intact and milled efavirenz. Thermal analysis which performed by DSC showed a reduction on endothermic peak after milling process which related to decreasing of crystallinity. Solubility test of intact and milled efavirenz was conducted in distilled water free CO2with 0.25% sodium lauryl sulfate media and measured using high-performance liquid chromatography method with acetonitrile: distilled water (80:20 as mobile phases. The solubility was significantly increased (P < 0.05 after milling processes, which the intact efavirenz was 27.12 ± 2.05, while the milled efavirenz for 30, 60, and 180 min were 75.53 ± 1.59, 82.34 ± 1.23, and 104.75 ± 0.96 μg/mL, respectively. Conclusions: Based on the results, the solubility of efavirenz improved after milling process.

  20. Glomerular Podocytes Express Type 1 Adenylate Cyclase: Inactivation Results in Susceptibility to Proteinuria

    Science.gov (United States)

    Xiao, Zhijie; He, Liqun; Takemoto, Minoru; Jalanko, Hannu; Chan, Guy C.; Storm, Daniel R.; Betsholtz, Christer; Tryggvason, Karl; Patrakka, Jaakko

    2011-01-01

    Background/Aims The organization of actin cytoskeleton in podocyte foot processes plays a critical role in the maintenance of the glomerular filtration barrier. The cAMP pathway is an important regulator of the actin network assembly in cells. However, the role of the cAMP pathway in podocytes is not well understood. Type 1 adenylate cyclase (Adcy1), previously thought to be specific for neuronal tissue, is a member of the family of enzymes that catalyses the formation of cAMP. In this study, we characterized the expression and role of Adcy1 in the kidney. Methods Expression of Adcy1 was studied by RT-PCR, Northern blotting and in situ hybridization. The role of Adcy1 in podocytes was investigated by analyzing Adcy1 knockout mice (Adcy1–/–). Results and Conclusion: Adcy1 is expressed in the kidney specifically by podocytes. In the kidney, Adcy1 does not have a critical role in normal physiological functioning as kidney histology and function are normal in Adcy1–/– mice. However, albumin overload resulted in severe albuminuria in Adcy1–/– mice, whereas wild-type control mice showed only mild albumin leakage to urine. In conclusion, we have identified Adcy1 as a novel podocyte signaling protein that seems to have a role in compensatory physiological processes in the glomerulus. PMID:21196775