WorldWideScience

Sample records for adenylate cyclase activity

  1. Adenyl cyclase.

    Science.gov (United States)

    Coppe, D; Steer, M L

    1978-02-01

    Certain hormones regulate the activity of their target cells by stimulating adenyl cyclase, which is an enzyme located within the target cell's plasma membrane. Adenyl cyclase catalyzes the formation of cyclic AMP, which is released into the cell and modulates cell functions. In this communication the characteristics of adenyl cyclase are reviewed. The coupling between hormone receptors and this enzyme is discussed as is the ability of agents such as hormones, ATP, magnesium, calcium, guanine nucleotides and prostaglandins to alter cyclase activity. Several diseases that result from derangements of the adenyl cyclase system are known and the molecular bases for these diseases are discussed in this review.

  2. 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...... with moderate or severe migraine headache had elevated PACAP in the external jugular vein during headache (n = 15), that was reduced 1 h after treatment with sumatriptan 6 mg (n = 11), and further reduced interictally (n = 9). The data suggest PACAP, or its receptors, are a promising target for migraine...

  3. Lymphocyte adenylate cyclase activity in immunosuppressed patients

    NARCIS (Netherlands)

    Michel, M. C.; Brodde, O. E.

    1989-01-01

    In order to determine whether alterations of adenylate cyclase are involved in the immunosuppressive effect of glucocorticoid/cyclosporin treatment we measured basal, prostaglandin E1-, and forskolin-stimulated cAMP production in lymphocyte membranes from kidney transplant patients undergoing

  4. Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site.

    Science.gov (United States)

    Morris, S A; Bilezikian, J P

    1983-02-01

    The diterpene forskolin has been reported to activate adenylate cyclase in a manner consistent with an interaction at the catalytic unit. However, some of its actions are more consistent with an interaction at the coupling unit that links the hormone receptor to the adenylate cyclase activity. This report adds support to the latter possibility. Under conditions that lead to stimulation of adenylate cyclase in turkey erythrocyte membranes by GTP, forskolin also becomes more active. Additional evidence to support an influence of forskolin upon adenylate cyclase via the GTP-coupling protein N includes the following: (i) forskolin, at submaximal concentrations, leads to enhanced sensitivity and responsiveness of isoproterenol-dependent adenylate cyclase activity in turkey erythrocyte membranes; (ii) under specified conditions, the nucleotide GDP, an inhibitor of the stimulating nucleotide GTP and its analog, guanyl imidodiphosphate (Gpp(NH)p), also markedly inhibits the action of forskolin; (iii) both Gpp(NH)p and forskolin are associated with a decrease in agonist affinity for the beta-adrenergic receptor. However, actions of forskolin in the turkey erythrocyte are not identical to those of GTP: (i) forskolin is never as potent as Gpp(NH)p in activating adenylate cyclase; (ii) the magnitude of synergism between isoproterenol and forskolin is not equal to that observed with isoproterenol and Gpp(NH)p; (iii) at high concentrations, forskolin inhibits antagonist binding to the beta-receptor. Forskolin appears to have several sites of action in the turkey erythrocyte membrane, including an influence upon the adenylate cyclase regulatory protein N.

  5. Determination of the turn-off reaction for the hormone-activated adenylate cyclase.

    Science.gov (United States)

    Cassel, D; Eckstein, F; Lowe, M; Selinger, Z

    1979-10-10

    Previous work suggested that hormonal activation of adenylate cyclase involves the introduction of GTP to the regulatory site, and subsequent hydrolysis of the bound GTP terminates the activation. In many tissues the turn-off GTPase reaction cannot be readily measured because of a high background of nonspecific GTP hydrolysis. To circumvent this problem a general assay for the turn-off reaction has now been developed. The adenylate cyclase is first activated by hormone and GTP and the introduction of GTP is then stopped either by addition of an excess of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) or by addition of a receptor blocking agent. The decay of adenylate cyclase activity brought on by these inhibitors is used to calculate the rate constant of the turn-off reaction. In turkey erythrocyte and rat parotid membranes the rate constant of the decay process as determined with GDP beta S is similar to that determined with the beta-adrenergic blocker propranolol. The rate constants (min-1 at 30 degrees C) for various adenylate cyclase preparations are 10 for turkey erythrocyte, 7.5 for rat parotid, and 6.2 for the rat liver enzyme. The finding of similar rate constants in the various preparations indicates that GTP hydrolysis at the regulatory site is a general mechanism for terminating the activation of adenylate cyclase.

  6. 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...

  7. 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

  8. Enhanced adenylate cyclase activity of turkey erythrocytes following treatment with beta-adrenergic receptor antagonists.

    Science.gov (United States)

    Peters, J R; Nambi, P; Sibley, D R; Lefkowitz, R J

    1984-12-15

    The turkey erythrocyte contains a beta 1-adrenergic receptor-linked adenylate cyclase system. We have examined the effects of pretreatment with receptor antagonists on adenylate cyclase activity and the individual components in the pathway of enzyme activation in this system. Isoproterenol-stimulated adenylate cyclase activity is increased by 30% (P less than 0.01) over control in membranes derived from cells preincubated with the antagonist propranolol. The effect is stereospecific and dose-related with a EC50 of 100 nM for the (-) isomer. The time course of effect is rapid being complete by 90 min. Non-receptor mediated stimulation of adenylate cyclase activity by manganese ion, forskolin and NaF is similarly enhanced following propranolol pretreatment. Sensitization of adenylate cyclase activity also occurs following pretreatment with a number of antagonists but is not seen after preincubation with pindolol or practolol. Quantitation of beta-adrenergic receptor (R) density using [125I]cyanopindolol indicates no difference between membranes derived from control and antagonist pretreated cells. Coupling of R with the guanine nucleotide regulatory protein (N) as assessed by high affinity agonist binding is unchanged following pretreatment. The efficacy of 5'-guanylylimidodiphosphate Gpp(NH)p in producing a shift of agonist binding curves associated with destabilization of high affinity H-R-N complexes, is also the same (EC50 = 0.2 microM) in membranes from control and antagonist treated cells. The isoproterenol stimulated rate of release of [3H]GDP from membranes preloaded with [3H]GTP as an index of formation of an active form of the N protein is similarly unaffected by antagonist preincubation. We conclude that the mechanism of the observed sensitization of turkey erythrocyte adenylate cyclase by beta-adrenergic antagonists is receptor mediated and likely involves facilitation of N interaction with the catalytic subunit of the enzyme.

  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. 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

  11. Evidence for the existence of a sulfhydryl group in the adenylate cyclase active site.

    Science.gov (United States)

    Skurat, A V; Yurkova, M S; Baranova, L A; Gulyaev, N N; Bulargina, T V; Severin, E S

    1985-03-01

    6-Cloro-9-beta-d-ribofuranosylpurine 5'-triphosphate (CIRTP) and 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (SRTP) irreversibly inhibit adenylate cyclase from rat brain. Adenosine 5'-[beta, gamma -imido] triphosphate protects the enzyme against inactivation by CIRTP and SRTP and acts as a competitive inhibitor with respect to ATP with the Ki value 2 X 10(-4) M. Study of the pH-dependence of the rate of the enzyme inactivation by CIRTP showed that pK for the group modified by this compound is equal to 7.45. Inactivation is first order with respect to the enzyme; the saturation effect is observed at the increased concentration of CIRTP. The k2 and KI values for irreversible inhibition of brain adenylate cyclase by CIRTP were 0.25 min-1 and 1.9 X 10(-4) M, respectively. Adenylate cyclase inhibition by SRTP is also time-dependent. Partial protection against the enzyme inactivation was observed. Dithiothreitol restores the activity of SRTP-inactivated adenylate cyclase. The results obtained indicate the presence of an -SH group in the purine amino group binding area of the enzyme active site.

  12. Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

    International Nuclear Information System (INIS)

    McKee, R.L.; Pelton, J.T.; Trivedi, D.; Johnson, D.G.; Coy, D.H.; Sueiras-Diaz, J.; Hruby, V.J.

    1986-01-01

    In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4]glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists

  13. Functional exchange of components between light-activated photoreceptor phosphodiesterase and hormone-activated adenylate cyclase systems

    Energy Technology Data Exchange (ETDEWEB)

    Bitensky, M.W.; Wheeler, M.A.; Rasenick, M.M.; Yamazaki, A.; Stein, P.J.; Halliday, K.R.; Wheeler, G.L.

    1982-06-01

    Previous studies have noted profound similarities between the regulation of light-activated (3',5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in retinal rods and hormone-activated adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) in a variety of tissues. We report ere the functional exchange of components isolated from the photoreceptor system, which displayed predicted functional characteristics when incubated with recipient adenylate cyclase systems from rat cerebral cortical and hypothalamic synaptic membranes and frog erythrocyte ghosts. We demonstrate functional exchange of photoreceptor components at each of three loci: the hormone receptor, the GTP-binding protein (GBP), and the catalytic moiety of adenylate cyclase. Illuminated (but not unilluminated) rhodopsin was found to mimic the hormone-receptor complex, causing GTP-dependent activation of adenylate cyclase. The photoreceptor GBP complexed with guanosine 5'-(..beta..,..gamma..)imidotriphosphate (p(NH)ppG) produced a marked activation of recipient adenylate cyclase systems. Much smaller activation was observed when GBP was not complexed with p(NH)ppG. A heat-stable photoreceptor phosphodiesterase inhibitor reduced both basal and Mn/sup 2 +/-activated adenylate cyclase activites and this inhibition was reversed by photoreceptor GBPp(NH)ppG. These data demonstrate a remarkable functional compatibility between subunits of both systems and furthermore imply that specialized peptide domains responsible for protein-protein interactions are highly conserved.

  14. 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.

  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. The hormonal regulation of adenylate cyclase.

    Science.gov (United States)

    Feder, D; Im, M J; Pfeuffer, T; Hekman, M; Helmreich, E J; Levitzki, A

    1986-01-01

    The regulation of adenylate cyclase by hormones and by GTP regulatory proteins was investigated in native membrane systems and in systems reconstituted from purified components. These studies can be summarized as follows. The stimulatory beta 1-adrenoceptor catalyses the activation of a complex between the GTP stimulatory protein GS and the catalytic unit C. The agonist-receptor complex can activate a few cyclase units in native membrane systems as well as in reconstituted systems. GS from turkey erythrocytes is functionally different from rabbit liver GS, the latter being more amenable to activation by guanyl nucleotides in the absence of hormone. The coupling between the beta 1-adrenoceptor GS and C is efficient when compared with the coupling obtained in native membrane systems. GTP/GDP exchange at the alpha S subunit requires the presence of the beta gamma subunits. A mechanism for the inhibition of adenylate cyclase by the inhibitory GTP regulatory protein Gi is suggested.

  17. 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.)

  18. Mechanism of adenylate cyclase activation through the beta-adrenergic receptor: catecholamine-induced displacement of bound GDP by GTP.

    Science.gov (United States)

    Cassel, D; Selinger, Z

    1978-09-01

    The fate of the guanyl nucleotide bound to the regulatory site of adenylate cyclase was studied on a preparation of turkey erythrocyte membranes that was incubated with [3H]GTP plus isoproterenol and subsequently washed to remove hormone and free guanyl nucleotide. Further incubation of this preparation in the presence of beta-adrenergic agonists resulted in the release from the membrane of tritiated nucleotide, identified as [3H]GDP. The catecholamine-induced release of [3H]GDP was increased 2 to 3 times in the presence of the unlabeled guanyl nucleotides GTP, guanosine 5'-(beta,gamma-imino)triphosphate [gpp(NH)p], GDP, and GMP, whereas adenine nucleotides had little effect. In the presence of Gpp(NH)p, isoproterenol induced the release of [3H]GDP and the activation of adenylate cyclase, both effects following similar time courses. The findings indicate that the inactive adenylate cyclase possesses tightly bound (GDP, produced by the hydrolysis of GTP at the regulatory site. The hormone stimulates adenylate cyclase activity by inducing an "opening" of the guanyl nucleotide site, resulting in dissociation of the bound GDP and binding of the activating guanosine triphosphate.

  19. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    Energy Technology Data Exchange (ETDEWEB)

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.; Seidler, F.J. (Duke Univ. Medical Center, Durham, NC (USA))

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a {beta}-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of {beta}-adrenergic receptors: in fact, ({sup 125}I)pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.

  20. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    International Nuclear Information System (INIS)

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.; Seidler, F.J.

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a β-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of β-adrenergic receptors: in fact, [ 125 I]pindolol binding was significantly decreased in the nicotine group. These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level

  1. 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.

  2. Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion

    DEFF Research Database (Denmark)

    Sasaki, S; Watanabe, J; Ohtaki, H

    2017-01-01

    BACKGROUND: Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required...... and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. OBJECTIVES: To investigate the effect of PACAP on eccrine sweat secretion. METHODS: Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression....... Pituitary adenylate cyclase-activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP-specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin...

  3. 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...

  4. Ligand requirements for the relaxation of adenylate cyclase from activated and inhibited states.

    Science.gov (United States)

    Lad, P M; Reisinger, D M; Smiley, P A

    1983-06-21

    The turkey erythrocyte adenylate cyclase system binds tightly the inhibitory nucleotide GDP, and a pretreatment step with isoproterenol and GMP is required to restore activation. Under identical pretreatment conditions, the release of labeled nucleotide is complete within 1 min whereas the restoration of activation by Gpp(NH)p requires 15 min. A study of the ligand requirements of the slow step shows the following: (a) The role of GMP is that of an obligatory allosteric regulator. (b) Cholera toxin modification of the system abolishes the requirement for GMP with a considerable enhancement in the reaction rate. (c) GMP is without effect on the relaxation process with the activator Gpp(NH)p as the resident nucleotide. In sharp contrast, ethylenediamine-tetraacetic acid (without effect in a GDP-occupied complex) markedly potentiates alterations from the Gpp(NH)p-occupied state. (d) Formation of a GDP/guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) hybrid leads to the suppression of both F- and Gpp(NH)p activation. F- activation is restored by isoproterenol alone, while GMP is still required to restore Gpp(NH)p activation. The results suggest that covalent modification or nucleotide analogue occupancy of the regulatory complex can modify the allosteric role for GMP, with consequences for the rate of the slow step.

  5. 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

  6. Site I Inactivation Impacts Calmodulin Calcium Binding and Activation of Bordetella pertussis Adenylate Cyclase Toxin.

    Science.gov (United States)

    Johns, Christian W; Finley, Natosha L

    2017-11-30

    Site I inactivation of calmodulin (CaM) was used to examine the importance of aspartic acid 22 at position 3 in CaM calcium binding, protein folding, and activation of the Bordetella pertussis adenylate cyclase toxin domain (CyaA-ACD). NMR calcium titration experiments showed that site I in the CaM mutant (D22A) remained largely unperturbed, while sites II, III, and IV exhibited calcium-induced conformational changes similar to wild-type CaM (CaMWt). Circular dichroism analyses revealed that D22A had comparable α -helical content to CaMWt, and only modest differences in α -helical composition were detected between CaMWt-CyaA-ACD and D22A-CyaA-ACD complexes. However, the thermal stability of the D22A-CyaA-ACD complex was reduced, as compared to the CaMWt-CyaA-ACD complex. Moreover, CaM-dependent activity of CyaA-ACD decreased 87% in the presence of D22A. Taken together, our findings provide evidence that D22A engages CyaA-ACD, likely through C -terminal mediated binding, and that site I inactivation exerts functional effects through the modification of stabilizing interactions that occur between N -terminal CaM and CyaA-ACD.

  7. 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.

  8. 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

  9. Renoprotection by pituitary adenylate cyclase-activating polypeptide in multiple myeloma and other kidney diseases.

    Science.gov (United States)

    Li, Min; Maderdrut, Jerome L; Lertora, Juan J L; Arimura, Akira; Batuman, Vecihi

    2008-01-10

    Renal involvement in patients with multiple myeloma complicates their treatment and shortens their life-span. The main renal lesion is a tubulointerstitial transformation with fibrosis, frequently associated with cast formation in the distal nephron that results from co-precipitation of pathological immunoglobulin light chains with Tamm-Horsfall proteins. The human renal proximal tubular reabsorption of excessive light chains by endocytosis causes cellular protein overload and activates the transcription factor nuclear factor kappa B (NFkappaB). The activation of NFkappaB promotes the synthesis of inflammatory cytokines and activates signaling pathways, such as mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2, Jun kinase, and p38 MAPK, thus promoting interstitial inflammation and fibrosis. We tested the concept that pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/vasoactive intestinal peptide family, could prevent the development of cast nephropathies. PACAP38 inhibited myeloma light chain-induced proinflammatory cytokine expression with greater potency than dexamethasone, and attenuated the resulting cell damage in the renal proximal tubule epithelial cells. The results indicated that its effects are mediated through inhibition of phosphorylation of p38 MAPK and nuclear translocation of the p50 subunit of NFkappaB via both the PAC(1) and VPAC(1) receptors. PACAP was also shown to be efficacious in other common in vivo animal models for kidney hypertrophies, including streptozotocin-induced diabetic nephropathy and gentamicin-induced nephrotoxicity. Thus, our studies suggest that PACAP38 could be used as a cytoprotective agent that would be effective in the treatment of renal tubule injury in multiple myeloma and other chronic kidney diseases.

  10. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP knockout mice

    Directory of Open Access Journals (Sweden)

    Satoko eHattori

    2012-10-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1. Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J x 129SvEv for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased social interaction in Crawley’s three-chamber social approach test, although PACAP KO had no significant impact on social interaction in a home cage. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze and the T-maze, while they did not show any significant abnormalities in the left-right discrimination task in the T-maze. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially

  11. Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion.

    Science.gov (United States)

    Sasaki, S; Watanabe, J; Ohtaki, H; Matsumoto, M; Murai, N; Nakamachi, T; Hannibal, J; Fahrenkrug, J; Hashimoto, H; Watanabe, H; Sueki, H; Honda, K; Miyazaki, A; Shioda, S

    2017-02-01

    Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required. Pituitary adenylate cyclase-activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP-specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. To investigate the effect of PACAP on eccrine sweat secretion. Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression and localization of PACAP and its receptors in mouse and human eccrine sweat glands. We injected PACAP subcutaneously into the footpads of mice and used the starch-iodine test to visualize sweat-secreting glands. Immunostaining showed PACAP and PAC1R expression by secretory cells from mouse and human sweat glands. PACAP immunoreactivity was also localized in nerve fibres around eccrine sweat glands. PACAP significantly promoted sweat secretion at the injection site, and this could be blocked by the PAC1R-antagonist PACAP6-38. VIP, an agonist of VPAC1R and VPAC2R, failed to induce sweat secretion. This is the first report demonstrating that PACAP may play a crucial role in sweat secretion via its action on PAC1R located in eccrine sweat glands. The mechanisms underlying the role of PACAP in sweat secretion may provide new therapeutic options to combat sweating disorders. © 2016 British Association of Dermatologists.

  12. Pituitary adenylate cyclase activating polypeptide modulates catecholamine storage and exocytosis in PC12 cells.

    Directory of Open Access Journals (Sweden)

    Yan Dong

    Full Text Available A number of efforts have been made to understand how pituitary adenylate cyclase activating polypeptide (PACAP functions as a neurotrophic and neuroprotective factor in Parkinson's disease (PD. Recently its effects on neurotransmission and underlying mechanisms have generated interest. In the present study, we investigate the effects of PACAP on catecholamine storage and secretion in PC12 cells with amperometry and transmission electron microscopy (TEM. PACAP increases quantal release induced by high K+ without significantly regulating the frequency of vesicle fusion events. TEM data indicate that the increased volume of the vesicle is mainly the result of enlargement of the fluidic space around the dense core. Moreover, the number of docked vesicles isn't modulated by PACAP. When cells are acutely treated with L-DOPA, the vesicular volume and quantal release both increase dramatically. It is likely that the characteristics of amperometric spikes from L-DOPA treated cells are associated with increased volume of individual vesicles rather than a direct effect on the mechanics of exocytosis. Treatment with PACAP versus L-DOPA results in different profiles of the dynamics of exocytosis. Release via the fusion pore prior to full exocytosis was observed with the same frequency following treatment with PACAP and L-DOPA. However, release events have a shorter duration and higher average current after PACAP treatment compared to L-DOPA. Furthermore, PACAP reduced the proportion of spikes having rapid decay time and shortened the decay time of both fast and slow spikes. In contrast, the distributions of the amperometric spike decay for both fast and slow spikes were shifted to longer time following L-DOPA treatment. Compared to L-DOPA, PACAP may produce multiple favorable effects on dopaminergic neurons, including protecting dopaminergic neurons against neurodegeneration and potentially regulating dopamine storage and release, making it a promising

  13. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

    Directory of Open Access Journals (Sweden)

    Matthew M. Hurley

    2016-08-01

    Full Text Available While pituitary adenylate cyclase activating polypeptide (PACAP signaling in the hypothalamic ventromedial nuclei (VMN has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger and hedonic-related (palatability drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding; surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding. In contrast, inhibition of the nucleus accumbens (NAc, through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive.

  14. Pituitary adenylate cyclase-activating polypeptide (PACAP) in fetal cord blood.

    Science.gov (United States)

    Winters, Stephen J; King, Jeffrey C; Brees, Carol K; Moore, Joseph P

    2014-09-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is a centrally-acting peptide with highest concentration within the limbic area of the brain. PACAP is also expressed in and affects the functions of vascular and nervous tissues, endocrine glands, and the placenta. PACAP appears to be associated with the 'fight-or-flight' response to emergency partly through its effect on adrenal production of cortisol and catecholamines. We sought to explore the impact of labor as a stressor on the level of PACAP in the fetus, and hypothesized that PACAP levels would be increased when labor, abnormal fetal heart tracing, or fetal growth impairment was evident. Fetal cord venous blood samples were collected immediately after delivery from a random group of women undergoing either vaginal or Cesarean delivery. The blood was placed in chilled EDTA-aprotinin containing tubes, centrifuged, and stored at -80°C for PACAP immunoassay. Delivery data were abstracted for analysis. The level of PACAP in cord blood was similar in both males and females. There was a trend toward higher levels in the cord blood of fetuses delivered by Cesarean compared to those delivered vaginally. PACAP levels were unrelated to birth weight, Apgar scores, and the presence or absence of labor prior to delivery. While PACAP and its receptor are expressed in placenta, and PACAP protein is found in cord blood, no effect of labor stress on PACAP was found. Further research is needed to understand the role of PACAP in gestation and parturition. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Adenylate cyclases involvement in pathogenicity, a minireview.

    Science.gov (United States)

    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

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

  16. Adenylate Cyclase-the More Membrane Associated, the Less Radiosensitive

    OpenAIRE

    ATTILA, TAKATS; VU HOA, BINH; KATALIN, OFFENMULLER; KATALIN, BODO; LORAND, BERTOK; GYORGY J., KOTELES; Frederic Joliot-Curie National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology; "Frederic Joliot-Curie" National Research Institute for Radiobiology

    1993-01-01

    A dose dependent but not parallel decreases were observed both in SH content and catalytic activity of "free" catalytic subunit after irradiation (0-3200 Gy), while SR groups of membrane-associated adenylate cyclase were insensitive (under 3200 Gy). An initial "radioactivation" of membrane-associated enzyme was found under 800 Gy, then an inhibition above 1600 Gy. The SH alkylating agent, N-ethylmaleimide resulted in a complete inactivation, both of membrane associated form of adenylate cycla...

  17. Receptors, adenylate cyclase, depression, and lithium.

    Science.gov (United States)

    Belmaker, R H

    1981-04-01

    Although numerous studies have suggested that depression may be associated with a reduction in synaptic noradrenaline in the brain, direct beta-adrenergic receptor agonists have not been tested in the treatment of depression until recently. Moreover, newer theories of antidepressant action suggest that a reduction in beta-adrenergic receptor sensitivity is a better correlate of antidepressant treatment than noradrenaline turnover changes. It is possible to evaluate the beta-adrenergic receptor-adenylate cyclase complex in the human periphery by measuring the plasma cyclic AMP rise after adrenergic agonists. A clinical trial of the beta-2 adrenergic agonist salbutamol in depression provided an opportunity to test whether adrenergic receptor subsensitivity does occur during clinical antidepressant treatment. Plasma cyclic AMP before treatment with salbutamol rose 26% in response to salbutamol 0.25 mg iv. After 1 and 3 weeks of oral salbutamol treatment, depression scores declined significantly in 11 depressed patients, while the plasma cyclic AMP response to iv salbutamol declined over 60%. The beta-adrenergic adenylate cyclase remained subsensitive 4 days after cessation of salbutamol therapy. The results support the concept that receptor sensitivity changes occur during human antidepressant therapy. Data are presented that Li, too, markedly reduces activity of beta-adrenergic adenylate cyclase in humans. The effect was evaluated by studying the effect of Li at therapeutic serum concentrations on the plasma cyclic AMP response to subcutaneous epinephrine. The Li effect is specific, since the plasma cyclic AMP response to glucagon is not inhibited. The plasma cyclic GMP response to subcutaneous epinephrine, suggested as a model for presynaptic alpha-noradrenergic mechanisms, is also partially inhibited by Li therapy. Since cyclic AMP and cyclic GMP may be viewed as balancing substances, their interaction may provide a mechanism for Li's dual clinical effects in mania

  18. Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A

    2012-01-01

    for the demonstration of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (NOS), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), VIP and PACAP common receptors (VPAC1, VPAC2), and PACAP receptor (PAC1). In addition, double labeling...

  19. A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies.

    Science.gov (United States)

    Jayakumar, Thanasekaran; Chen, Wei-Fan; Lu, Wan-Jung; Chou, Duen-Suey; Hsiao, George; Hsu, Chung-Yi; Sheu, Joen-Rong; Hsieh, Cheng-Ying

    2013-06-01

    Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15-75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca(2+) mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH(●)) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH(●) formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This

  20. The Escherichia coli adenylate cyclase complex. Stimulation by potassium and phosphate.

    Science.gov (United States)

    Liberman, E; Reddy, P; Gazdar, C; Peterkofsky, A

    1985-04-10

    In Escherichia coli, adenylate cyclase activity in toluene-treated cells can be inhibited by glucose while the activity in a broken cell preparation cannot. Adenylate cyclase activity in the permeabilized but not in broken cells is stimulated somewhat specifically and additively by potassium and phosphate. Kinetic studies show sigmoid substrate-velocity curves for the toluene-treated cells but hyperbolic curves for the broken cells. The stimulatory effects of potassium and phosphate on adenylate cyclase activity in tolulene-treated cells are associated with increases in the Vmax and Km for ATP. While the enzyme activity in toluene-treated cells shows a preference for magnesium over manganese, the reverse is observed in broken cells. Stimulation of adenylate cyclase activity in toluene-treated cells requires the presence of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS proteins can be phosphorylated in a P-enolpyruvate-dependent reaction. The stimulatory effects of ions will not occur if the PTS proteins are not phosphorylated. Since potassium phosphate stimulates both adenylate cyclase and PTS activities in toluene-treated cells, it is proposed that the effect of potassium phosphate on adenylate cyclase activity is mediated through an effect on the PTS. A model for dual regulation by glucose of adenylate cyclase activity is proposed. This model involves regulation of both the condition of the PTS proteins as well as the cellular concentration of phosphate.

  1. 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

  2. High skeletal muscle adenylate cyclase in malignant hyperthermia.

    OpenAIRE

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

    1981-01-01

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

  3. Kynurenic Acid Inhibits the Electrical Stimulation Induced Elevated Pituitary Adenylate Cyclase-Activating Polypeptide Expression in the TNC

    Directory of Open Access Journals (Sweden)

    Tamás Körtési

    2018-01-01

    Full Text Available BackgroundMigraine is a primary headache of imprecisely known mechanism, but activation of the trigeminovascular system (TS appears to be essential during the attack. Intensive research has recently focused on pituitary adenylate cyclase-activating polypeptide (PACAP and the kynurenine systems as potential pathogenic factors.AimWe investigated the link between these important mediators and the effects of kynurenic acid (KYNA and its synthetic analog (KYNA-a on PACAP expression in the rat trigeminal nucleus caudalis (TNC in a TS stimulation model related to migraine mechanisms.MethodsAdult male Sprague-Dawley rats were pretreated with KYNA, KYNA-a, the NMDA receptor antagonist MK-801, or saline (vehicle. Next, the trigeminal ganglion (TRG was electrically stimulated, the animals were transcardially perfused following 180 min, and the TNC was removed. In the TNC samples, 38 amino acid form of PACAP (PACAP1–38-like radioimmunoactivity was measured by radioimmunoassay, the relative optical density of preproPACAP was assessed by Western blot analysis, and PACAP1–38 mRNA was detected by real-time PCR.Results and conclusionElectrical TRG stimulation resulted in significant increases of PACAP1–38-LI, preproPACAP, and PACAP1–38 mRNA in the TNC. These increases were prevented by the pretreatments with KYNA, KYNA-a, and MK-801. This is the first study to provide evidence for a direct link between PACAP and the kynurenine system during TS activation.

  4. Interactions of fluoride and guanine nucleotides with thyroid adenylate cyclase.

    Science.gov (United States)

    Goldhammer, A; Wolff, J

    1982-02-18

    The activation of bovine thyroid adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Gpp(NH)p has been studied using steady-state kinetic methods. This activation is complex and may be characterized by two Gpp(NH)p binding sites of different affinities with measured constants: Ka1 = 0.1 micro M and Ka2 = 2.9 micro M. GDP beta S does not completely inhibit the Gpp(NH)p activation: analysis of the data is consistent with a single GDP beta S inhibitory site which is competitive with the weaker Gpp(NH)p site. Guanine nucleotide effects upon F- activation of adenylate cyclase have been studied. When App(NH)p is the substrate, 10 micro M GTP along with 10 mM NaF gives higher activity than NaF alone, while GDP together with NaF inhibits the activity by 50% relative to NaF. These features are not observed when the complex is assayed with ATP in the presence of a nucleotide regenerating system or when analogs Gpp)NH)p or GDP beta S are used along with NaF. These effects were studied in three other membrane systems using App(NH)p as substrate: rat liver, rat ovary and turkey erythrocyte. No consistent pattern of guanine nucleotide effects upon fluoride activation could be observed in the different membrane preparations. Previous experiments showed that the size of soluble thyroid adenylate cyclase changed whether membranes were preincubated with Gpp(NH)p or NaF. This size change roughly corresponded to the molecular weight of the nucleotide regulatory protein. This finding, coupled with the present data, suggests that two guanine nucleotide binding sites may be involved in regulating thyroid cyclase and that these sites may be on different protein chains.

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

    Science.gov (United States)

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

    2010-01-01

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

  6. The effects of isatin (indole-2, 3-dione on pituitary adenylate cyclase-activating polypeptide-induced hyperthermia in rats

    Directory of Open Access Journals (Sweden)

    Tóth Gábor

    2002-02-01

    Full Text Available Abstract Background Previous studies have demonstrated that centrally administered natriuretic peptides and pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38 have hyperthermic properties. Isatin (indole-2, 3-dione is an endogenous indole that has previously been found to inhibit hyperthermic effects of natriuretic peptides. In this study the aim was to investigate the effects of isatin on thermoregulatory actions of PACAP-38, in rats. Results One μg intracerebroventricular (icv. injection of PACAP-38 had hyperthermic effect in male, Wistar rats, with an onset of the effect at 2 h and a decline by the 6th h after administration. Intraperitoneal (ip. injection of different doses of isatin (25-50 mg/kg significantly decreased the hyperthermic effect of 1 μg PACAP-38 (icv., whereas 12.5 mg/kg isatin (ip. had no inhibiting effect. Isatin alone did not modify the body temperature of the animals. Conclusion The mechanisms that participate in the mediation of the PACAP-38-induced hyperthermia may be modified by isatin. The capability of isatin to antagonize the hyperthermia induced by all members of the natriuretic peptide family and by PACAP-38 makes it unlikely to be acting directly on receptors for natriuretic peptides or on those for PACAP in these hyperthermic processes.

  7. 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

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

    Science.gov (United States)

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

    2013-09-01

    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.

  9. Absorption and fluorescence characteristics of photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-01-02

    Graphical abstract: Protein color center emissions were observed in the wavelength range from 340 nm to 900 nm from nano-clusters of the photo-activated adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi. Highlights: Black-Right-Pointing-Pointer Adenylyl cyclase nPAC in aqueous pH 7.5 buffer dissolved only to nano-clusters. Black-Right-Pointing-Pointer Nano-cluster size was determined by light attenuation (scattering) measurements. Black-Right-Pointing-Pointer The size of the nano-clusters was growing by coalescing during observation period. Black-Right-Pointing-Pointer In nPAC nano-clusters color centers were present in emission range of 360-900 nm. Black-Right-Pointing-Pointer The nPAC color center emission is compared with fluorescent protein emission. - Abstract: The spectroscopic characteristics of BLUF (BLUF = sensor of blue light using flavin) domain containing soluble adenylate cyclase (nPAC = Naegleria photo-activated cyclase) samples from the amoeboflagellate Naegleria gruberi NEG-M strain is studied at room temperature. The absorption and fluorescence spectroscopic development in the dark was investigated over two weeks. Attenuation coefficient spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation distributions were measured. Thawing of frozen nPAC samples gave solutions with varying protein nano-cluster size and varying flavin, tyrosine, tryptophan, and protein color-center emission. Protein color-center emission was observed in the wavelength range of 360-900 nm with narrow emission bands of small Stokes shift and broad emission bands of large Stokes shift. The emission spectra evolved in time with protein nano-cluster aging.

  10. 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

  11. Vanadate stimulates adenylate cyclase via the guanine nucleotide regulatory protein by a mechanism differing from that of fluoride.

    Science.gov (United States)

    Krawietz, W; Downs, R W; Spiegel, A M; Aurbach, G D

    1982-03-01

    Vanadate stimulates adenylate cyclase activity in turkey erythrocyte membranes. The maximal stimulation is 7-fold over basal at 3 mM vanadate; higher concentrations are inhibitory. A suboptimal concentration of fluoride (1 mM) together with vanadate (3 mM) activates adenylate cyclase in a non-additive manner; cyclase activation by optimal fluoride (10 mM) is inhibited by vanadate (3 mM). There is no stimulation by vanadate of adenylate cyclase activity (measured either with Mg2+ or Mn2+) in CYC- S49 lymphoma cell membranes. Vanadate (3 mM) shows no effect on binding of Beta-adrenergic agonists or antagonists to the [3H] (-)-dihydroalprenolol binding site in turkey erythrocyte membranes. These results suggest that the effect of vanadate on Adenylate cyclase is mediated through the nucleotide regulatory protein and may act by a mechanism similar to fluoride. However, in cholera toxic-treated membranes as well as in GDP-beta-S plus isoproterenol-treated membranes, fluoride-stimulated adenylate cyclase activity is significantly reduced, but vanadate stimulation is not. Our results suggest that although the actions of vanadate and fluoride in adenylate cyclase may each involve the nucleotide regulatory unit, the exact mechanisms of activation by the two anions differ.

  12. 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)

  13. Involvement of endogenous antioxidant systems in the protective activity of pituitary adenylate cyclase-activating polypeptide against hydrogen peroxide-induced oxidative damages in cultured rat astrocytes.

    Science.gov (United States)

    Douiri, Salma; Bahdoudi, Seyma; Hamdi, Yosra; Cubì, Roger; Basille, Magali; Fournier, Alain; Vaudry, Hubert; Tonon, Marie-Christine; Amri, Mohamed; Vaudry, David; Masmoudi-Kouki, Olfa

    2016-06-01

    Astroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the

  14. 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

  15. 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.

  16. 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

  17. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

    Directory of Open Access Journals (Sweden)

    Tamás Juhász

    2015-07-01

    Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.

  18. 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

  19. Structure of the turkey erythrocyte adenylate cyclase system.

    Science.gov (United States)

    Nielsen, T B; Lad, P M; Preston, M S; Kempner, E; Schlegel, W; Rodbell, M

    1981-02-01

    Target analysis of the turkey erythrocyte adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] system showed that the molecular weight of the ground state enzyme increases from 92,000 with MnATP as substrate and no stimulatory ligands to 226,000 when activated by fluoride ion or by 5'-guanyl imidodiphosphate (p[NH]ppG) subsequent to clearance of previously bound GDP. The identical increment in size (130,000) suggests that the same regulatory unit is involved in the activation by both effectors. When assayed with isoproterenol and p[NH]ppG, the enzyme system displayed a further increment in size of 90,000 daltons. Based on binding of the antagonist 125I-labeled hydroxybenzylpindolol, the beta-adrenergic receptor is about 90,000 daltons or the same as that seen for activation of the enzyme by isoproterenol through the beta-adrenergic-receptor. Because single targets were seen for the ground state enzyme system under all conditions, it would appear that the various regulatory and catalytic components are structurally linked prior to activation by hormone, guanine nucleotides, and fluoride ion. Furthermore, based on reported subunit sizes of the nucleotide regulatory and receptor components are composed of multiple subunits, either homologous or heterologous in structure.

  20. 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

  1. Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present and biochemically active in the central nervous system of the pond snail Lymnaea stagnalis.

    Science.gov (United States)

    Pirger, Zsolt; Laszlo, Zita; Hiripi, Laszlo; Hernadi, Laszlo; Toth, Gabor; Lubics, Andrea; Reglodi, Dora; Kemenes, Gyorgy; Mark, Laszlo

    2010-11-01

    PACAP is a highly conserved adenylate cyclase (AC) activating polypeptide, which, along with its receptors (PAC1-R, VPAC1, and VPAC2), is expressed in both vertebrate and invertebrate nervous systems. In vertebrates, PACAP has been shown to be involved in associative learning, but it is not known if it plays a similar role in invertebrates. To prepare the way for a detailed investigation into the possible role of PACAP and its receptors in a suitable invertebrate model of learning and memory, here, we undertook a study of their expression and biochemical role in the central nervous system of the pond snail Lymnaea stagnalis. Lymnaea is one of the best established invertebrate model systems to study the molecular mechanisms of learning and memory, including the role of cyclic AMP-activated signaling mechanisms, which crucially depend on the learning-induced activation of AC. However, there was no information available on the expression of PACAP and its receptors in sensory structures and central ganglia of the Lymnaea nervous system known to be involved in associative learning or whether or not PACAP can actually activate AC in these ganglia. Here, using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and immunohistochemistry, we established the presence of PACAP-like peptides in the cerebral ganglia and the lip region of Lymnaea. The MALDI-TOF data indicated an identity with mammalian PACAP-27 and the presence of a squid-like PACAP-38 highly homologous to vertebrate PACAP-38. We also showed that PACAP, VIP, and maxadilan stimulated the synthesis of cAMP in Lymnaea cerebral ganglion homogenates and that this effect was blocked by the appropriate general and selective PACAP receptor antagonists.

  2. 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.

  3. Bordetella pertussis commits human dendritic cells to promote a Th1/Th17 response through the activity of adenylate cyclase toxin and MAPK-pathways.

    Directory of Open Access Journals (Sweden)

    Giorgio Fedele

    Full Text Available The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA, an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC, an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT or a mutant lacking CyaA (BpCyaA-, or the BpCyaA- strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.

  4. Intravenous infusion of pituitary adenylate cyclase-activating polypeptide (PACAP) in a patient with multiple myeloma and myeloma kidney: a case study.

    Science.gov (United States)

    Li, Min; Maderdrut, Jerome L; Lertora, Juan J L; Batuman, Vecihi

    2007-09-01

    We have recently shown significant renoprotective effects with the administration of pituitary adenylate cyclase-activating polypeptide (PACAP) in models of myeloma kidney. PACAP markedly inhibited the production of proinflammatory cytokines stimulated by immunoglobulin light chains in human renal proximal tubule epithelial cells and in the kidneys of rats infused with myeloma light chains. PACAP was also shown to suppress the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. In this case study, an 81-year-old male patient with active multiple myeloma and myeloma kidney was infused intravenously with synthetic human PACAP38 at a rate of 4 pmol/kg/min for 120 min. The continuous infusion increased the level of PACAP38 in blood, with a plateau at about 0.2 nM during the infusion. The level of PACAP in the blood rapidly declined after the cessation of administration with a half-life of about 5-10 min. The continuous infusion did not significantly alter the basal glucose level, blood gases or blood pressure. There was a large reduction in free lambda light chains in urine after the start of the treatment with PACAP. These studies show that PACAP can be safely used in humans and suggest that it could be used as a novel therapeutic agent for the treatment of multiple myeloma and myeloma kidney.

  5. Does treatment with beta-adrenoceptor antagonists in vivo alter human adenylate cyclase responsiveness in vitro?

    NARCIS (Netherlands)

    Michel, M. C.; Klüppel, M.; Philipp, T.; Brodde, O. E.

    1991-01-01

    1. Treatment with beta-adrenoceptor antagonists in vivo can alter adenylate cyclase responsiveness in the human heart. We have determined the effects of treatment with four different beta-adrenoceptor antagonists in vivo on the responsiveness of lymphocyte and platelet adenylate cyclase in vitro in

  6. Pituitary adenylate cyclase-activating polypeptide (PACAP) contributes to the proliferation of hematopoietic progenitor cells in murine bone marrow via PACAP-specific receptor.

    Science.gov (United States)

    Xu, Zhifang; Ohtaki, Hirokazu; Watanabe, Jun; Miyamoto, Kazuyuki; Murai, Norimitsu; Sasaki, Shun; Matsumoto, Minako; Hashimoto, Hitoshi; Hiraizumi, Yutaka; Numazawa, Satoshi; Shioda, Seiji

    2016-02-29

    Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1(+) were large in size, had oval nuclei, and merged with CD34(+) cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1(-/-) mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1(-/-) and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1.

  7. 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

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

    Science.gov (United States)

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

    2014-04-01

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-12-01

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

  11. 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

  12. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    Science.gov (United States)

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC. Copyright © 2017 Elsevier B.V. All

  13. 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.

  14. 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.

  15. Pituitary adenylate cyclase-activating polypeptide (PACAP has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models

    Directory of Open Access Journals (Sweden)

    Gabor Maasz

    2017-02-01

    Full Text Available Pituitary adenylate cyclase-activating polypeptide (PACAP rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin, metabolic enzyme (S-COMT, MB-COMT and MAO-B and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP.

  16. Alternative Splicing of the Pituitary Adenylate Cyclase-Activating Polypeptide Receptor PAC1: Mechanisms of Fine Tuning of Brain Activity

    Directory of Open Access Journals (Sweden)

    Janna eBlechman

    2013-05-01

    Full Text Available Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.

  17. Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in the prefrontal cortex modulates cued fear learning, but not spatial working memory, in female rats.

    Science.gov (United States)

    Kirry, Adam J; Herbst, Matthew R; Poirier, Sarah E; Maskeri, Michelle M; Rothwell, Amy C; Twining, Robert C; Gilmartin, Marieke R

    2018-05-01

    A genetic polymorphism within the gene encoding the pituitary adenylate cyclase- activating polypeptide (PACAP) receptor type I (PAC1R) has recently been associated with hyper-reactivity to threat-related cues in women, but not men, with post-traumatic stress disorder (PTSD). PACAP is a highly conserved peptide, whose role in mediating adaptive physiological stress responses is well established. Far less is understood about the contribution of PACAP signaling in emotional learning and memory, particularly the encoding of fear to discrete cues. Moreover, a neurobiological substrate that may account for the observed link between PAC1R and PTSD in women, but not men, has yet to be identified. Sex differences in PACAP signaling during emotional learning could provide novel targets for the treatment of PTSD. Here we investigated the contribution of PAC1R signaling within the prefrontal cortex to the acquisition of cued fear in female and male rats. We used a variant of fear conditioning called trace fear conditioning, which requires sustained attention to fear cues and depends on working-memory like neuronal activity within the prefrontal cortex. We found that cued fear learning, but not spatial working memory, was impaired by administration of a PAC1R antagonist directly into the prelimbic area of the prefrontal cortex. This effect was specific to females. We also found that levels of mRNA for the PAC1R receptor in the prelimbic cortex were greater in females compared with males, and were highest during and immediately following the proestrus stage of the estrous cycle. Together, these results demonstrate a sex-specific role of PAC1R signaling in learning about threat-related cues. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Role of Pituitary Adenylate-Cyclase Activating Polypeptide and Tac1 gene derived tachykinins in sensory, motor and vascular functions under normal and neuropathic conditions.

    Science.gov (United States)

    Botz, Bálint; Imreh, András; Sándor, Katalin; Elekes, Krisztián; Szolcsányi, János; Reglődi, Dóra; Quinn, John P; Stewart, James; Zimmer, Andreas; Hashimoto, Hitoshi; Helyes, Zsuzsanna

    2013-05-01

    Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP) and Tac1 gene-encoded tachykinins (substance P: SP, neurokinin A: NKA) are expressed in capsaicin-sensitive nerves, but their role in nociception, inflammation and vasoregulation is unclear. Therefore, we investigated the function of these neuropeptides and the NK1 tachykinin receptor (from Tacr1 gene) in the partial sciatic nerve ligation-induced traumatic mononeuropathy model using gene deficient (PACAP(-/-), Tac1(-/-), and Tacr1(-/-)) mice. Mechanonociceptive threshold of the paw was measured with dynamic plantar aesthesiometry, motor coordination with Rota-Rod and cutaneous microcirculation with laser Doppler imaging. Neurogenic vasodilation was evoked by mustard oil stimulating sensory nerves. In wildtype mice 30-40% mechanical hyperalgesia developed one week after nerve ligation, which was not altered in Tac1(-/-) and Tacr1(-/-) mice, but was absent in PACAP(-/-) animals. Motor coordination of the PACAP(-/-) and Tac1(-/-) groups was significantly worse both before and after nerve ligation compared to their wildtypes, but it did not change in Tacr1(-/-) mice. Basal postoperative microcirculation on the plantar skin of PACAP(-/-) mice did not differ from the wildtypes, but was significantly lower in Tac1(-/-) and Tacr1(-/-) ones. In contrast, mustard oil-induced neurogenic vasodilation was significantly smaller in PACAP(-/-) mice, but not in Tacr1(-/-) and Tac1(-/-) animals. Both PACAP and SP/NKA, but not NK1 receptors participate in normal motor coordination. Tachykinins maintain basal cutaneous microcirculation. PACAP is a crucial mediator of neuropathic mechanical hyperalgesia and neurogenic vasodilation. Therefore identifying its target and developing selective, potent antagonists, might open promising new perspectives for the treatment of neuropathic pain and vascular complications. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Bi-directional effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on fear-related behavior and c-Fos expression after fear conditioning in rats.

    Science.gov (United States)

    Meloni, Edward G; Venkataraman, Archana; Donahue, Rachel J; Carlezon, William A

    2016-02-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is implicated in stress regulation and learning and memory. PACAP has neuromodulatory actions on brain structures within the limbic system that could contribute to its acute and persistent effects in animal models of stress and anxiety-like behavior. Here, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannula for infusion of PACAP-38 (0.5, 1, or 1.5 μg) or vehicle followed 30 min later by fear conditioning. Freezing was measured early (1, 4, and 7 days) or following a delay (7, 10, and 13 days) after conditioning. PACAP (1.5 μg) produced a bi-phasic response in freezing behavior across test days: relative to controls, PACAP-treated rats showed a reduction in freezing when tested 1 or 7 days after fear conditioning that evolved into a significant elevation in freezing by the third test session in the early, but not delayed, group. Corticosterone (CORT) levels were significantly elevated in PACAP-treated rats following fear conditioning, but not at the time of testing (Day 1). Brain c-Fos expression revealed PACAP-dependent alterations within, as well as outside of, areas typically implicated in fear conditioning. Our findings raise the possibility that PACAP disrupts fear memory consolidation by altering synaptic plasticity within neurocircuits normally responsible for encoding fear-related cues, producing a type of dissociation or peritraumatic amnesia often seen in people early after exposure to a traumatic event. However, fear memories are retained such that repeated testing and memory reactivation (e.g., re-experiencing) causes the freezing response to emerge and persist at elevated levels. PACAP systems may represent an axis on which stress and exposure to trauma converge to promote maladaptive behavioral responses characteristic of psychiatric illnesses such as post-traumatic stress disorder (PTSD). Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Serotonin-Sensitive Adenylate Cyclase in Neural Tissue and Its Similarity to the Serotonin Receptor: A Possible Site of Action of Lysergic Acid Diethylamide

    Science.gov (United States)

    Nathanson, James A.; Greengard, Paul

    1974-01-01

    An adenylate cyclase (EC 4.6.1.1) that is activated specifically by low concentrations of serotonin has been identified in homogenates of the thoracic ganglia of an insect nervous system. The activation of this enzyme by serotonin was selectively inhibited by extremely low concentrations of D-lysergic acid diethylamide (LSD), 2-bromo-LSD, and cyproheptadine, agents which are known to block certain serotonin receptors in vivo. The inhibition was competitive with respect to serotonin, and the calculated inhibitory constant of LSD for this serotonin-sensitive adenylate cyclase was 5 nM. The data are consistent with a model in which the serotonin receptor of neural tissue is intimately associated with a serotonin-sensitive adenylate cyclase which mediates serotonergic neurotransmission. The results are also compatible with the possibility that some of the physiological effects of LSD may be mediated through interaction with serotonin-sensitive adenylate cyclase. PMID:4595572

  1. 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

  2. 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

  3. Ex vivo and in vivo studies of CME-1, a novel polysaccharide purified from the mycelia of Cordyceps sinensis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP.

    Science.gov (United States)

    Lu, Wan-Jung; Chang, Nen-Chung; Jayakumar, Thanasekaran; Liao, Jiun-Cheng; Lin, Mei-Jiun; Wang, Shwu-Huey; Chou, Duen-Suey; Thomas, Philip Aloysius; Sheu, Joen-Rong

    2014-12-01

    CME-1, a novel water-soluble polysaccharide, was purified from the mycelia of Cordyceps sinensis, and its chemical structure was characterized to contain mannose and galactose in a ratio of 4:6 (27.6 kDa). CME-1 was originally observed to exert a potent inhibitory effect on tumor migration and a cytoprotective effect against oxidative stress. Activation of platelets caused by arterial thrombosis is relevant to various cardiovascular diseases (CVDs). However, no data are available concerning the effects of CME-1 on platelet activation. Hence, the purpose of this study was to examine the ex vivo and in vivo antithrombotic effects of CME-1 and its possible mechanisms in platelet activation. The aggregometry, immunoblotting, flow cytometric analysis and platelet functional analysis were used in this study. CME-1 (2.3-7.6 μM) exhibited highly potent activity in inhibiting human platelet aggregation when stimulated by collagen, thrombin, and arachidonic acid but not by U46619. CME-1 inhibited platelet activation accompanied by inhibiting Akt, mitogen-activated protein kinases (MAPKs), thromboxane B2 (TxB2) and hydroxyl radical (OH(●)) formation. However, CME-1 interrupted neither FITC-triflavin nor FITC-collagen binding to platelets. CME-1 markedly increased cyclic AMP levels, but not cyclic GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ, an inhibitor of guanylate cyclase, obviously reversed the CME-1-mediated effects on platelet aggregation and vasodilator-stimulated phosphoprotein (VASP), Akt, p38 MAPK phosphorylation, and TxB2 formation. CME-1 substantially prolonged the closure time of whole blood and the occlusion time of platelet plug formation. This study demonstrates for the first time that CME-1 exhibits highly potent antiplatelet activity that may initially activate adenylate cyclase/cyclic AMP and, subsequently, inhibit intracellular signals (such as Akt and

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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

  10. 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

  11. A Role for Calcium-Activated Adenylate Cyclase and Protein Kinase A in the Lens Src Family Kinase and Na,K-ATPase Response to Hyposmotic Stress.

    Science.gov (United States)

    Shahidullah, Mohammad; Mandal, Amritlal; Delamere, Nicholas A

    2017-09-01

    Na,K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na,K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na,K-ATPase response. Intact porcine lenses were exposed to hyposmotic Krebs solution (200 mOsm) then the epithelium was assayed for cAMP, SFK phosphorylation (activation) or Na,K-ATPase activity. An increase of cAMP was observed in the epithelium of lenses exposed to hyposmotic solution. In lenses exposed to hyposmotic solution SFK phosphorylation in the epithelium approximately doubled as did Na,K-ATPase activity and both responses were prevented by H89, a protein kinase A inhibitor. The magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist HC067047 added to prevent TRPV4-mediated calcium entry, and by a cytoplasmic Ca2+ chelator BAPTA-AM. The Na,K-ATPase activity response in the epithelium of lenses exposed to hyposmotic solution was abolished by BAPTA-AM. As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell-permeable cAMP analog. 8-pCPT-cAMP caused robust SFK activation. Using Western blot, two calcium-activated adenylyl cyclases, ADCY3 and ADCY8, were detected in lens epithelium. Calcium-activated adenylyl cyclases are expressed in the lens epithelium and SFK activation is linked to a rise of cAMP that occurs upon hyposmotic challenge. The findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, SFK activation, and a subsequent increase of Na,K-ATPase activity.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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

  2. 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

  3. How adenylate cyclase choreographs the pas de deux of the receptors heteromerization dance.

    Science.gov (United States)

    Woods, A S; Jackson, S N

    2013-05-15

    Our work suggests that heteromer formation, mainly involves linear motifs (LMs) found in disordered regions of proteins. Local disorder imparts plasticity to LMs. Most molecular recognition of proteins occurs between short linear segments, known as LMs. Interaction of short continuous epitopes is not constrained by sequence and has the advantage of resulting in interactions with micromolar affinities which suit transient, reversible complexes such as receptor heteromers. Electrostatic interactions between epitopes of the G-protein coupled receptors (GPCR) involved, are the key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating Ser/Thr in an epitope containing a casein kinase 1/2-consensus site. Our data suggest that dopaminergic neurotransmission, through cAMP-dependent protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg-rich epitope, affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers' interactions and their binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at. Published by Elsevier Ltd.

  4. 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

  5. Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    International Nuclear Information System (INIS)

    Grasso, P.; Reichert, L.E. Jr.

    1990-01-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel

  6. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    Energy Technology Data Exchange (ETDEWEB)

    Grasso, P.; Reichert, L.E. Jr. (Albany Medical College, NY (USA))

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  7. 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

  8. 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

  9. Calcium influx rescues adenylate cyclase-hemolysin from rapid cell membrane removal and enables phagocyte permeabilization by toxin pores.

    Directory of Open Access Journals (Sweden)

    Radovan Fiser

    Full Text Available Bordetella adenylate cyclase toxin-hemolysin (CyaA penetrates the cytoplasmic membrane of phagocytes and employs two distinct conformers to exert its multiple activities. One conformer forms cation-selective pores that permeabilize phagocyte membrane for efflux of cytosolic potassium. The other conformer conducts extracellular calcium ions across cytoplasmic membrane of cells, relocates into lipid rafts, translocates the adenylate cyclase enzyme (AC domain into cells and converts cytosolic ATP to cAMP. We show that the calcium-conducting activity of CyaA controls the path and kinetics of endocytic removal of toxin pores from phagocyte membrane. The enzymatically inactive but calcium-conducting CyaA-AC⁻ toxoid was endocytosed via a clathrin-dependent pathway. In contrast, a doubly mutated (E570K+E581P toxoid, unable to conduct Ca²⁺ into cells, was rapidly internalized by membrane macropinocytosis, unless rescued by Ca²⁺ influx promoted in trans by ionomycin or intact toxoid. Moreover, a fully pore-forming CyaA-ΔAC hemolysin failed to permeabilize phagocytes, unless endocytic removal of its pores from cell membrane was decelerated through Ca²⁺ influx promoted by molecules locked in a Ca²⁺-conducting conformation by the 3D1 antibody. Inhibition of endocytosis also enabled the native B. pertussis-produced CyaA to induce lysis of J774A.1 macrophages at concentrations starting from 100 ng/ml. Hence, by mediating calcium influx into cells, the translocating conformer of CyaA controls the removal of bystander toxin pores from phagocyte membrane. This triggers a positive feedback loop of exacerbated cell permeabilization, where the efflux of cellular potassium yields further decreased toxin pore removal from cell membrane and this further enhances cell permeabilization and potassium efflux.

  10. 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

  11. 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

  12. Kinetics of interaction between beta-receptors, GTP protein, and the catalytic unit of turkey erythrocyte adenylate cyclase.

    Science.gov (United States)

    Tolkovsky, A M; Braun, S; Levitzki, A

    1982-01-01

    The kinetics of turkey erythrocyte membrane adenylate cyclase activation by beta-agonists and guanyl-5'-yl imidodiphosphate is explored as a function of the concentration of the GTP regulatory protein and of the catalytic unit. It was found that the overall kinetics of activation is first order and is independent of the concentration of the GTP regulatory unit N, the catalytic unit C, and of hormone over a very wide concentration range. It was established that the rate-limiting step does not involve GDP dissociation from the inactive N unit or the association between activated N' and C. Also, it was found that guanyl-5'-yl imidodiphosphate binding occurs in a random fashion and is not hormone dependent. These results enable us to exclude models of the sequential type in which N in its inactive form is bound to receptor R, is released in an active form N' upon hormone activation, and then binds to C, activating the latter. An acceptable model that accounts for all of the data conforms to the original formulation of "collision coupling" in which N is tightly associated to C at all times.

  13. Mechanism of control of the turkey erythrocyte beta-adrenoceptor dependent adenylate cyclase by guanyl nucleotides: a minimum model.

    Science.gov (United States)

    Braun, S; Tolkovsky, A M; Levitzki, A

    1982-01-01

    Treatment of native turkey erythrocyte membranes with GMP and epinephrine produces a highly active but metastable form of adenylate cyclase which decays slowly to basal native state. The decay process is greatly facilitated by GTP and GDP beta S1, and is further enhanced by 1-epinephrine. This decay process is prevented reversibly by GMP. GppNHp, like GMP, prevents the decay process first reversibly, but with time stabilizes the highly active state in a persistently active state. The expression of the catalytic activity of the enzyme in the metastable state can also be inhibited reversibly by GTP, GDP beta S and GMP at all times during the decay process. The GppNHp stabilized form is not susceptible to nucleotide inhibition. Thus, two forms of the guanyl nucleotide unit are postulated to exist: an "open" and a "closed" form. In the presence of hormone and GTP, the enzyme shuttles between these two forms continuously. GMP and GppNHp favor the complete conversion to the "open" form in the presence of beta-agonist. Evidence is also presented for the existence of two GTP dependent processes which exhibit different apparent affinities towards the nucleotide: A high affinity GTP binding process is essential for the fruitful coupling between receptor and enzyme, and a low affinity GTPase site which is responsible for the termination of the hormonal signal.

  14. Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development.

    Directory of Open Access Journals (Sweden)

    Maria Cristina Antal

    Full Text Available Adenylate cyclase type III (AC3 is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells. In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors.

  15. Effects of forskolin on cerebral blood flow: implications for a role of adenylate cyclase

    International Nuclear Information System (INIS)

    Wysham, D.G.; Brotherton, A.F.; Heistad, D.D.

    1986-01-01

    We have studied cerebral vascular effects of forskolin, a drug which stimulates adenylate cyclase and potentiates dilator effects of adenosine in other vascular beds. Our goals were to determine whether forskolin is a cerebral vasodilator and whether it potentiates cerebral vasodilator responses to adenosine. We measured cerebral blood flow with microspheres in anesthetized rabbits. Forskolin (10 micrograms/kg per min) increased blood flow (ml/min per 100 gm) from 39 +/- 5 (mean +/- S.E.) to 56 +/- 9 (p less than 0.05) in cerebrum, and increased flow to myocardium and kidney despite a decrease in mean arterial pressure. Forskolin did not alter cerebral oxygen consumption, which indicates that the increase in cerebral blood flow is a direct vasodilator effect and is not secondary to increased metabolism. We also examined effects of forskolin on the response to infusion of adenosine. Cerebral blood flow was measured during infusion of 1-5 microM/min adenosine into one internal carotid artery, under control conditions and during infusion of forskolin at 3 micrograms/kg per min i.v. Adenosine alone increased ipsilateral cerebral blood flow from 32 +/- 3 to 45 +/- 5 (p less than 0.05). Responses to adenosine were not augmented during infusion of forskolin. We conclude that forskolin is a direct cerebral vasodilator and forskolin does not potentiate cerebral vasodilator responses to adenosine

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. D-1 dopaminergic and beta-adrenergic stimulation of adenylate cyclase in a clone derived from the human astrocytoma cell line G-CCM.

    Science.gov (United States)

    Balmforth, A J; Ball, S G; Freshney, R I; Graham, D I; McNamee, H B; Vaughan, P F

    1986-09-01

    Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 microM, respectively. The Ka apparent value for dopamine was increased by the D-1 antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 microM, respectively, but was unaffected by propranolol (1 microM). Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 microM) or cis-flupenthixol (1 microM). Propranolol (10 microM), but not cis-flupenthixol (1 microM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 microM) and sulpiride (1 microM). These results suggest that clone D384 contains both D-1 dopaminergic and beta-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via beta-receptors, and noradrenaline through both receptors.

  1. Multiple pertussis toxin substrates as candidates for regulatory G proteins of adenylate cyclase coupled to the somatostatin receptor in primary rat astrocytes.

    Science.gov (United States)

    Gebicke-Haerter, P J; Seregi, A; Wurster, S; Schobert, A; Allgaier, C; Hertting, G

    1988-10-01

    The involvement of G proteins in receptor mediated astroglial cAMP formation was studied. Isoproterenol or prostaglandin E2 stimulated adenylate cyclase of primary astroglial cells was inhibited by somatostatin. Preincubation of cells with increasing concentrations of islet activating protein (IAP) diminished somatostatin inhibition of adenylate cyclase. At an IAP concentration of 50 ng/ml somatostatin inhibition was completely abolished. Studies on IAP catalyzed 32P-ADP-ribosylation of astroglial cell particulate material revealed an incorporation of radiolabel into three polypeptides in the molecular weight range of 41,000-39,000 Dalton. Pretreatment of intact cells with IAP reduced radiolabeling of this molecular species in a concentration dependent manner. No further radiolabeling above background level was detectable after pretreatment of cultures with 10 ng IAP/ml or more. At present, the occurrence of at least three IAP substrates (G proteins) does not permit an identification of the somatostatin receptor coupled G protein. Rather, the finding reveals that astrocytes are endowed with multiple variants of GTP binding proteins likely to be coupled to different receptors.

  2. 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.

  3. 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.

  4. Brain histamine H1- and H2-receptors and histamine-sensitive adenylate cyclase: effects of antipsychotics and antidepressants

    International Nuclear Information System (INIS)

    Coupet, J.; Szuches-Myers, V.A.

    1981-01-01

    Several classes of psychoactive compounds have been investigated for their effects on histamine-sensitive adenylate cyclase in cell-free preparations from the guinea-pig cerebral cortex. Their inhibitory actions on this enzyme system have been compared with their abilities to displace [ 3 H]pyrilamine and [ 3 H]cimetidine from histamine H 1 - and H 2 -receptor sites, respectively. The results of these studies show that compounds which inhibited the histamine-sensitive cyclase were also displacers of either [ 3 H]pyrilamine or [ 3 H]cimetidine or both 3 H-ligands from their binding sites. In spite of the lack of a correlation between binding and cyclase antagonism it was observed that compounds that displace both ligands showed greater inhibition of the cyclase than those that have affinities for sites labeled by one or the other ligand. It was concluded that antihistamines, the antipsychotics and the antidepressants share a common property through their antagonism of H 1 -receptors and that may be responsible for their sedative side effect. (Auth.)

  5. Stimulatory GTP regulatory unit Ns and the catalytic unit of adenylate cyclase are tightly associated: mechanistic consequences.

    Science.gov (United States)

    Arad, H; Rosenbusch, J P; Levitzki, A

    1984-11-01

    Turkey erythrocyte membranes were solubilized in the mild detergent octylpenta(oxyethylene) [CH3(CH2)7-(OCH2CH2)5OH], which possesses a high critical micelle concentration (approximately equal to 6 mM) and forms small, dynamic micelles. Both the native enzyme Ns(GDP) X C and the p[NH]ppG-preactivated species N's X p[NH]ppG X C' were found to possess the same molecular mass of 215,000 +/- 17,000 daltons. Both enzyme species migrate as a tight complex between Ns and C on both gel permeation columns and on DEAE-Sephacel columns in detergent. The two functional units, Ns and C, remain associated even in dilute detergent solutions and throughout a 300- to 400-fold purification in octylpoly(oxyethylene). These results strongly support the view that Ns and C do not come apart during the process of enzyme activation by the beta-adrenergic receptor. Furthermore, these results strongly support our previous assertion that the beta-adrenergic receptor activation of adenylate cyclase is by a simple "collision coupling" between the receptor and NsC. These results are not compatible with shuttle mechanisms that postulate that Ns physically migrates from the receptor R to the catalytic unit C and back during the activation cycle, as suggested by Citri and Schramm [Citri, Y. & Schramm, M. (1980) Nature (London) 287, 297-300] and by De Lean et al. [De Lean, A., Stadel, J. M. & Lefkowitz, R. J. (1980) J. Biol. Chem. 255, 5108-5117].

  6. The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

    Directory of Open Access Journals (Sweden)

    Laura B Duvall

    Full Text Available The neuropeptide Pigment Dispersing Factor (PDF is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+ LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

  7. 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

  8. Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells.

    Science.gov (United States)

    Chang, Mei-Chi; Lin, Szu-I; Lin, Li-Deh; Chan, Chiu-Po; Lee, Ming-Shu; Wang, Tong-Mei; Jeng, Po-Yuan; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

    2016-04-01

    Prostaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. Primary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate-activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. PGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. These results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  9. Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity.

    Science.gov (United States)

    Fedele, Giorgio; Schiavoni, Ilaria; Adkins, Irena; Klimova, Nela; Sebo, Peter

    2017-09-21

    Adenylate cyclase toxin (CyaA) is released in the course of B. pertussis infection in the host's respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC), macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3',5'-cyclic adenosine monophosphate (cAMP), which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.

  10. Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity

    Directory of Open Access Journals (Sweden)

    Giorgio Fedele

    2017-09-01

    Full Text Available Adenylate cyclase toxin (CyaA is released in the course of B. pertussis infection in the host’s respiratory tract in order to suppress its early innate and subsequent adaptive immune defense. CD11b-expressing dendritic cells (DC, macrophages and neutrophils are professional phagocytes and key players of the innate immune system that provide a first line of defense against invading pathogens. Recent findings revealed the capacity of B. pertussis CyaA to intoxicate DC with high concentrations of 3′,5′-cyclic adenosine monophosphate (cAMP, which ultimately skews the host immune response towards the expansion of Th17 cells and regulatory T cells. CyaA-induced cAMP signaling swiftly incapacitates opsonophagocytosis, oxidative burst and NO-mediated killing of bacteria by neutrophils and macrophages. The subversion of host immune responses by CyaA after delivery into DC, macrophages and neutrophils is the subject of this review.

  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. Galanin can inhibit insulin release by a mechanism other than membrane hyperpolarization or inhibition of adenylate cyclase.

    Science.gov (United States)

    Sharp, G W; Le Marchand-Brustel, Y; Yada, T; Russo, L L; Bliss, C R; Cormont, M; Monge, L; Van Obberghen, E

    1989-05-05

    -dependent Ca2+ channels. One suggested mechanism for the action of galanin is inhibition of adenylate cyclase. However, it was found that galanin inhibits insulin release even in the presence of 8-Br-cAMP, an agent which effectively bypasses adenylate cyclase. Therefore, an additional mechanism for the inhibitory effect of galanin must be present. All of the effects of galanin were sensitive to pertussis toxin. These data suggest two G-protein-dependent actions of galanin, one to hyperpolarize the plasma membrane and one at a distal point in stimulus-secretion coupling, close to the exocytotic event.

  13. Photo-dynamics of the BLUF domain containing soluble adenylate cyclase (nPAC) from the amoeboflagellate Naegleria gruberi NEG-M strain

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-08-25

    Graphical abstract: The photoactivated adenylyl cyclase (nPAC) from Naegleria gruberi was expressed heterologously in Escherichia coli and its photo-cycling dynamics was studied by optical absorption and fluorescence spectroscopy. Highlights: {yields} Photo-activated adenylyl cyclase (nPAC) from Naegleria gruberi NEG-M was expressed. {yields} Photodynamics of BLUF domain in BLUF sensor - cyclase actuator protein was studied. {yields} Photo-excitation caused BLUF photo-cycling and permanent protein re-conformation. {yields} Re-conformed protein enabled photo-induced flavin reduction by proton transfer. {yields} Fluorescence of flavin in dark- and light-adapted state of nPAC was characterized. - Abstract: The amoeboflagellate Naegleria gruberi NEG-M comprises a BLUF (blue light sensor using flavin) regulated adenylate cyclase (nPAC). The nPAC gene was expressed heterologously in Escherichia coli and the photo-dynamics of the nPAC protein was studied by optical absorption and fluorescence spectroscopy. Blue-light exposure of nPAC caused a typical BLUF-type photo-cycle behavior (spectral absorption red-shift, fluorescence quenching, absorption and fluorescence recovery in the dark). Additionally, time-delayed reversible photo-induced one-electron reduction of fully oxidized flavin (Fl{sub ox}) to semi-reduced flavin (FlH{sup {center_dot}}) occurred. Furthermore, photo-excitation of FlH{sup {center_dot}} caused irreversible electron transfer to fully reduced anionic flavin (FlH{sup -}). A photo-induced electron transfer from Tyr or Trp to flavin (Tyr{sup {center_dot}+}-Fl{sup {center_dot}-} or Trp{sup {center_dot}+}-Fl{sup {center_dot}-} radical ion-pair formation) is thought to cause H-bond restructuring responsible for BLUF-type photo-cycling and permanent protein re-conformation enabling photo-induced flavin reduction by proton transfer. Some photo-degradation of Fl{sub ox} to lumichrome was observed. A model of the photo-dynamics of nPAC is developed.

  14. On the role of adenylate cyclase, tyrosine kinase, and tyrosine phosphatase in the response of nerve and glial cells to photodynamic impact

    Science.gov (United States)

    Kolosov, Mikhail S.; Bragin, D. E.; Dergacheva, Olga Y.; Vanzha, O.; Oparina, L.; Uzdensky, Anatoly B.

    2004-08-01

    The role of different intercellular signaling pathways involving adenylate cyclase (AC), receptor tyrosine kinase (RTK), tyrosine and serine/threonine protein phosphatases (PTP or PP, respectively) in the response of crayfish mechanoreceptor neuron (MRN) and surrounding glial cells to photodynamic effect of aluminum phthalocyanine Photosens have been studied. AC inhibition by MDL-12330A decreased neuron lifetime, whereas AC activation by forskolin increase it. Thus, increase in cAMP produced by activated AC protects SRN against photodynamic inactivation. Similarly, RTK inhibition by genistein decreased neuron lifetime, while inhibition of PTP or PP that remove phosphate groups from proteins, prolonged neuronal activity. AC inhibition reduced photoinduced damage of the plasma membrane, and, therefore, necrosis in neuronal and glial cells. RTK inhibition protected only neurons against PDT-induced membrane permeabilization while glial cells became lesser permeable under ortovanadate-mediated PTP inhibition. AC activation also prevented PDT-induced apoptosis in glial cells. PP inhibition enhanced apoptotic processes in photosensitized glial cells. Therefore, both intercellular signaling pathways involving AC and TRK are involved in the maintenance of neuronal activity, integrity of the neuronal and glial plasma membranes and in apoptotic processes in glia under photosensitization.

  15. Pharmacological characterization of the dopamine-sensitive adenylate cyclase in cockroach brain: evidence for a distinct dopamine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Orr, G.L.; Gole, J.W.D.; Notman, H.J.; Downer, R.G.H.

    1987-12-21

    Dopamine increases cyclic AMP production in crude membrane preparations of cockroach brain with plateaus in cyclic AMP production occurring between 1-10 ..mu..M and 10 mM. Maximal production of cyclic AMP is 2.25 fold greater than that of control values. Octopamine also increases cyclic AMP production with a Ka of 1.4 ..mu..M and maximal production 3.5 fold greater than that of control. 5-Hydroxytryptamine does not increase cyclic AMP production. The effects of octopamine and dopamine are fully additive. The vertebrate dopamine agonists ADTN and epinine stimulate the dopamine-sensitive adenylate cyclase (AC) with Ka values of 4.5 and 0.6 ..mu..M respectively and with maximal effectiveness 1.7 fold greater than that of control. The selective D/sub 2/-dopamine agonist LY-171555 stimulates cyclic AMP production to a similar extent with a Ka of 50 ..mu..M. Other dopamine agonists have no stimulatory effects. With the exception of mianserin, /sup 3/H-piflutixol is displaced from brain membranes by dopamine antagonists with an order of potency similar to that observed for the inhibition of dopamine-sensitive AC. The results indicate that the octopamine- and dopamine-sensitive AC in cockroach brain can be distinguished pharmacologically and the dopamine receptors coupled to AC have pharmacological characteristics distinct from vertebrate D/sup 1/- and D/sup 2/-dopamine receptors. 33 references, 3 figures, 2 tables.

  16. Pharmacological characterization of the dopamine-sensitive adenylate cyclase in cockroach brain: evidence for a distinct dopamine receptor

    International Nuclear Information System (INIS)

    Orr, G.L.; Gole, J.W.D.; Notman, H.J.; Downer, R.G.H.

    1987-01-01

    Dopamine increases cyclic AMP production in crude membrane preparations of cockroach brain with plateaus in cyclic AMP production occurring between 1-10 μM and 10 mM. Maximal production of cyclic AMP is 2.25 fold greater than that of control values. Octopamine also increases cyclic AMP production with a Ka of 1.4 μM and maximal production 3.5 fold greater than that of control. 5-Hydroxytryptamine does not increase cyclic AMP production. The effects of octopamine and dopamine are fully additive. The vertebrate dopamine agonists ADTN and epinine stimulate the dopamine-sensitive adenylate cyclase (AC) with Ka values of 4.5 and 0.6 μM respectively and with maximal effectiveness 1.7 fold greater than that of control. The selective D 2 -dopamine agonist LY-171555 stimulates cyclic AMP production to a similar extent with a Ka of 50 μM. Other dopamine agonists have no stimulatory effects. With the exception of mianserin, 3 H-piflutixol is displaced from brain membranes by dopamine antagonists with an order of potency similar to that observed for the inhibition of dopamine-sensitive AC. The results indicate that the octopamine- and dopamine-sensitive AC in cockroach brain can be distinguished pharmacologically and the dopamine receptors coupled to AC have pharmacological characteristics distinct from vertebrate D 1 - and D 2 -dopamine receptors. 33 references, 3 figures, 2 tables

  17. Lack of adenylate cyclase 1 (AC1): consequences on corticospinal tract development and on locomotor recovery after spinal cord injury.

    Science.gov (United States)

    Nait Taleb Ali, H; Morel, M P; Doulazmi, M; Scotto-Lomassese, S; Gaspar, P; Dusart, I; Bennis, M

    2014-02-26

    Cyclic AMP (cAMP) signalling pathways are involved in axonal growth and regeneration. The calcium-calmodulin- stimulated adenylate cyclase 1 (AC1), a regulator of cAMP levels, is strongly expressed in the corticospinal motor neurons (CSMN) in cerebral cortex layer V during development, but its role in the development of the corticospinal tract (CST) is unknown. Here, we analyse the organization of the CST pathway using anterograde and retrograde tracers in the barrelless (brl) mouse that carries an inactivating mutation of the AC1 gene. We show that in brl mice the general organization of the CST is normal but there is an increase in the number of axons in the ipsilateral contingent in the dorsal and ventral medial funiculi of the cervical spinal cord. The density of CSMN in layer V of the motor cortex is increased in brl compared to wild-type mice. Thus, lack of AC1 likely perturbs late phases of CSMN and CST development. Next, we examine the motor recovery after a spinal cord injury (SCI). We find that brl mice show enhanced locomotor functions as assessed by the BMS (Basso mouse scale) as early as 6h and up to 6 weeks after SCI, indicating a smaller responsiveness of brl mice to SCI. It is therefore possible that developmental effects on motor systems might decrease the locomotor effects consecutive to a SCI. This point is particularly important with regards to the use of transgenic animals for testing SCI recovery. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Effect of modulators of cytoskeletal function on desensitization and recovery of PGE2-responsive ovarian adenylate cyclase.

    Science.gov (United States)

    Zor, U; Strulovici, B; Lamprecht, S A; Amsterdam, A; Oplatka, A; Lindner, H R

    1979-12-01

    Exposure of cultured Graafian follicles to PGE2 for 20 h resulted in a loss of the cyclic AMP response to fresh hormone. This desensitization was prevented by addition to the medium of D2O (25--50%) or Li+ (0.6--6 mM), agents believed to stabilize microtubules, as well as by phalloidin (1.0--10 microM), believed to stabilize the polymerized state of actin, in a dose-dependent manner. The spontaneous recovery of responsiveness to PGE2 upon incubation of refractory follicles for 6 h in hormone-free medium was prevented by addition to the medium of cytochalasin B (CB; 3 microgram/ml) or of the actin-binding myosin subfragment HMM S-1 (80 microgram/ml) or of anti-actin serum; viz. by agents likely to interfere with microfilament function. D2O (50%) caused morphological damage to the inner layer of the membrana granulosa and severe depression of protein synthesis. The other drugs used (phalloidin, LiCl and cytochalasin B) had no such effects. Resensitization of refractory follicles was also prevented by cycloheximide (10 micrograms/ml) and by actinomycin D (10 micrograms/ml). It is speculated that the recovery process may involve the insertion of a newly synthesized protein, such as PG-receptor, into the membrane by a mechanism dependent on microfilament action. These findings provide suggestive evidence for the hypothesis that cytoskeletal elements associated with the cell membrane take part in the modulation of the adenylate cyclase response to hormones.

  19. Pituitary adenylate cyclase activating polypeptide (PACAP), stress, and sex hormones.

    Science.gov (United States)

    King, S Bradley; Toufexis, Donna J; Hammack, Sayamwong E

    2017-09-01

    Stressor exposure is associated with the onset and severity of many psychopathologies that are more common in women than men. Moreover, the maladaptive expression and function of stress-related hormones have been implicated in these disorders. Evidence suggests that PACAP has a critical role in the stress circuits mediating stress-responding, and PACAP may interact with sex hormones to contribute to sex differences in stress-related disease. In this review, we describe the role of the PACAP/PAC1 system in stress biology, focusing on the role of stress-induced alterations in PACAP expression and signaling in the development of stress-induced behavioral change. Additionally, we present more recent data suggesting potential interactions between stress, PACAP, and circulating estradiol in pathological states, including PTSD. These studies suggest that the level of stress and circulating gonadal hormones may differentially regulate the PACAPergic system in males and females to influence anxiety-like behavior and may be one mechanism underlying the discrepancies in human psychiatric disorders.

  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. Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

    Science.gov (United States)

    Angely, Christelle; Nguyen, Ngoc-Minh; Andre Dias, Sofia; Planus, Emmanuelle; Pelle, Gabriel; Louis, Bruno; Filoche, Marcel; Chenal, Alexandre; Ladant, Daniel; Isabey, Daniel

    2017-08-01

    The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure 95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 μm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in

  2. Phosphoproteomics of cAMP signaling of Bordetella adenylate cyclase toxin in mouse dendritic cells

    Czech Academy of Sciences Publication Activity Database

    Novák, Jakub; Fabrik, I.; Linhartová, Irena; Link, M.; Černý, Ondřej; Stulík, J.; Šebo, Peter

    2017-01-01

    Roč. 7, NOV 24 (2017), č. článku 16298. ISSN 2045-2322 R&D Projects: GA MZd(CZ) NV16-28126A; GA ČR(CZ) GA13-14547S Institutional support: RVO:61388971 Keywords : GTPASE-ACTIVATING-PROTEIN * SALT-INDUCIBLE KINASES * GENE ONTOLOGY Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.259, year: 2016

  3. Oligomerization is involved in pore formation by Bordetella adenylate cyclase toxin

    Czech Academy of Sciences Publication Activity Database

    Vojtová, Jana; Basler, Marek; Osička, Radim; Knapp, O.; Maier, E.; Černý, J.; Benada, Oldřich; Benz, R.; Šebo, Peter

    2009-01-01

    Roč. 23, - (2009), s. 2831-2843 ISSN 0892-6638 R&D Projects: GA AV ČR IAA500200914; GA MŠk 1M0506 Grant - others:-(XE) LSHB-CT-2003-503582 THERAVAC Institutional research plan: CEZ:AV0Z50200510 Keywords : blue native electrophoresis * planar lipid bilayer membranes * pore-forming activity Subject RIV: EE - Microbiology, Virology Impact factor: 6.401, year: 2009

  4. Adenylate cyclase toxin translocates across target cell membrane without forming a pore

    Czech Academy of Sciences Publication Activity Database

    Osičková, Adriana; Mašín, Jiří; Fayolle, C.; Krůšek, Jan; Basler, Marek; Pospíšilová, Eva; Leclerc, C.; Osička, Radim; Šebo, Peter

    2010-01-01

    Roč. 75, č. 6 (2010), s. 1550-1562 ISSN 0950-382X R&D Projects: GA AV ČR IAA500200914; GA ČR GA310/08/0447; GA MŠk 1M0506; GA MŠk 2B06161 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z50200510 Keywords : ANTIGEN PRESENTATION PATHWAY * BORDETELLA- PERTUSSIS * INVASIVE ACTIVITY Subject RIV: EE - Microbiology, Virology Impact factor: 4.819, year: 2010

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

  6. 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

  7. Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors

    Directory of Open Access Journals (Sweden)

    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.

  8. Ca2+ influx and tyrosine kinases trigger Bordetella adenylate cyclase toxin (ACT endocytosis. Cell physiology and expression of the CD11b/CD18 integrin major determinants of the entry route.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

    Full Text Available Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3, its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca(2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

  9. Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

    Science.gov (United States)

    Etxebarria, Aitor; González-Bullón, David; Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2013-01-01

    Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface. PMID:24058533

  10. [Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3 by using suppression subtractive hybridization approach].

    Science.gov (United States)

    Zhenlong, Cao; Jiangye, Hao; Yanfen, Zhou; Zhe, Zhang; Zhihua, Ni; Yuanxiang, Hu; Weili, Liu; Yongchao, Li; Daniel, R Storm; Runlin, Z Ma; Zhenshan, Wang

    2014-06-01

    Adenylate cyclase 3 (AC3) is one of the major players in the olfactory signaling within the main olfactory epithelium (MOE) of mice. However, we are not ascertained whether deficiency of AC3 will lead to the differential expression of related genes in the MOE. Forward and reverse subtractive libraries were constructed by suppression subtractive hybridization (SSH) approach, with MOEs from AC3(-/-) and AC3(+/+) mice. These two libraries were primarily screened by Dot blot, differential expressed clones were sequenced and analyzed by bioinformatics, and differential expressed genes were verified by qRT-PCR. A total of 386 differentially expressed clones were picked out after Dot blot. The DNA sequences of 80 clones randomly selected were determined, and 62 clones were identified by blasting in GenBank. We found that 24 up-regulated clones were corresponded to genes of kcnk3, mapk7, megf11, and 38 down-regulated clones were corresponded to tmem88b, c-mip, skp1a, mlycd, etc. Their functions were annotated with Gene Ontology (GO) and found to be mainly focused on molecular binding, cell cycle, processes of biology and cells. Five genes (kcnk3, c-mip, mlycd, tmem88b and trappc5) were verified by qRT-PCR with individuals of AC3(+/+) and AC3(-/-) mice. The data indicate that kcnk3 gene is up-regulated significantly, increasing 1.27 folds compared to control mice, whereas c-mip, mlycd, tmem88b and trappc5 are down-regulated significantly, decreasing 20%, 7%, 32% and 29% compared to the AC3(+/+)mice. The functions of these genes are closely related with K(+) channels, cell differentiation, metabolism of fats, membrane transportation, and so on. It is tempting to speculate that these genes might work together with AC3 to orchestrate the olfactory transduction signaling in the MOE.

  11. Y1 receptors for neuropeptide Y are coupled to mobilization of intracellular calcium and inhibition of adenylate cyclase

    DEFF Research Database (Denmark)

    Aakerlund, L; Gether, U; Fuhlendorff, J

    1990-01-01

    Two types of binding sites have previously been described for neuropeptide Y (NPY), called Y1 and Y2 receptors. The intracellular events following Y1 receptor activation was studied in the human neuroblastoma cell line SK-N-MC. Both NPY and the specific Y1 receptor ligand, [Leu31,Pro34]-NPY, caused...

  12. Pituitary Adenlylate Cyclase Activating Peptide Protects Adult Neural Stem Cells from a Hypoglycaemic milieu.

    Directory of Open Access Journals (Sweden)

    Shiva Mansouri

    Full Text Available Hypoglycaemia is a common side-effect of glucose-lowering therapies for type-2 diabetic patients, which may cause cognitive/neurological impairment. Although the effects of hypoglycaemia in the brain have been extensively studied in neurons, how hypoglycaemia impacts the viability of adult neural stem cells (NSCs has been poorly investigated. In addition, the cellular and molecular mechanisms of how hypoglycaemia regulates NSCs survival have not been characterized. Recent work others and us have shown that the pituitary adenylate cyclase-activating polypeptide (PACAP and the glucagon-like peptide-1 receptor (GLP-1R agonist Exendin-4 stimulate NSCs survival against glucolipoapoptosis. The aim of this study was to establish an in vitro system where to study the effects of hypoglycaemia on NSC survival. Furthermore, we determine the potential role of PACAP and Exendin-4 in counteracting the effect of hypoglycaemia. A hypoglycaemic in vitro milieu was mimicked by exposing subventricular zone-derived NSC to low levels of glucose. Moreover, we studied the potential involvement of apoptosis and endoplasmic reticulum stress by quantifying protein levels of Bcl-2, cleaved caspase-3 and mRNA levels of CHOP. We show that PACAP via PAC-1 receptor and PKA activation counteracts impaired NSC viability induced by hypoglycaemia. The protective effect induced by PACAP correlated with endoplasmic reticulum stress, Exendin-4 was ineffective. The results show that hypoglycaemia decreases NSC viability and that this effect can be substantially counteracted by PACAP via PAC-1 receptor activation. The data supports a potential therapeutic role of PAC-1 receptor agonists for the treatment of neurological complications, based on neurogenesis impairment by hypoglycaemia.

  13. Pituitary adenylate cyclase-activating peptide stimulates neurite growth in PC12 cells.

    Science.gov (United States)

    Hernandez, A; Kimball, B; Romanchuk, G; Mulholland, M W

    1995-01-01

    The ability of PACAP-38 to stimulate morphological development was studied using rat pheochromocytoma PC12 cells. PACAP-38 produced concentration-dependent increases in percentage of cells exhibiting neurite extension. Similar increases were produced by forskolin (28 +/- 2% at 96 h) and 8-bromo cAMP (30 +/- 2%). Vasoactive intestinal peptide and alpha-calcitonin gene-related peptide were without effect. PACAP-38 produced significant increases in PC12 cell cAMP content and inositol phosphate turnover. Intracellular [Ca2+] increased from 169 +/- 14 nM to 560 +/- 58 nM in response to 1 microM PACAP-38. PACAP-stimulated neurite outgrowth was abolished by RpcAMPS, an inhibitor of cAMP-dependent kinases but was unaffected by the protein kinase C antagonist H7.

  14. 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

    is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel...

  15. Pituitary adenylate cyclase activating polypeptide plays a role in olfactory memory formation in chicken.

    Science.gov (United States)

    Józsa, Rita; Hollósy, Tibor; Tamás, Andrea; Tóth, Gábor; Lengvári, István; Reglodi, Dóra

    2005-11-01

    PACAP plays an important role during development of the nervous system and is also involved in memory processing. The aim of the present study was to investigate the function of PACAP in chicken embryonic olfactory memory formation by blocking PACAP at a sensitive period in ovo. Chicken were exposed daily to strawberry scent in ovo from embryonic day 15. Control eggs were treated only with saline, while other eggs received a single injection of the PACAP antagonist PACAP6-38 at day 15. The consumption of scented and unscented water was measured daily after hatching. Animals exposed to strawberry scent in ovo showed no preference. However, chickens exposed to PACAP6-38, showed a clear preference for plain water, similarly to unexposed chicken. Our present study points to PACAP's possible importance in embryonic olfactory memory formation.

  16. Acylation of lysine 983 is sufficient for toxin activity of Bordetella pertussis adenylate cyclase

    Czech Academy of Sciences Publication Activity Database

    Basar, T.; Havlíček, Vladimír; Bezoušková, Silvia; Hackett, M.; Šebo, Peter

    2001-01-01

    Roč. 276, č. 1 (2001), s. 348-354 ISSN 0021-9258 R&D Projects: GA ČR GA310/98/0432; GA ČR GV310/96/K102; GA AV ČR IAA5020907; GA MŠk ME 167; GA MŠk VS96149 Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EE - Microbiology, Virology Impact factor: 7.258, year: 2001

  17. Inflammasome Activation by Adenylate Cyclase Toxin Directs Th17 Responses and Protection against Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Dunne, A.; Ross, P. J.; Pospíšilová, Eva; Mašín, Jiří; Meaney, A.; Sutton, C. E.; Iwakura, Y.; Tschopp, J.; Šebo, Peter; Mills, K. H. G.

    2010-01-01

    Roč. 187, č. 3 (2010), s. 1711-1719 ISSN 0022-1767 R&D Projects: GA ČR GA310/08/0447; GA AV ČR IAA500200914 Institutional research plan: CEZ:AV0Z50200510 Keywords : ADAPTIVE IMMUNE-RESPONSES * IL-17-PRODUCING T-CELLS * HOST-DEFENSE Subject RIV: EC - Immunology Impact factor: 5.745, year: 2010

  18. 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

  19. 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

  20. 31P NMR Spectroscopy Revealed Adenylate kinase-like Activity and Phosphotransferase-like Activity from F1-ATPase of Escherichia coli

    International Nuclear Information System (INIS)

    Kim, Hyun Won

    2011-01-01

    Adenylate kinase-like activity and phosphotransferase-like activity from F 1 -ATPase of Escherichia coli was revealed by 31 P NMR spectroscopy. Incubation of F 1 -ATPase with ADP in the presence of Mg 2+ shows the appearance of 31 P resonances from AMP and Pi, suggesting generation of AMP and ATP by adenylate kinase-like activity and the subsequent hydrolysis to Pi. Incubation of F1-ATPase with ADP in the presence of methanol shows additional peak from methyl phosphate, suggesting phosphotransferase-like activity of F 1 -ATPase. Both adenylate kinase-like activity and phosphotransferase-like activity has not been reported from F 1 -ATPase of Escherichia coli. 31 P NMR could be a valuable tool for the investigation of phosphorous related enzyme

  1. 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

    samples (plasma PACAP38 and vasoactive intestinal polypeptide and serum tryptase), and vital signs (blood pressure, heart rate, respiratory frequency, and end-tidal pressure of CO2) was recorded before and up to 5 h after infusion. Twenty-two patients [mean age 24 years (range 19-36)] completed the study...... on both days. Sixteen patients (73%) reported migraine-like attacks after PACAP38 and four after vasoactive intestinal polypeptide (18%) infusion (P = 0.002). Three of four patients, who reported migraine-like attacks after vasoactive intestinal polypeptide, also reported attacks after PACAP38. Both...... the start of PACAP38 infusion only in those patients who later reported migraine attacks. Blood levels of vasoactive intestinal polypeptide and tryptase were unchanged after PACAP38 infusion. In conclusion, PACAP38-induced migraine was associated with sustained dilatation of extracranial arteries...

  2. Innervation of the rat pineal gland by pituitary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nerve fibres

    DEFF Research Database (Denmark)

    Møller, Morten; Fahrenkrug, Jan; Hannibal, J.

    1999-01-01

    Calcitonin gene-related peptide, vasoactive intestinal peptide, neuropeptide Y, colocalization, trigeminal ganglion, rat (Wistar)......Calcitonin gene-related peptide, vasoactive intestinal peptide, neuropeptide Y, colocalization, trigeminal ganglion, rat (Wistar)...

  3. Pore-Forming and Enzymatic Activities of Bordetella pertussis Adenylate Cyclase Toxin Synergize in Promoting Lysis of Monocytes

    Czech Academy of Sciences Publication Activity Database

    Basler, Marek; Mašín, Jiří; Osička, Radim; Šebo, Peter

    2006-01-01

    Roč. 74, č. 5 (2006), s. 2207-2214 ISSN 0019-9567 R&D Projects: GA AV ČR IAA5020406; GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50200510 Keywords : bordetella pertussis * cyaa * cytotoxicity Subject RIV: EE - Microbiology, Virology Impact factor: 4.004, year: 2006

  4. Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8(+) and CD4(+) T cells

    Czech Academy of Sciences Publication Activity Database

    Švédová, Martina; Mašín, Jiří; Fišer, Radovan; Černý, Ondřej; Tomala, Jakub; Freudenberg, M.; Tučková, Ludmila; Kovář, Marek; Dadaglio, G.; Adkins, Irena; Šebo, Peter

    2016-01-01

    Roč. 94, č. 4 (2016), s. 322-333 ISSN 0818-9641 R&D Projects: GA ČR(CZ) GA13-14547S; GA ČR(CZ) GAP302/12/0460 Institutional support: RVO:61388971 Keywords : N-TERMINAL KINASE * BORDETELLA- PERTUSSIS * MEMBRANE TRANSLOCATION Subject RIV: EE - Microbiology, Virology Impact factor: 4.557, year: 2016

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    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. Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells

    Science.gov (United States)

    Ng, KY; Yeung, BHS; Wong, YH; Wise, H

    2013-01-01

    Background and Purpose Hyper-nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other Gs-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity. Experimental Approach Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. Key Results Pharmacological analysis showed the presence of Gs-coupled β2-adrenoceptors and CGRP receptors, but not β1-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. Conclusions and Implications Gs-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses. PMID:22924655

  8. Effects of abstinence and family history for alcoholism on platelet adenylyl cyclase activity.

    Science.gov (United States)

    Menninger, J A; Barón, A E; Tabakoff, B

    1998-12-01

    Platelet adenylyl cyclase (AC) activity was measured in 32 alcohol-dependent subjects and 27 control subjects who were categorized as either family history-positive (FHP) or family history-negative (FHN) for alcoholism. The interview and blood sample collections were performed shortly after cessation of heavy drinking in the alcoholic group, and repeat blood samples were obtained at the end of the first and second weeks of monitored abstinence. Control subjects received the same interview and provided blood samples at the time of the interview. When subjects were not segregated for FHP or FHN status, there were no statistically significant differences in basal, cesium fluoride (CsF)-, or forskolin-stimulated mean AC activities between the controls and the alcoholics, at study entry or with 1 or 2 weeks of abstinence. On the other hand, over the 2-week course of sobriety from heavy drinking, the CsF-stimulated AC activity of FHP alcohol-dependent subjects decreased significantly (p = 0.03). FHP alcohol-dependent subjects after 2 weeks of sobriety had significantly lower mean CsF-stimulated AC activity than FHN controls (p = 0.04), whereas the FHN alcoholic subjects' CsF-stimulated AC activity did not differ significantly from FHN controls at this point in time. When all subjects were pooled and then categorized as either FHP or FHN, there was a significant difference in mean CsF-stimulated AC activity (p = 0.02) between the FHP and FHN subject groups. Genetic factors and abstinence appear to have roles in determining low platelet AC activity in alcoholic and nonalcoholic subjects. CsF-stimulated platelet AC activity, in particular, appears to act as a trait marker for a genetic vulnerability to developing alcoholism, but recent heavy drinking in male alcoholics is a factor that can mask differences between FHP and FHN subjects.

  9. A HCO(3)(-)-dependent mechanism involving soluble adenylyl cyclase for the activation of Ca²⁺ currents in locus coeruleus neurons.

    Science.gov (United States)

    Imber, Ann N; Santin, Joseph M; Graham, Cathy D; Putnam, Robert W

    2014-12-01

    Hypercapnic acidosis activates Ca²⁺ channels and increases intracellular Ca²⁺ levels in neurons of the locus coeruleus, a known chemosensitive region involved in respiratory control. We have also shown that large conductance Ca²⁺-activated K⁺ channels, in conjunction with this pathway, limits the hypercapnic-induced increase in firing rate in locus coeruleus neurons. Here, we present evidence that the Ca²⁺ current is activated by a HCO(3)(-)-sensitive pathway. The increase in HCO(3)(-) associated with hypercapnia activates HCO(3)(-)-sensitive adenylyl cyclase (soluble adenylyl cyclase). This results in an increase in cyclic adenosine monophosphate levels and activation of Ca²⁺ channels via cyclic adenosine monophosphate-activated protein kinase A. We also show the presence of soluble adenylyl cyclase in the cytoplasm of locus coeruleus neurons, and that the cyclic adenosine monophosphate analogue db-cyclic adenosine monophosphate increases Ca²⁺i. Disrupting this pathway by decreasing HCO(3)(-) levels during acidification or inhibiting either soluble adenylyl cyclase or protein kinase A, but not transmembrane adenylyl cyclase, can increase the magnitude of the firing rate response to hypercapnia in locus coeruleus neurons from older neonates to the same extent as inhibition of K⁺ channels. 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.

  10. Bicarbonate disruption of the pulmonary endothelial barrier via activation of endogenous soluble adenylyl cyclase, isoform 10.

    Science.gov (United States)

    Obiako, Boniface; Calchary, Wendy; Xu, Ningyong; Kunstadt, Ryan; Richardson, Bianca; Nix, Jessica; Sayner, Sarah L

    2013-07-15

    It is becoming increasingly apparent that cAMP signals within the pulmonary endothelium are highly compartmentalized, and this compartmentalization is critical to maintaining endothelial barrier integrity. Studies demonstrate that the exogenous soluble bacterial toxin, ExoY, and heterologous expression of the forskolin-stimulated soluble mammalian adenylyl cyclase (AC) chimera, sACI/II, elevate cytosolic cAMP and disrupt the pulmonary microvascular endothelial barrier. The barrier-disruptive effects of cytosolic cAMP generated by exogenous soluble ACs are in contrast to the barrier-protective effects of subplasma membrane cAMP generated by transmembrane AC, which strengthens endothelial barrier integrity. Endogenous soluble AC isoform 10 (AC10 or commonly known as sAC) lacks transmembrane domains and localizes within the cytosolic compartment. AC10 is uniquely activated by bicarbonate to generate cytosolic cAMP, yet its role in regulation of endothelial barrier integrity has not been addressed. Here we demonstrate that, within the pulmonary circulation, AC10 is expressed in pulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs), yet expression in PAECs is lower. Furthermore, pulmonary endothelial cells selectively express bicarbonate cotransporters. While extracellular bicarbonate generates a phosphodiesterase 4-sensitive cAMP pool in PMVECs, no such cAMP response is detected in PAECs. Finally, addition of extracellular bicarbonate decreases resistance across the PMVEC monolayer and increases the filtration coefficient in the isolated perfused lung above osmolality controls. Collectively, these findings suggest that PMVECs have a bicarbonate-sensitive cytosolic cAMP pool that disrupts endothelial barrier integrity. These studies could provide an alternative mechanism for the controversial effects of bicarbonate correction of acidosis of acute respiratory distress syndrome patients.

  11. Characterisation of two genes for guanylate cyclase activator protein (GCAP1 and GCAP2) in the Japanese pufferfish, Fugu rubripes.

    Science.gov (United States)

    Wilkie, Susan E; Stinton, Inez; Cottrill, Phillippa; Deery, Evelyne; Newbold, Richard; Warren, Martin J; Bhattacharya, Shomi S; Hunt, David M

    2002-08-19

    cDNA and genomic clones encoding guanylate cyclase activating proteins (GCAP1 and GCAP2) in the Japanese puffer fish (Fugu rubripes) were identified by probing, respectively, a retinal cDNA library and a whole genomic cosmid library with human GCAP1 and GCAP2 cDNA probes. Clones were identified as GCAP1 and GCAP2 on the basis of amino acid identity with the equivalent frog sequences and their placement into GCAP1 and GCAP2 clades within a GCAP phylogenetic tree. The Fugu genes have an identical four exon/three intron structure to GCAP1 and GCAP2 genes from other vertebrates but the introns are smaller, with the result that the four exons spread over approximately 1 kb of DNA in each case. The two genes are separated on to separate cosmids. However, the results of Southern analysis of the cosmids and of genomic DNA are consistent with a tail-to-tail gene arrangement, as in other species, but with a surprisingly large intergenic separation of around 18.7 kb. Recombinant Fugu GCAP1 failed to activate human retinal guanylate cyclase (retGC) in vitro although CD spectroscopy shows that the protein is folded with a similar secondary structure to that of human GCAP1. The failure to activate may be due therefore to a lack of molecular compatibility in this heterologous assay system.

  12. ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Baoyu; Sysoeva, Tatyana A.; Chowdhury, Saikat; Guo, Liang; Nixon, B.Tracy; (IIT); (Penn)

    2009-10-06

    Except for apyrases, ATPases generally target only the {gamma}-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10 000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.

  13. Probing the Molecular Mechanism of Human Soluble Guanylate Cyclase Activation by NO in vitro and in vivo.

    Science.gov (United States)

    Pan, Jie; Yuan, Hong; Zhang, Xiaoxue; Zhang, Huijuan; Xu, Qiming; Zhou, Yajun; Tan, Li; Nagawa, Shingo; Huang, Zhong-Xian; Tan, Xiangshi

    2017-02-23

    Soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. NO binds to the heme of sGC to catalyze the synthesis of the second messenger cGMP, which plays a critical role in several physiological processes. However, the molecular mechanism for sGC to mediate the NO signaling remains unclear. Here fluorophore FlAsH-EDT 2 and fluorescent proteins were employed to study the NO-induced sGC activation. FlAsH-EDT 2 labeling study revealed that NO binding to the H-NOX domain of sGC increased the distance between H-NOX and PAS domain and the separation between H-NOX and coiled-coil domain. The heme pocket conformation changed from "closed" to "open" upon NO binding. In addition, the NO-induced conformational change of sGC was firstly investigated in vivo through fluorescence lifetime imaging microscopy. The results both in vitro and in vivo indicated the conformational change of the catalytic domain of sGC from "open" to "closed" upon NO binding. NO binding to the heme of H-NOX domain caused breaking of Fe-N coordination bond, initiated the domain moving and conformational change, induced the allosteric effect of sGC to trigger the NO-signaling from H-NOX via PAS &coiled-coil to the catalytic domain, and ultimately stimulates the cyclase activity of sGC.

  14. Mechanism of A2 adenosine receptor activation. I. Blockade of A2 adenosine receptors by photoaffinity labeling

    International Nuclear Information System (INIS)

    Lohse, M.J.; Klotz, K.N.; Schwabe, U.

    1991-01-01

    It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine [(R)-AHPIA] into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling

  15. 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

  16. 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.

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

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

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

  18. Pituitary adenylate cyclase-activating peptide (PACAP) and PAC1 receptor in the testis of cartilaginous fish Torpedo marmorata: A molecular and phylogenetic study.

    Science.gov (United States)

    Agnese, Marisa; Valiante, Salvatore; Rosati, Luigi; Andreuccetti, Piero; Prisco, Marina

    2016-01-01

    The role of PACAP in spermatogenesis and steroidogenesis has been largely investigated in last years in mammals; conversely, a few studies have been performed in non mammalian vertebrates. In this paper we investigated the sequence, expression and localization of PACAP and its PAC1 receptor in the testis of the benthic elasmobranch Torpedo marmorata, the marbled electric ray. Cloning a partial PACAP cDNA, we demonstrated for the first time in elasmobranches that PACAP shows a highly conserved sequence, compared with the PACAP of other chordates (tunicates and vertebrates). Moreover, the phylogenetic analysis revealed that PACAP has been well preserved during evolution and that a negative selection acts on PACAP sequence, leading to the conservation of the coding sites. The phylogenetic consensus tree showed also that Torpedo PACAP is more related with the amphibian PACAP than with the teleost one. Finally, we demonstrated that in T. marmorata PACAP and its PAC1 receptor are synthesized directly in the testis, where they show a wider localization than mammals, suggesting that this neuropeptide is involved in the control of Torpedo spermatogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. cAMP signalling of Bordetella adenylate cyclase toxin through the SHP-1 phosphatase activates the BimEL-Bax pro-apoptotic cascade in phagocytes

    Czech Academy of Sciences Publication Activity Database

    Ahmad, Jawid Nazir; Černý, Ondřej; Linhartová, Irena; Mašín, Jiří; Osička, Radim; Šebo, Peter

    2016-01-01

    Roč. 18, č. 3 (2016), s. 384-398 ISSN 1462-5814 R&D Projects: GA ČR GA13-14547S; GA ČR(CZ) GAP302/11/0580; GA ČR GAP302/12/0460 Institutional support: RVO:61388971 Keywords : FORKHEAD TRANSCRIPTION FACTOR * HUMAN T-CELLS * DENDRITIC CELLS Subject RIV: EE - Microbiology, Virology Impact factor: 4.554, year: 2016

  20. Bordetella Adenylate Cyclase Toxin Differentially Modulates Toll-Like Receptor-Stimulated Activation, Migration and T Cell Stimulatory Capacity of Dendritic Cells

    Czech Academy of Sciences Publication Activity Database

    Adkins, Irena; Kamanová, Jana; Kocourková, A.; Švédová, Martina; Tomala, Jakub; Janová, H.; Mašín, Jiří; Chládková, Barbara; Bumba, Ladislav; Kovář, Marek; Ross, P. J.; Tučková, Ludmila; Spíšek, R.; Mills, K. H. G.; Šebo, Peter

    2014-01-01

    Roč. 9, č. 8 (2014) E-ISSN 1932-6203 R&D Projects: GA ČR GA310/08/0447; GA ČR GP310/09/P582; GA ČR GAP301/11/0325; GA MŠk 1M0506 Institutional support: RVO:61388971 Keywords : RESPIRATORY-INFECTION * INTERLEUKIN-10 PRODUCTION * PROTECTIVE IMMUNITY Subject RIV: EE - Microbiology, Virology Impact factor: 3.234, year: 2014

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

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

  2. CD-NP: a novel engineered dual guanylyl cyclase activator with anti-fibrotic actions in the heart.

    Directory of Open Access Journals (Sweden)

    Fernando L Martin

    Full Text Available Natriuretic peptides (NPs are cardioprotective through the activation of guanylyl cyclase (GC receptors A and B. CD-NP, also known as cenderitide, is a novel engineered NP that was designed to uniquely serve as a first-in-class dual GC receptor agonist. Recognizing the aldosterone suppressing actions of GC-A activation and the potent inhibitory actions on collagen synthesis and fibroblast proliferation through GC-B activation, the current study was designed to establish the anti-fibrotic actions of CD-NP, administered subcutaneously, in an experimental rat model of early cardiac fibrosis induced by unilateral nephrectomy (UNX. Our results demonstrate that a two week subcutaneous infusion of CD-NP significantly suppresses left ventricular fibrosis and circulating aldosterone, while preserving both systolic and diastolic function, in UNX rats compared to vehicle treated UNX rats. Additionally we also confirmed, in vitro, that CD-NP significantly generates the second messenger, cGMP, through both the GC-A and GC-B receptors. Taken together, this novel dual GC receptor activator may represent an innovative anti-fibrotic therapeutic agent.

  3. The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants

    KAUST Repository

    Kwezi, Lusisizwe

    2011-04-19

    Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 μM and Vmax of 1800 nmol min-1 mg-1 of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg2+ or Mn2+. Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Retigeric acid B attenuates the virulence of Candida albicans via inhibiting adenylyl cyclase activity targeted by enhanced farnesol production.

    Directory of Open Access Journals (Sweden)

    Wenqiang Chang

    Full Text Available Candida albicans, the most prevalent fungal pathogen, undergoes yeast-to-hyphal switch which has long been identified as a key fungal virulence factor. We showed here that the lichen-derived small molecule retigeric acid B (RAB acted as an inhibitor that significantly inhibited the filamentation of C. albicans, leading to the prolonged survival of nematodes infected by C. albicans. Quantitative real-time PCR analysis and intracellular cAMP measurement revealed RAB regulated the Ras1-cAMP-Efg1 pathway by reducing cAMP level to inhibit the hyphae formation. Confocal microscopic observation showed RAB induced the expression of Dpp3, synthesizing more farnesol, which was confirmed by gas chromatography-mass spectroscopy detection. An adenylyl cyclase activity assay demonstrated RAB could repress the activity of Cdc35 through stimulating farnesol synthesis, thus causing a decrease in cAMP synthesis, leading to retarded yeast-to-hyphal transition. Moreover, reduced levels of intracellular cAMP resulted in the inhibition of downstream adhesins. Together, these findings indicate that RAB stimulates farnesol production that directly inhibits the Cdc35 activity, reducing the synthesis of cAMP and thereby causing the disruption of the morphologic transition and attenuating the virulence of C. albicans. Our work illustrates the underlying mechanism of RAB-dependent inhibition of the yeast-to-hyphal switch and provides a potential application in treating the infection of C. albicans.

  5. Rat striatal muscarinic receptors coupled to the inhibition of adenylyl cyclase activity: potent block by the selective m4 ligand muscarinic toxin 3 (MT3).

    Science.gov (United States)

    Olianas, M C; Adem, A; Karlsson, E; Onali, P

    1996-05-01

    1. In rat striatal membranes, muscarinic toxin 3 (MT3), a selective ligand of the cloned m4 receptor subtype, antagonized the acetylcholine (ACh) inhibition of forskolin- and dopamine D1 receptor-stimulated adenylyl cyclase activities with pA2 values of 8.09 and 8.15, respectively. 2. In radioligand binding experiments, MT3 increased the Kd but did not change the Bmax value of [3H]-N-methylscopolamine (3H]-NMS) binding to rat striatal muscarinic receptors. The toxin displaced the major portion of the [3H]-NMS binding sites with a Ki of 8.0 nM. 3. In rat myocardium, MT3 antagonized the ACh inhibition of adenylyl cyclase with a Ki value of 860 nM. 4. In rat cerebral cortical membranes prelabelled with [3H]-myo-inositol, MT3 counteracted the methacholine stimulation of [3H]-inositol phosphates formation with a Ki value of 113 nM. 5. The present study shows that MT3 is a potent antagonist of the striatal muscarinic receptors coupled to inhibition of adenylyl cyclase activity. This finding provides strong evidence for the classification of these receptors as pharmacologically equivalent to the m4 gene product (M4). On the other hand, the weaker potencies of MT3 in antagonizing the muscarinic responses in cerebral cortex and in the heart are consistent with the reported lower affinities of the toxin for the cloned m1 and m2 receptor subtypes, respectively.

  6. Dependence of electrical activity and calcium influx-controlled prolactin release on adenylyl cyclase signaling pathway in pituitary lactotrophs

    Czech Academy of Sciences Publication Activity Database

    Gonzalez-Iglesias, A. E.; Jiang, Y.; Tomič, M.; Kretschmannová, K.; Andric, S. A.; Zemková, Hana

    2006-01-01

    Roč. 20, č. 9 (2006), s. 2231-2246 ISSN 0888-8809 R&D Projects: GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : adenylyl cyclase * intracellular calcium concentration * lactotrophs Subject RIV: FH - Neurology Impact factor: 4.967, year: 2006

  7. Pituitary adenylyl cyclase activating polypeptide inhibits gli1 gene expression and proliferation in primary medulloblastoma derived tumorsphere cultures

    International Nuclear Information System (INIS)

    Cohen, Joseph R; Resnick, Daniel Z; Niewiadomski, Pawel; Dong, Hongmei; Liau, Linda M; Waschek, James A

    2010-01-01

    Hedgehog (HH) signaling is critical for the expansion of granule neuron precursors (GNPs) within the external granular layer (EGL) during cerebellar development. Aberrant HH signaling within GNPs is thought to give rise to medulloblastoma (MB) - the most commonly-observed form of malignant pediatric brain tumor. Evidence in both invertebrates and vertebrates indicates that cyclic AMP-dependent protein kinase A (PKA) antagonizes HH signalling. Receptors specific for the neuropeptide pituitary adenylyl cyclase activating polypeptide (PACAP, gene name ADCYAP1) are expressed in GNPs. PACAP has been shown to protect GNPs from apoptosis in vitro, and to interact with HH signaling to regulate GNP proliferation. PACAP/ptch1 double mutant mice exhibit an increased incidence of MB compared to ptch1 mice, indicating that PACAP may regulate HH pathway-mediated MB pathogenesis. Primary MB tumorsphere cultures were prepared from thirteen ptch1 +/- /p53 +/- double mutant mice and treated with the smoothened (SMO) agonist purmorphamine, the SMO antagonist SANT-1, the neuropeptide PACAP, the PKA activator forskolin, and the PKA inhibitor H89. Gene expression of gli1 and [ 3 H]-thymidine incorporation were assessed to determine drug effects on HH pathway activity and proliferation, respectively. PKA activity was determined in cell extracts by Western blotting using a phospho-PKA substrate antibody. Primary tumor cells cultured for 1-week under serum-free conditions grew as tumorspheres and were found to express PAC1 receptor transcripts. Gli1 gene expression was significantly reduced by SANT-1, PACAP and forskolin, but was unaffected by purmorphamine. The attenuation of gli1 gene expression by PACAP was reversed by the PKA inhibitor H89, which also blocked PKA activation. Treatment of tumorsphere cultures with PACAP, forskolin, and SANT-1 for 24 or 48 hours reduced proliferation. Primary tumorspheres derived from ptch1 +/- /p53 +/- mice exhibit constitutive HH pathway activity

  8. Pituitary adenylyl cyclase activating polypeptide inhibits gli1 gene expression and proliferation in primary medulloblastoma derived tumorsphere cultures

    Directory of Open Access Journals (Sweden)

    Dong Hongmei

    2010-12-01

    Full Text Available Abstract Background Hedgehog (HH signaling is critical for the expansion of granule neuron precursors (GNPs within the external granular layer (EGL during cerebellar development. Aberrant HH signaling within GNPs is thought to give rise to medulloblastoma (MB - the most commonly-observed form of malignant pediatric brain tumor. Evidence in both invertebrates and vertebrates indicates that cyclic AMP-dependent protein kinase A (PKA antagonizes HH signalling. Receptors specific for the neuropeptide pituitary adenylyl cyclase activating polypeptide (PACAP, gene name ADCYAP1 are expressed in GNPs. PACAP has been shown to protect GNPs from apoptosis in vitro, and to interact with HH signaling to regulate GNP proliferation. PACAP/ptch1 double mutant mice exhibit an increased incidence of MB compared to ptch1 mice, indicating that PACAP may regulate HH pathway-mediated MB pathogenesis. Methods Primary MB tumorsphere cultures were prepared from thirteen ptch1+/-/p53+/- double mutant mice and treated with the smoothened (SMO agonist purmorphamine, the SMO antagonist SANT-1, the neuropeptide PACAP, the PKA activator forskolin, and the PKA inhibitor H89. Gene expression of gli1 and [3H]-thymidine incorporation were assessed to determine drug effects on HH pathway activity and proliferation, respectively. PKA activity was determined in cell extracts by Western blotting using a phospho-PKA substrate antibody. Results Primary tumor cells cultured for 1-week under serum-free conditions grew as tumorspheres and were found to express PAC1 receptor transcripts. Gli1 gene expression was significantly reduced by SANT-1, PACAP and forskolin, but was unaffected by purmorphamine. The attenuation of gli1 gene expression by PACAP was reversed by the PKA inhibitor H89, which also blocked PKA activation. Treatment of tumorsphere cultures with PACAP, forskolin, and SANT-1 for 24 or 48 hours reduced proliferation. Conclusions Primary tumorspheres derived from ptch1+/-/p53

  9. Alterations in adenylate kinase activity in human PBMCs after in vitro exposure to electromagnetic field: comparison between extremely low frequency electromagnetic field (ELF) and therapeutic application of a musically modulated electromagnetic field (TAMMEF).

    Science.gov (United States)

    Albanese, Antonietta; Battisti, Emilio; Vannoni, Daniela; Aceto, Emilia; Galassi, Gianmichele; Giglioni, Stefania; Tommassini, Valentina; Giordano, Nicola

    2009-01-01

    This study investigated the effects of electromagnetic fields on enzymes involved in purine metabolism in human peripheral blood mononuclear cells in vitro. Cells were obtained from 20 volunteers. We tested both low-energy, extremely low frequency (ELF; 100-Hz) electromagnetic fields and the Therapeutic Application of Musically Modulated Electromagnetic Fields (TAMMEFs); the latter is characterized by variable frequencies, intensities, and wave shapes. Adenylate kinase activity was increased after ELF field exposure but decreased slightly after TAMMEF exposure. Neither of the two electromagnetic field affected the activities of the purine metabolism enzymes ecto-5'-nucleotidase, adenosine deaminase, and adenosine kinase. We concluded that ELF fields may influence cellular electrical charge stability; stimulation of adenylate kinase activity could restore the cell to a state of equilibrium. In contrast, TAMMEF fields may be useful for maintaining and regulating the cellular electrical charge.

  10. Adenylic dinucleotides produced by CD38 are negative endogenous modulators of platelet aggregation.

    Science.gov (United States)

    Magnone, Mirko; Basile, Giovanna; Bruzzese, Debora; Guida, Lucrezia; Signorello, Maria Grazia; Chothi, Madhu Parakkottil; Bruzzone, Santina; Millo, Enrico; Qi, Ai-Dong; Nicholas, Robert A; Kassack, Matthias U; Leoncini, Giuliana; Zocchi, Elena

    2008-09-05

    Diadenosine 5',5'''-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC50 values ranging between 5 and 15 microm. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y11, exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC50 value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.

  11. A HCO3−-dependent mechanism involving soluble adenylyl cyclase for the activation of Ca2+ currents in locus coeruleus neurons

    Science.gov (United States)

    Imber, Ann N.; Santin, Joseph M.; Graham, Cathy D.; Putnam, Robert W.

    2014-01-01

    Hypercapnic acidosis activates Ca2+ channels and increases intracellular Ca2+ levels in neurons of the locus coeruleus (LC), a known chemosensitive region involved in respiratory control. We have also shown that large conductance Ca2+-activated K+ channels (BK), in conjunction with this pathway, limits the hypercapnic-induced increase in firing rate in LC neurons. Here, we present evidence that the Ca2+ current is activated by a HCO3−-sensitive pathway. The increase in HCO3− associated with hypercapnia activates HCO3−-sensitive adenylyl cyclase (sAC). This results in an increase in cAMP levels and activation of Ca2+ channels via cAMP-activated protein kinase A (PKA). We also show the presence of sAC in the cytoplasm of LC neurons, and that the cAMP analogue db-cAMP increases Ca2+i. Disrupting this pathway by decreasing HCO3− levels during acidification or inhibiting either sAC or PKA, but not transmembrane adenylyl cyclase (tmAC), can increase the magnitude of the firing rate response to hypercapnia in LC neurons from older neonates to the same extent as inhibition of BK channels. PMID:25092170

  12. Glycosylated Triterpenoid Saponin : a Specific Inhibitor of Diguanylate Cyclase from Acetobacter xylinum.Biological Activity and Distribution

    OpenAIRE

    Patricia, Ohana; Deborah P., Delmer; Gail, Volman; Moshe, Benziman; Department of Biological Chemistry, Institute of Life Scineces, The Hebrew University of Jerusalem; Section of Plant Biology, University of California Davis; Department of Biological Chemistry, Institute of Life Scineces, The Hebrew University of Jerusalem; Department of Biological Chemistry, Institute of Life Scineces, The Hebrew University of Jerusalem

    1998-01-01

    In a recent paper (Ohana et al.1998), we described the purification and structural charactrization of a novel glycosidic triterpenoid saponin (GTS), a specific inhibitor of diguanylate cyclase (dgc), the key regulatory enzyme of the cellulose synthesizing apparatus of the bacterium Acetobacter xylinum. This compound and an identical or very similar one were isolated from pea (Pisum sativum), and A.xylinum respectively. We now present the effects of GTS on the kinetic properties of dgc. The ob...

  13. Modulation of adenylyl cyclase activity by baclofen in the developing rat brain: difference between cortex, thalamus and hippocampus

    Czech Academy of Sciences Publication Activity Database

    Hejnová, Lucie; Ihnatovych, Ivanna; Novotný, Jiří; Kubová, Hana; Mareš, Pavel; Svoboda, Petr

    2002-01-01

    Roč. 330, č. 1 (2002), s. 9-12 ISSN 0304-3940 R&D Projects: GA ČR GA309/99/0207; GA ČR GA309/01/0255 Institutional research plan: CEZ:AV0Z5011922 Keywords : GABA(B) receptors * baclofen * adenylyl cyclase Subject RIV: FH - Neurology Impact factor: 2.100, year: 2002

  14. [Modifications of enzyme activities in rat liver plasma membrane after carbon tetrachloride poisoning].

    Science.gov (United States)

    Ribotta, P B; Parola, M; Carasso, M C; Caristo, G; Paradisi, L

    1981-03-15

    Adenylate Cyclase activity is increased in the liver of animals treated with CCl4 (250 ul/100g body wt.) after 30 min. The maximum increase is observed 2 hours after administration of the hepatotoxin. Whereas, 3',5'-nucleotidephosphodiesterase decreases significantly throughout all the experiments. Our results present evidence that there is relationship between Adenylate Cyclase and Phosphodiesterase activity and suggest that intracellular calcium ion may mediate a regulation of the synthesis and degradation of cyclic nucleotides. It is difficult to determine the exact role Ca2+ plays in regulating these two opposing reactions. Thus, in the near future the work in this laboratory will be to define carefully the effects of the CCl4 on ions on these two systems.

  15. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

    DEFF Research Database (Denmark)

    Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

    1983-01-01

    , and Mg2+ ions, catalyzes the synthesis of ADP from three different substrates which all lead to enzyme-bound glycyl adenylate, that is, ATP, adenosine 5'-[ß,¿-methylene]triphosphate and Ap4A. It was furthermore demonstrated that the only pathway by which a synthetase-catalyzed degradation of Ap4A can...... of adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')triphospho(5')adenosine (Ap3A), also catalyzes the formation of ADP from inorganic phosphate and the enzyme-bound glycyl adenylate. Accordingly it was shown that E. coli glycyl-tRNA synthetase, in the presence of inorganic phosphate, glycine...... of the applied enzyme, the study also showed that the preparation catalyzes a glycine-independent transfer of the ¿-phosphate group from ATP to nucleoside 5'-diphosphates. The importance of the observed reaction between inorganic phosphate and enzyme-bound aminoacyl adenylate in relation to the remaining...

  16. Effect of high potassium on dopamine receptor activity in bovine retina

    International Nuclear Information System (INIS)

    Ackerman, J.M.

    1989-01-01

    In the present study, the hypothesis that dopamine released by light caused a subsensitivity of the dopamine receptor was investigated. Bovine eyes were obtained from a slaughterhouse, and retinas were dissected in a dark room. Filter binding assays were developed to measure agonist and antagonist binding to the dopamine receptor using [ 3 H]dopamine and [ 3 H]SCH 23390, respectively, in a retinal membrane fraction. Adenylate cyclase activation was measured by the production of [ 32 P]cyclic AMP from 32 ATP. In desensitization experiments, bovine retinas were incubated for fifteen minutes with 56 mM potassium, which also causes a release of dopamine in retinas were washed, and membranes were prepared. The stimulation of adenylate cyclase evoked by dopamine and radiolabeled agonist and antagonist binding were measured. In the receptor binding characterization studies, the dissociation constant and the maximum number of binding sites were obtained for [ 3 H]dopamine and [ 3 H]SCH 23390 binding

  17. GUANYLYL CYCLASE/NATRIURETIC PEPTIDES RECEPTOR-A SIGNALING ANTAGONIZES PHOSPHOINOSITIDE HYDROLYSIS, Ca2+ RELEASE, AND ACTIVATION OF PROTEIN KINASE C

    Directory of Open Access Journals (Sweden)

    Kailash N Pandey

    2014-08-01

    Full Text Available Thus far, three related natriuretic peptides (NPs and three distinct sub-types of cognate NP receptors have been identified and characterized based on the specific ligand binding affinities, guanylyl cyclase activity, and generation of intracellular cGMP. Atrial and brain natriuretic peptides (ANP and BNP specifically bind and activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA, and C-type natriuretic peptide (CNP shows specificity to activate guanylyl cyclase/natriuretic peptide receptor-B (GC-B/NPRB. All three NPs bind to natriuretic peptide receptor-C (NPRC, which is also known as clearance or silent receptor. The NPRA is considered the principal biologically active receptor of NP family; however, the molecular signaling mechanisms of NP receptors are not well understood. The activation of NPRA and NPRB produces the intracellular second messenger cGMP, which serves as the major signaling molecule of all three NPs. The activation of NPRB in response to CNP also produces the intracellular cGMP; however, at lower magnitude than that of NPRA, which is activated by ANP and BNP. In addition to enhanced accumulation of intracellular cGMP in response to all three NPs, the levels of cAMP, Ca2+ and inositol triphosphate (IP3 have also been reported to be altered in different cells and tissue types. Interestingly, ANP has been found to lower the concentrations of cAMP, Ca2+, and IP3; however, NPRC has been proposed to increase the levels of these metabolic signaling molecules. The mechanistic studies of decreased and/or increased levels of cAMP, Ca2+, and IP3 in response to NPs and their receptors have not yet been clearly established. This review focuses on the signaling mechanisms of ANP/NPRA and their biological effects involving an increased level of intracellular accumulation of cGMP and a decreased level of cAMP, Ca2+, and IP3 in different cells and tissue systems.

  18. 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.

  19. Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity.

    Science.gov (United States)

    Graeff, R M; Walseth, T F; Fryxell, K; Branton, W D; Lee, H C

    1994-12-02

    Cyclic nucleotides such as cAMP and cGMP are second messengers subserving various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD+ and is a mediator of Ca2+ mobilization in various cellular systems. The synthesis and degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyclase and cADPR hydrolase. CD38, a differentiation antigen of B lymphocytes, has recently been shown to be a bifunctional enzyme catalyzing both the formation and hydrolysis of cADPR. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD+, identical to that catalyzed by NADase. The difficulties in detecting the formation of cADPR have led to frequent identification of CD38 as a classical NADase. In this study, we show that both ADP-ribosyl cyclase and CD38, but not NADase, can cyclize nicotinamide guanine dinucleotide (NGD+) producing a new nucleotide. Analyses by high performance liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except with guanine replacing adenine. Compared to cADPR, cGDPR is a more stable compound showing 2.8 times more resistance to heat-induced hydrolysis. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Spectroscopic analyses show that cGDPR is fluorescent and has an absorption spectrum different from both NGD+ and GDPR, providing a very convenient way for monitoring its enzymatic formation. The use of NGD+ as substrate for assaying the cyclization reaction was found to be applicable to pure enzymes as well as crude tissue extracts making it a useful diagnostic tool for distinguishing CD38-like enzymes from degradative NADases.

  20. Identification and characterization of muscarinic receptors potentiating the stimulation of adenylyl cyclase activity by corticotropin-releasing hormone in membranes of rat frontal cortex.

    Science.gov (United States)

    Onali, P; Olianas, M C

    1998-08-01

    In membranes of the rat frontal cortex, acetylcholine (ACh) and other cholinergic agonists were found to potentiate the stimulation of adenylyl cyclase activity elicited by corticotropin-releasing hormone (CRH). Oxotremorine-M, carbachol and methacholine were as effective as ACh, whereas oxotremorine and arecoline were much less effective. The facilitating effect of Ach was potently blocked by the M1 antagonists R-trihexyphenidyl, telenzepine and pirenzepine and by the M3 antagonists hexahydro-sila-difenidol and p-fluorohexahydro-sila-difenidol, whereas the M2 and M4 antagonists himbacine, methoctramine, AF-DX 116 and AQ-RA 741 were less potent. The mamba venom toxin MT-1, which binds with high affinity to M1 receptors, was also a potent blocker. The pharmacological profile of the muscarinic potentiation of CRH receptor activity was markedly different from that displayed by the muscarinic inhibition of forskolin-stimulated adenylyl cyclase, which could be detected in the same membrane preparations. Moreover, the intracerebral injection of pertussis toxin impaired the muscarinic inhibition of cyclic AMP formation and reduced the Ach stimulation of [35S]GTPgammaS binding to membrane G proteins but failed to affect the facilitating effect on CRH receptor activity. The latter response was also insensitive to the phospholipase C inhibitor U-73122, the protein kinase inhibitor staurosporine and to the inhibitors of arachidonic acid metabolism indomethacin and nordihydroguaiaretic acid. These data demonstrate that in the rat frontal cortex, muscarinic receptors of the M1 subtype potentiate CRH transmission by interacting with pertussis toxin-insensitive G proteins.

  1. Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation.

    Science.gov (United States)

    Staswick, Paul E; Tiryaki, Iskender; Rowe, Martha L

    2002-06-01

    Jasmonic acid (JA) and related cyclopentanones are critical plant signaling molecules, but their mode of action at the molecular level is unclear. A map-based approach was used to identify the defective gene in the Arabidopsis JA response mutant jar1-1. JAR1 is 1 of 19 closely related Arabidopsis genes that are similar to the auxin-induced soybean GH3 gene. Analysis of fold predictions for this protein family suggested that JAR1 might belong to the acyl adenylate-forming firefly luciferase superfamily. These enzymes activate the carboxyl groups of a variety of substrates for their subsequent biochemical modification. An ATP-PPi isotope exchange assay was used to demonstrate adenylation activity in a glutathione S-transferase-JAR1 fusion protein. Activity was specific for JA, suggesting that covalent modification of JA is important for its function. Six other Arabidopsis genes were specifically active on indole-3-acetic acid (IAA), and one was active on both IAA and salicylic acid. These findings suggest that the JAR1 gene family is involved in multiple important plant signaling pathways.

  2. Ethanol extract of the seed of Zizyphus jujuba var. spinosa potentiates hippocampal synaptic transmission through mitogen-activated protein kinase, adenylyl cyclase, and protein kinase A pathways.

    Science.gov (United States)

    Jo, So Yeon; Jung, In Ho; Yi, Jee Hyun; Choi, Tae Joon; Lee, Seungheon; Jung, Ji Wook; Yun, Jeanho; Lee, Young Choon; Ryu, Jong Hoon; Kim, Dong Hyun

    2017-03-22

    As the seed of Zizyphus jujuba var. spinosa (Bunge) Hu ex H.F. Chow (Rhamnaceae) has been used to sleep disturbances in traditional Chinese and Korean medicine, many previous studies have focused on its sedative effect. Recently, we reported the neuroprotective effect of the effect of Z. jujuba var. spinosa. However, its effects on synaptic function have not yet been studied. In this project, we examined the action of ethanol extract of the seed of Z. jujuba var. spinosa (DHP1401) on synaptic transmission in the hippocampus. To investigate the effects of DHP1401, field recordings were conducted using hippocampal slices (400µm). Object recognition test was introduced to examine whether DHP1401 affect normal recognition memory. DHP1401 (50μg/ml) induced a significant increase in synaptic activity in Shaffer collateral pathway in a concentration-dependent manner. This increase of synaptic responses was blocked by NBQX, a broad spectrum α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, but not IEM-1460, a Ca 2+ -permeable AMPAR blocker. Moreover, U0126, a mitogen-activated protein kinase inhibitor, SQ22536, an adenylyl cyclase inhibitor, and PKI, a protein kinase A inhibitor, blocked DHP1401-induced increase in synaptic transmission. Finally, DHP1401 facilitated object recognition memory. These results suggest that DHP1401 increase synaptic transmission through increase of synaptic AMPAR transmission via MAPK, AC and PAK. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  3. Prolonged exposure of chromaffin cells to nitric oxide down-regulates the activity of soluble guanylyl cyclase and corresponding mRNA and protein levels

    Directory of Open Access Journals (Sweden)

    Torres Magdalena

    2002-09-01

    Full Text Available Abstract Background Soluble guanylyl cyclase (sGC is the main receptor for nitric oxide (NO when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO, resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability.

  4. High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

    Directory of Open Access Journals (Sweden)

    Gregory Kohn

    Full Text Available BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i, which could be cytotoxic because of its high affinity for Ca(2+, thereby interfering with Ca(2+ signaling.

  5. 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.

  6. Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

    Energy Technology Data Exchange (ETDEWEB)

    Tumbale, Percy [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Williams, Jessica S. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Schellenberg, Matthew J. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology; Kunkel, Thomas A. [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology and Lab. of Molecular Genetics; Williams, R. Scott [National Inst. of Environmental Health Sciences, Research Triangle Park, NC (United States). Lab. of Structural Biology and Lab. Molecular Genetics

    2013-12-22

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.

  7. Aprataxin resolves adenylated RNA-DNA junctions to maintain genome integrity

    Science.gov (United States)

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R. Scott

    2014-01-01

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions employed by ATP-dependent DNA ligases1,2. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5′-adenylated (5′-AMP) DNA lesions3–6 (Fig. 1a). Aprataxin (Aptx) reverses DNA-adenylation but the context for deadenylation repair is unclear. Here we examine the importance of Aptx to RNaseH2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5′-ends containing a ribose characteristic of RNaseH2 incision. Aptx efficiently repairs adenylated RNA-DNA, and acting in an RNA-DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure-function studies of human Aptx/RNA-DNA/AMP/Zn complexes define a mechanism for detecting and reversing adenylation at RNA-DNA junctions. This involves A-form RNA-binding, proper protein folding and conformational changes, all of which are impacted by heritable APTX mutations in Ataxia with Oculomotor Apraxia 1 (AOA1). Together, these results suggest that accumulation of adenylated RNA-DNA may contribute to neurological disease. PMID:24362567

  8. In vitro histamine H/sub 2/-antagonist activity of the novel compound HUK 978

    Energy Technology Data Exchange (ETDEWEB)

    Coombes, J.D.; Norris, D.B.; Rising, T.J.; Ross, B.C.; Steward, A.

    1985-11-04

    Histamine stimulated adenylate cyclase from guinea-pig fundic mucosa and /sup 3/H-tiotidine binding in guinea-pig cerebral cortex were used to assess the in-vitro histamine H/sub 2/-activity of the novel H/sub 2/-antagonist HUK 978. The results showed that HUK 978 was a more potent H/sub 2/-antagonist than either cimetidine or ranitidine. HUK 978 was also shown to be devoid of activity at the histamine H-/sub 1/-receptor, the muscarinic receptor and the ..cap alpha.. and ..beta..-adrenergic receptors.

  9. Classification of the adenylation and acyl-transferase activity of NRPS and PKS systems using ensembles of substrate specific hidden Markov models.

    Directory of Open Access Journals (Sweden)

    Barzan I Khayatt

    Full Text Available There is a growing interest in the Non-ribosomal peptide synthetases (NRPSs and polyketide synthases (PKSs of microbes, fungi and plants because they can produce bioactive peptides such as antibiotics. The ability to identify the substrate specificity of the enzyme's adenylation (A and acyl-transferase (AT domains is essential to rationally deduce or engineer new products. We here report on a Hidden Markov Model (HMM-based ensemble method to predict the substrate specificity at high quality. We collected a new reference set of experimentally validated sequences. An initial classification based on alignment and Neighbor Joining was performed in line with most of the previously published prediction methods. We then created and tested single substrate specific HMMs and found that their use improved the correct identification significantly for A as well as for AT domains. A major advantage of the use of HMMs is that it abolishes the dependency on multiple sequence alignment and residue selection that is hampering the alignment-based clustering methods. Using our models we obtained a high prediction quality for the substrate specificity of the A domains similar to two recently published tools that make use of HMMs or Support Vector Machines (NRPSsp and NRPS predictor2, respectively. Moreover, replacement of the single substrate specific HMMs by ensembles of models caused a clear increase in prediction quality. We argue that the superiority of the ensemble over the single model is caused by the way substrate specificity evolves for the studied systems. It is likely that this also holds true for other protein domains. The ensemble predictor has been implemented in a simple web-based tool that is available at http://www.cmbi.ru.nl/NRPS-PKS-substrate-predictor/.

  10. 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.

  11. Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers

    Czech Academy of Sciences Publication Activity Database

    Hasan, Shakir; Kulkarni, N.N.; Asbjarnarson, A.; Linhartová, Irena; Osička, Radim; Šebo, Peter; Gudmundsson, H.

    2018-01-01

    Roč. 86, č. 3 (2018), č. článku e00445-17. ISSN 0019-9567 R&D Projects: GA ČR GA15-09157S; GA MZd(CZ) NV16-28126A; GA MŠk(CZ) LM2015064 Institutional support: RVO:61388971 Keywords : Bordetella pertussis * airway epithelia * CyaA Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 3.593, year: 2016

  12. A Gateway((R)) -compatible bacterial adenylate cyclase-based two-hybrid system

    Czech Academy of Sciences Publication Activity Database

    Ouellette, S. P.; Gauliard, E.; Antošová, Zuzana; Ladant, D.

    2014-01-01

    Roč. 6, č. 3 (2014), s. 259-267 ISSN 1758-2229 Institutional support: RVO:67985823 Keywords : bacterial two-hybrid system * protein–protein interactions * cell division * Gateway((R))(GW) cloning system Subject RIV: EE - Microbiology, Virology Impact factor: 3.293, year: 2014

  13. Channel formation in model membranes by the adenylate cyclase toxin of Bordetella pertussis: Effect if Calcium

    Czech Academy of Sciences Publication Activity Database

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

    2003-01-01

    Roč. 42, - (2003), s. 8077-8084 ISSN 0006-2960 R&D Projects: GA AV ČR IPP1050128 Institutional research plan: CEZ:AV0Z5020903 Keywords : bordetella pertussis * calcium Subject RIV: EE - Microbiology, Virology Impact factor: 3.922, year: 2003

  14. Membrane Restructuring by Bordetella pertussis Adenylate Cyclase Toxin, a Member of the RTX Toxin Family

    Czech Academy of Sciences Publication Activity Database

    Martín, C.; Requero, M. A.; Mašín, Jiří; Konopásek, Ivo; Goni, F. M.; Šebo, Peter; Ostolaza, H.

    2004-01-01

    Roč. 186, č. 12 (2004), s. 3760-3765 ISSN 0021-9193 R&D Projects: GA AV ČR IAA5020907 Grant - others:GA European Union contract(XX) QLK2-1999-0.0556 Keywords : atc * lipid Subject RIV: EE - Microbiology, Virology Impact factor: 4.146, year: 2004

  15. Internation of Bordetella pertussis Adenylate Cyclase with CD11b/CD18

    Czech Academy of Sciences Publication Activity Database

    El-Azami-El-Idrisi, M.; Bauche, C.; Loucká, Jiřina; Osička, Radim; Šebo, Peter; Ladant, D.; Leclerc, C.

    2003-01-01

    Roč. 278, č. 40 (2003), s. 38514-38521 ISSN 0021-9258 R&D Projects: GA AV ČR IPP1050128; GA ČR GA310/01/0934; GA AV ČR IAA5020907 Grant - others:GA by National Institutes of Health Grant(XX) 55000334; GA QLK2-CT-1999(XX) 00556 Institutional research plan: CEZ:AV0Z5020903 Keywords : cyaa * rtx * cd11b Subject RIV: EE - Microbiology, Virology Impact factor: 6.482, year: 2003

  16. Delivery of multiple epitopes by recombinant detoxified adenylate cyclase of Bordetella pertussis induces protective antiviral immunity

    Czech Academy of Sciences Publication Activity Database

    Fayolle, C.; Osičková, Adriana; Osička, Radim; Henry, T.; Rojas, M. J.; Saron, M. F.; Šebo, Peter; Leclers, C.

    2001-01-01

    Roč. 75, č. 16 (2001), s. 7330-7338 ISSN 0022-538X R&D Projects: GA ČR GA310/98/0432; GA MŠk ME 167; GA MŠk VS96149 Institutional research plan: CEZ:AV0Z5020903 Keywords : antiviral immunity * Bordetella pertusis Subject RIV: EE - Microbiology, Virology Impact factor: 5.622, year: 2001

  17. Gustatory Habituation in "Drosophila" Relies on "Rutabaga" (Adenylate Cyclase)-Dependent Plasticity of GABAergic Inhibitory Neurons

    Science.gov (United States)

    Paranjpe, Pushkar; Rodrigues, Veronica; VijayRaghavan, K.; Ramaswami, Mani

    2012-01-01

    In some situations, animals seem to ignore stimuli which in other contexts elicit a robust response. This attenuation in behavior, which enables animals to ignore a familiar, unreinforced stimulus, is called habituation. Despite the ubiquity of this phenomenon, it is generally poorly understood in terms of the underlying neural circuitry. Hungry…

  18. The importance of conserved amino acids in heme-based globin-coupled diguanylate cyclases.

    Directory of Open Access Journals (Sweden)

    Xuehua Wan

    Full Text Available Globin-coupled diguanylate cyclases contain globin, middle, and diguanylate cyclase domains that sense O2 to synthesize c-di-GMP and regulate bacterial motility, biofilm formation, and virulence. However, relatively few studies have extensively examined the roles of individual residues and domains of globin-coupled diguanylate cyclases, which can shed light on their signaling mechanisms and provide drug targets. Here, we report the critical residues of two globin-coupled diguanylate cyclases, EcGReg from Escherichia coli and BpeGReg from Bordetella pertussis, and show that their diguanylate cyclase activity requires an intact globin domain. In the distal heme pocket of the globin domain, residues Phe42, Tyr43, Ala68 (EcGReg/Ser68 (BpeGReg, and Met69 are required to maintain full diguanylate cyclase activity. The highly conserved amino acids His223/His225 and Lys224/Lys226 in the middle domain of EcGReg/BpeGReg are essential to diguanylate cyclase activity. We also identified sixteen important residues (Leu300, Arg306, Asp333, Phe337, Lys338, Asn341, Asp342, Asp350, Leu353, Asp368, Arg372, Gly374, Gly375, Asp376, Glu377, and Phe378 in the active site and inhibitory site of the diguanylate cyclase domain of EcGReg. Moreover, BpeGReg266 (residues 1-266 and BpeGReg296 (residues 1-296, which only contain the globin and middle domains, can inhibit bacterial motility. Our findings suggest that the distal residues of the globin domain affect diguanylate cyclase activity and that BpeGReg may interact with other c-di-GMP-metabolizing proteins to form mixed signaling teams.

  19. Chlorogenic Acid Inhibits Human Platelet Activation and Thrombus Formation

    Science.gov (United States)

    Fuentes, Eduardo; Caballero, Julio; Alarcón, Marcelo; Rojas, Armando; Palomo, Iván

    2014-01-01

    Background Chlorogenic acid is a potent phenolic antioxidant. However, its effect on platelet aggregation, a critical factor in arterial thrombosis, remains unclear. Consequently, chlorogenic acid-action mechanisms in preventing platelet activation and thrombus formation were examined. Methods and Results Chlorogenic acid in a dose-dependent manner (0.1 to 1 mmol/L) inhibited platelet secretion and aggregation induced by ADP, collagen, arachidonic acid and TRAP-6, and diminished platelet firm adhesion/aggregation and platelet-leukocyte interactions under flow conditions. At these concentrations chlorogenic acid significantly decreased platelet inflammatory mediators (sP-selectin, sCD40L, CCL5 and IL-1β) and increased intraplatelet cAMP levels/PKA activation. Interestingly, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent A2A receptor antagonist) attenuated the antiplatelet effect of chlorogenic acid. Chlorogenic acid is compatible to the active site of the adenosine A2A receptor as revealed through molecular modeling. In addition, chlorogenic acid had a significantly lower effect on mouse bleeding time when compared to the same dose of aspirin. Conclusions Antiplatelet and antithrombotic effects of chlorogenic acid are associated with the A2A receptor/adenylate cyclase/cAMP/PKA signaling pathway. PMID:24598787

  20. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    Science.gov (United States)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

    The spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay, is studied. It is found that the degrees of polymerization of the oligopeptides and polypeptides obtained is dependent on the amounts of polypeptides that were preadsorbed. It is concluded that a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances.

  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

    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.

  2. Soluble variants of human recombinant glutaminyl cyclase.

    Directory of Open Access Journals (Sweden)

    Cristiana Castaldo

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

  3. Phosphorolytic activity of Escherichia coli glycyl-tRNA synthetase towards its cognate aminoacyl adenylate detected by 31P-NMR spectroscopy and thin-layer chromatography

    DEFF Research Database (Denmark)

    Led, Jens Jørgen; Switon, Werner K.; Jensen, Kaj Frank

    1983-01-01

    The catalytic activity of highly purified Escherichia coli glycyl-tRNA synthetase has been studied by 31P-NMR spectroscopy and thin-layer chromatography on poly(ethyleneimine)-cellulose. It was found that this synthetase, besides the activation of its cognate amino acid and the syntheses...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-01-01

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

  5. T3SS effector ExoY reduces inflammasome-related responses by suppressing bacterial motility and delaying activation of NF-κB and caspase-1.

    Science.gov (United States)

    Jeon, Jisu; Kim, Yong-Jae; Shin, Heesung; Ha, Un-Hwan

    2017-10-01

    Type III-secreted effectors are essential for modulating host immune responses during the pathogenesis of Pseudomonas aeruginosa infections. Little is known about the impact of one of the effectors, ExoY, on inflammasome activation, which results in IL-1β production and pyroptotic cell death. In this study, we found that transcriptional expression of Il-1β was induced to a lesser extent in response to an exoY-harboring strain than to a deleted mutant. This suppressive effect of ExoY was verified by complementation assay as well as by direct translocation of exoY into host cells. In addition to the production of IL-1β, pyroptotic cell death was also diminished in response to an exoY-harboring strain. These inflammasome responses were mediated by the adenylate cyclase activity of ExoY, which plays a role in delaying the activation of NF-κB and caspase-1, a key component of inflammasome-mediated responses. Moreover, the negative effects of ExoY on these responses were in part conferred by the suppression of bacterial motility, which could reduce the degree of bacterial contact with cells. Together, these results demonstrate that the adenylate cyclase activity of P. aeruginosa ExoY can reduce inflammasome-related responses by influencing both the host and the bacterium itself by delaying the activation of inflammatory pathways and suppressing bacterial motility. © 2017 Federation of European Biochemical Societies.

  6. Cyclic AMP activates the mitogen-activated protein kinase cascade in PC12 cells

    DEFF Research Database (Denmark)

    Frödin, M; Peraldi, P; Van Obberghen, E

    1994-01-01

    Mitogen-activated protein (MAP) kinases are activated in response to a large variety of extracellular signals, including growth factors, hormones, and neurotransmitters, which activate distinct intracellular signaling pathways. Their activation by the cAMP-dependent pathway, however, has not been...... reported. In rat pheochromocytoma PC12 cells, we demonstrate here a stimulation of the MAP kinase isozyme extracellular signal-regulated kinase 1 (ERK1) following elevation of intracellular cAMP after exposure of the cells to isobutylmethylxanthine, cholera toxin, forskolin, or cAMP-analogues. cAMP acted...... synergistically with phorbol ester, an activator of protein kinase C, in the stimulation of ERK1. In accordance with this observation, the peptide neurotransmitter pituitary adenylate cyclase-activating polypeptide 38 (PACAP38), which stimulates cAMP production as well as phosphatidylinositol breakdown in PC12...

  7. Guanylate cyclase activator YC-1 potentiates apoptotic effect of licochalcone A on human epithelial ovarian carcinoma cells via activation of death receptor and mitochondrial pathways.

    Science.gov (United States)

    Lee, Chung Soo; Kwak, Sang Won; Kim, Yun Jeong; Lee, Seon Ae; Park, Eon Sob; Myung, Soon Chul; Kim, Wonyong; Lee, Min Sung; Lee, Jeong Jae

    2012-05-15

    Natural phenol licorice compounds have been shown to induce apoptosis in cancer cells. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) may enhance the sensitivity of cancer cells to anticancer drugs. However, the combined effect of licochalcone A and YC-1 on cell death in ovarian cancer cells has not been studied. We assessed the combined effect of licochalcone A and YC-1 on apoptosis in human epithelial ovarian carcinoma cell lines in relation to the cell death process. In the OVCAR-3 and SK-OV-3 cell lines, licochalocone A induced a decrease in Bid, Bcl-2, Bcl-xL and survivin protein levels; an increase in Bax levels; loss of the mitochondrial transmembrane potential; cytochrome c release; activation of caspases (-8, -9 and -3); cleavage of PARP-1; and an increase in the tumor suppressor p53 levels. YC-1 enhanced licochalcone A-induced apoptosis-related protein activation, nuclear damage and cell death. These results suggest that YC-1 may potentiate the apoptotic effect of licochalcone A on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activation. The combination of licochalcone A and YC-1 may confer a benefit in the treatment of human epithelial ovarian adenocarcinoma. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. [The influence of two-month treatment with bromocryptine on activity of the adenylyl cyclase signaling system in the myocardium and testes of rats with type 2 diabetes mellitus].

    Science.gov (United States)

    Derkach, K V; Bondareva, V M; Moyseyuk, I V; Shpakov, A O

    2014-01-01

    One of the common complications of type 2 diabetes mellitus (DM2) are cardiovascular diseases and dysfunctions of the reproductive system, indicating the urgency of developing new approaches to their correction. Last years for the treatment of DM2 began to use bromocryptine (BC), the agonist of type 2 dopamine receptors, which not only restores the energy metabolism, but also prevents the development of cardiovascular diseases. However, the mechanisms and targets of BC action are poorly understood. The purpose of this study was to investigate the effect of BC treatment on functional activity of adenylyl cyclase signaling system (ACSS) in the myocardium and testes of male rats with DM2, which is caused by high-fat diet and treatment with streptozotocin (25 mg/kg). The treatment with BC (60 days, orally at a dose of 0.6 mg/kg once every two days) was started 90 days after the beginning of high-fat diet. Diabetic rats had an increased body weight, elevated triglycerides level, impaired glucose tolerance, and insulin resistance. The treatment with BC resulted in the restoration of glycometabolic indicators and in the improvement of insulin sensitivity. Adenylyl cyclase (AC) stimulating effects of guanylylimidodiphosphate (GppNHp), relaxin, and agonists of β-adrenergic receptors (β3-AR)--isoproterenol and norepinephrine were decreased in the miocardium of the diabetic rats. The corresponding effects of the β-agonists BRL-37344 and CL-316243 was preserved. The inhibitory effect of somatostatin on forskolin-stimulated AC activity was attenuated, while the inhibitory effect of noradrenaline mediated through α2-AR increased. The treatment with BC resulted in the normalization of the adrenergic signaling in the myocardium and partially restoration of AC effects of relaxin and somatostatin. In the testes of diabetic rats, the basal and stimulated by GppNHp, forskolin, human chorionic gonadotropin and pituitary AC-activating polypeptide AC activity were decreased, and the

  9. Production and Characterization of Monoclonal Antibodies to Soluble Rat Lung Guanylate Cyclase

    Science.gov (United States)

    Brandwein, Harvey; Lewicki, John; Murad, Ferid

    1981-07-01

    Four monoclonal antibodies to rat lung soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2] have been produced by fusing spleen cells from immunized BALB/c mice with SP-2/0 myeloma cells. The antibodies were detected by their ability to bind immobilized guanylate cyclase and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibody. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid containing 2-5 mg of antibody per ml. The four antibodies obtained had titers of between 1:1580 and 1:3160 but were detectable at dilutions greater than 1:20,000. Soluble guanylate cyclase from several rat tissues were crossreactive with the four monoclonal antibodies, suggesting that the soluble enzyme from different rat tissues is antigenically similar. The antibodies also recognized soluble lung enzyme from rat, beef, and pig, while enzyme from rabbit was not crossreactive and mouse enzyme was recognized by only one of the antibodies. Particulate guanylate cyclase from a number of tissues had only minimal crossreactivity with the antibodies. Immunoprecipitated guanylate cyclase retained catalytic activity, could be activated with sodium nitroprusside, and was inhibited by cystamine. None of the antibodies were inhibitory under the conditions examined. These antibodies will be useful probes for the study of guanylate cyclase regulation and function under a variety of physiological conditions.

  10. Different effects of adenylyl cyclase activators and phosphodiesterases inhibitors on cervical cancer (HeLa) and breast cancer (MCF-7) cells proliferation.

    Science.gov (United States)

    Mahdian, Davood; Shafiee-Nick, Reza; Mousavi, Seyed Hadi

    2014-05-01

    Breast and cervical cancers are the most common cancers in Iran and worldwide. Hormonal stimulation of cyclic adenosine mono phosphate (cAMP) and the cAMP-dependent protein kinase PKA regulates cell growth by different mechanism. cAMP can stimulate cell growth in many cell types while inhibiting cell growth in others. In some cell lines have been shown that the proliferation of tumor cells is reduced by increasing cAMP in cells. In this study, we evaluate growth arrest of selective PDE3 and non-selective PDE inhibitors, which lead to increase level of cAMP in cervical (HeLa) and breast cancer (MCF7) cell lines have been studied. Cells were incubated with different concentrations of selective, non-selective PDE inhibitors, beta adrenergic receptor agonist and direct stimulator of adenylyl cyclase. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). Result showed that selective PDE inhibitors decreased cell viability in HeLa and MCF-7 cells as a time-dependent manner. Non-selective inhibitor and beta-adrenergic receptor agonist also decrease cell viability but they are less powerful than selective PDE3 inhibitors. Forskolin had no effect in viability of cells. Analysis of DNA fragmentation by flow cytometry showed apoptosis involved in selective PDE3 inhibitors induced toxicity in HeLa cell. Thus, the growth inhibitory effects of selective PDE3 inhibitors are more effective than non-selective inhibitor. Further studies are needed to investigate the mechanism of action is on the field.

  11. 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

  12. 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.

  13. Dephosphorylation of sperm guanylate cyclase during sea urchin fertilization

    Energy Technology Data Exchange (ETDEWEB)

    Ward, G.E.

    1985-01-01

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

  14. Role of MbtH-like Proteins in the Adenylation of Tyrosine during Aminocoumarin and Vancomycin Biosynthesis*

    Science.gov (United States)

    Boll, Björn; Taubitz, Tatjana; Heide, Lutz

    2011-01-01

    MbtH-like proteins consist of ∼70 amino acids and are encoded in the biosynthetic gene clusters of non-ribosomally formed peptides and other secondary metabolites derived from amino acids. Recently, several MbtH-like proteins have been shown to be required for the adenylation of amino acid in non-ribosomal peptide synthesis. We now investigated the role of MbtH-like proteins in the biosynthesis of the aminocoumarin antibiotics novobiocin, clorobiocin, and simocyclinone D8 and of the glycopeptide antibiotic vancomycin. The tyrosine-adenylating enzymes CloH, SimH, and Pcza361.18, involved in the biosynthesis of clorobiocin, simocyclinone D8, and vancomycin, respectively, required the presence of MbtH-like proteins in a 1:1 molar ratio, forming heterotetrameric complexes. In contrast, NovH, involved in novobiocin biosynthesis, showed activity in the absence of MbtH-like proteins. Comparison of the active centers of CloH and NovH showed only one amino acid to be different, i.e. Leu-383 versus Met-383. Mutation of this amino acid in CloH (L383M) indeed led to MbtH-independent adenylating activity. All investigated tyrosine-adenylating enzymes exhibited remarkable promiscuity for MbtH-like proteins from different pathways and organisms. YbdZ, the MbtH-like protein from the expression host Escherichia coli, was found to bind to adenylating enzymes during expression and to influence their biochemical properties markedly. Therefore, the use of ybdZ-deficient expression hosts is important in biochemical studies of adenylating enzymes. PMID:21890635

  15. 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.

  16. Soluble adenylyl cyclase in health and disease

    OpenAIRE

    Schmid, Andreas; Meili, Dimirela; Salathe, Matthias

    2014-01-01

    The second messenger cAMP is integral for many physiological processes. Soluble adenylyl cyclase (sAC) was recently identified as a widely expressed intracellular source of cAMP in mammalian cells. sAC is evolutionary, structurally, and biochemically distinct from the G-protein-responsive transmembranous adenylyl cyclases (tmAC). The structure of the catalytic unit of sAC is similar to tmAC, but sAC does not contain transmembranous domains, allowing localizations independent of the membranous...

  17. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Estrada, Paola; Manandhar, Miglena; Dong, Shi-Hui; Deveryshetty, Jaigeeth; Agarwal, Vinayak; Cronan, John E.; Nair, Satish K.

    2017-04-17

    Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

  18. Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera

    Directory of Open Access Journals (Sweden)

    Rouhiainen Leo

    2008-09-01

    Full Text Available Abstract Background Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (mcy. mcy genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer. Results A comparative analysis of the adenylation domains from the first module of McyB (McyB1 and McyC in the microcystin synthetase complex was performed on a large number of microcystin-producing strains from the Anabaena, Microcystis and Planktothrix genera. We found no decisive evidence for recombination between strains from different genera. However, we detected frequent recombination events in the mcyB and mcyC genes between strains within the same genus. Frequent interdomain recombination events were also observed between mcyB and mcyC sequences in Anabaena and Microcystis. Recombination and mutation rate ratios suggest that the diversification of mcyB and mcyC genes is driven by recombination events as well as point mutations in all three genera. Sequence analysis suggests that generally the adenylation domains of the first domain of McyB and McyC are under purifying selection. However, we found clear evidence for positive selection acting on a number of amino acid residues within these adenylation domains. These include residues important for active site selectivity of the adenylation domain, strongly suggesting selection for novel microcystin variants. Conclusion We provide the first clear evidence for positive selection acting on amino acid residues involved directly in the recognition and activation of amino acids incorporated into microcystin, indicating that the microcystin complement of a given strain may

  19. Neuronal Expression of Soluble Adenylyl Cyclase in the Mammalian Brain

    OpenAIRE

    Chen, Jonathan; Martinez, Jennifer; Milner, Teresa A.; Buck, Jochen; Levin, Lonny R.

    2013-01-01

    Cyclic 3’, 5’-adenosine monophosphate (cAMP) is a critical and ubiquitous second messenger involved in a multitude of signaling pathways. Soluble adenylyl cyclase (sAC) is a novel source of cAMP subject to unique localization and regulation. It was originally discovered in mammalian testis and found to be activated by bicarbonate and calcium. sAC has been implicated in diverse processes, including astrocyte-neuron metabolic coupling and axonal outgrowth of neurons. However, despite these func...

  20. Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    Science.gov (United States)

    Cámara, María de los Milagros; Bouvier, León A.; Canepa, Gaspar E.; Miranda, Mariana R.; Pereira, Claudio A.

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3′ UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes. PMID:23409202

  1. Adenylate kinase locus 1 polymorphism and feto-placental development.

    Science.gov (United States)

    Fulvia, Gloria-Bottini; Antonio, Pietroiusti; Anna, Neri; Patrizia, Saccucci; Ada, Amante; Egidio, Bottini; Andrea, Magrini

    2011-12-01

    Recently our group has found that the correlation between birth weight and placental weight - an index of a balanced feto-placental unit development - is influenced by genetic factors. Since adenylate kinase locus 1 (AK₁) is a polymorphic enzyme that plays an important role in the synthesis of nucleotides required for many metabolic functions, we have investigated the possible role of its genetic variability in the correlation between birth weight and placental weight. 342 consecutive healthy newborn infants from the population of Rome (Italy) and 286 puerperae from another population from Central Italy were studied. The correlation coefficient between birth weight and placental weight is much higher in infants with low activity AK₁2-1 phenotype than in those with high activity AK₁1 phenotype. The difference between AK₁ and AK₁2-1 is well marked only in newborns with a gestational age greater than 38 weeks and it is not influenced by sex, maternal age and maternal smoking. A similar pattern is observed with maternal AK₁ phenotype. These results suggest that the difference in enzymatic activity between AK₁ phenotypes influencing the equilibrium among ATP, ADP, AMP and adenosine could have an important role in a balanced development of feto-placental unit. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  2. Established and potential physiological roles of bicarbonatesensing soluble adenylyl cyclase (sAC) in aquatic animals

    OpenAIRE

    Tresguerres, M; Barott, KL; Barron, ME; Roa, JN

    2014-01-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO 3 - , and sAC has been confirmed to be a HCO 3 - sensor in a variety of mammalian cell types. In addition...

  3. An unusual plant triterpene synthase with predominant α-amyrin-producing activity identified by characterizing oxidosqualene cyclases from Malus × domestica.

    Science.gov (United States)

    Brendolise, Cyril; Yauk, Yar-Khing; Eberhard, Ellen D; Wang, Mindy; Chagne, David; Andre, Christelle; Greenwood, David R; Beuning, Lesley L

    2011-07-01

    The pentacyclic triterpenes, in particular ursolic acid and oleanolic acid and their derivatives, exist abundantly in the plant kingdom, where they are well known for their anti-inflammatory, antitumour and antimicrobial properties. α-Amyrin and β-amyrin are the precursors of ursolic and oleanolic acids, respectively, formed by concerted cyclization of squalene epoxide by a complex synthase reaction. We identified three full-length expressed sequence tag sequences in cDNA libraries constructed from apple (Malus × domestica 'Royal Gala') that were likely to encode triterpene synthases. Two of these expressed sequence tag sequences were essentially identical (> 99% amino acid similarity; MdOSC1 and MdOSC3). MdOSC1 and MdOSC2 were expressed by transient expression in Nicotiana benthamiana leaves and by expression in the yeast Pichia methanolica. The resulting products were analysed by GC and GC-MS. MdOSC1 was shown to be a mixed amyrin synthase (a 5 : 1 ratio of α-amyrin to β-amyrin). MdOSC1 is the only triterpene synthase so far identified in which the level of α-amyrin produced is > 80% of the total product and is, therefore, primarily an α-amyrin synthase. No product was evident for MdOSC2 when expressed either transiently or in yeast, suggesting that this putative triterpene synthase is either encoded by a pseudogene or does not express well in these systems. Transcript expression analysis in Royal Gala indicated that the genes are mostly expressed in apple peel, and that the MdOSC2 expression level was much lower than that of MdOSC1 and MdOSC3 in all the tissues tested. Amyrin content analysis was undertaken by LC-MS, and demonstrated that levels and ratios differ between tissues, but that the true consequence of synthase activity is reflected in the ursolic/oleanolic acid content and in further triterpenoids derived from them. Phylogenetic analysis placed the three triterpene synthase sequences with other triterpene synthases that encoded either

  4. Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli

    International Nuclear Information System (INIS)

    Kumar, S.

    1976-01-01

    Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi 80 cya + and phi 80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants

  5. Efficient Ex Vivo Stimulation of Mycobacterium tuberculosis-Specific T Cells by Genetically Detoxified Bordetella pertussis Adenylate Cyclase Antigen Toxodids

    Czech Academy of Sciences Publication Activity Database

    Wilkinson, K. A.; Šimšová, Marcela; Schölvinck, E.; Šebo, Peter; Leclerc, C.; Voredermeier, H. M.; Dickson, S. J.; Brown, J. R.; Davidson, R. N.; Pasvol, G.; Levin, M.; Wilkinson, R. J.

    2005-01-01

    Roč. 73, č. 5 (2005), s. 2991-2998 ISSN 0019-9567 R&D Projects: GA AV ČR IBS5020311; GA ČR GA310/01/0934 Institutional research plan: CEZ:AV0Z50200510 Keywords : mycobacterium tuberculosis * t cell * CyaA Subject RIV: EE - Microbiology, Virology Impact factor: 3.933, year: 2005

  6. Invasion of Dendritic Cells, Macrophages and Neutrophils by the Bordetella Adenylate Cyclase Toxin: A Subversive Move to Fool Host Immunity

    Czech Academy of Sciences Publication Activity Database

    Fedele, G.; Schiavoni, O.; Adkins, I.; Klímová, Nela; Šebo, Peter

    2017-01-01

    Roč. 9, č. 10 (2017), s. 1-15, č. článku 293. E-ISSN 2072-6651 R&D Projects: GA MZd(CZ) NV16-28126A; GA ČR(CZ) GA13-14547S; GA MŠk(CZ) LM2015064 Institutional support: RVO:61388971 Keywords : immune response * intracellular pathways * phagocytosis Subject RIV: EA - Cell Biology OBOR OECD: Other biological topics Impact factor: 3.030, year: 2016

  7. The conserved tyrosine residue 940 plays a key structural role in membrane interaction of Bordetella adenylate cyclase toxin

    Czech Academy of Sciences Publication Activity Database

    Mašín, Jiří; Roderová, Jana; Osičková, Adriana; Novák, Petr; Bumba, Ladislav; Fišer, Radovan; Šebo, Peter; Osička, Radim

    2017-01-01

    Roč. 7, AUG 24 (2017), s. 1-14, č. článku 9330. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA16-05919S; GA ČR GA15-09157S; GA ČR(CZ) GA15-11851S; GA ČR(CZ) GA13-14547S; GA MŠk(CZ) LM2015064; GA MŠk(CZ) LD15089; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : PERTUSSIS CYAA * BENZODIAZEPINE-RECEPTOR * ESCHERICHIA-COLI Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.259, year: 2016

  8. The adenylate cyclase toxin from Bordetella pertussis - a novel promising vehicke fer antigen delivery to dendritic cells

    Czech Academy of Sciences Publication Activity Database

    Šimšová, Marcela; Šebo, Peter; Leclerc, C.

    2004-01-01

    Roč. 293, - (2004), s. 571-576 ISSN 1438-4221 R&D Projects: GA ČR GA310/01/0934; GA AV ČR IAA5020907 Grant - others:GA QLK2-CT-1999(XX) 00556 Keywords : cyaa * cellular immune response * antigen delivery Subject RIV: EE - Microbiology, Virology Impact factor: 2.919, year: 2004

  9. Mass spectrometric analysis of recombinant adenylate cyclase toxin from Bordetella pertussis strain 18323/pHSP9+

    Czech Academy of Sciences Publication Activity Database

    Havlíček, Vladimír; Higgins, L.; Chen, W.; Halada, Petr; Šebo, Peter; Sakamoto, H.; Hackett, M.

    2001-01-01

    Roč. 36, - (2001), s. 384-391 ISSN 1076-5174 R&D Projects: GA AV ČR IAA5020907; GA MŠk ME 167; GA MŠk VS96141 Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EE - Microbiology, Virology Impact factor: 2.685, year: 2001

  10. Bordetella adenylate cyclase toxin induces a cascade of morphological changes of sheep erythrocytes and localizes into clusters in erythrocyte membranes

    Czech Academy of Sciences Publication Activity Database

    Vojtová, Jana; Kofroňová, Olga; Šebo, Peter; Benada, Oldřich

    2006-01-01

    Roč. 69, - (2006), s. 119-129 ISSN 1059-910X R&D Projects: GA AV ČR IAA5020406 Institutional research plan: CEZ:AV0Z50200510 Keywords : cyaa * scanning electron microscopy * transmission electron microscopy Subject RIV: EE - Microbiology, Virology Impact factor: 1.680, year: 2006

  11. The central regulation of plant physiology by adenylates.

    Science.gov (United States)

    Geigenberger, Peter; Riewe, David; Fernie, Alisdair R

    2010-02-01

    There have been many recent developments concerning the metabolic, transport and signalling functions of adenylates in plants, suggesting new roles for these compounds as central regulators of plant physiology. For example, altering the expression levels of enzymes involved in the equilibration, salvaging, synthesis and transport of adenylates leads to perturbations in storage, growth and stress responses, implying a role for adenylates as important signals. Furthermore, sensing of the internal energy status involves SNF1-related kinases, which control the expression and phosphorylation of key metabolic enzymes. ATP also acts as an apoplastic signalling molecule to control cell growth and pathogen responses. These new results could shed light on the emerging question of whether energy homeostasis in plant cells differs from mechanisms found in microbes and mammals. Copyright 2009 Elsevier Ltd. All rights reserved.

  12. Adenosine induces vasoconstriction through Gi-dependent activation of phospholipase C in isolated perfused afferent arterioles of mice

    DEFF Research Database (Denmark)

    Hansen, Pernille B; Castrop, Hayo; Briggs, Josie

    2003-01-01

    Adenosine induces vasoconstriction of renal afferent arterioles through activation of A1 adenosine receptors (A1AR). A1AR are directly coupled to Gi/Go, resulting in inhibition of adenylate cyclase, but the contribution of this signaling pathway to smooth muscle cell activation is unclear......)-cyclohexyladenosine. PTX pretreatment did not affect the constriction response to KCl, whereas the angiotensin II dose-response relationship was shifted rightward. Reverse transcription-PCR revealed expression of Gi but not Go in kidney cortex and preglomerular vessels. The phospholipase C inhibitor U73122 (4 micro M...... that the constriction response to adenosine in afferent arterioles is mediated by A1AR coupled to a PTX-sensitive Gi protein and subsequent activation of phospholipase C, presumably through betagamma subunits released from Galphai....

  13. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

    Science.gov (United States)

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-07-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages.

  14. 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

  15. Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

    Science.gov (United States)

    Kugel, Susann; Baunach, Martin; Baer, Philipp; Ishida-Ito, Mie; Sundaram, Srividhya; Xu, Zhongli; Groll, Michael; Hertweck, Christian

    2017-06-01

    Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

  16. Pharmacological profile of the abeorphine 201-678, a potent orally active and long lasting dopamine agonist

    Energy Technology Data Exchange (ETDEWEB)

    Jaton, A.L.; Giger, R.K.A.; Vigouret, J.M.; Enz, A.; Frick, W.; Closse, A.; Markstein, R.

    1986-01-13

    The central dopaminergic effects of an abeorphine derivative 201-678 were compared to those of apomorphine and bromocriptine in different model systems. After oral administration, this compound induced contralateral turning in rats with 6-hydroxydopamine induced nigral lesions and exhibited strong anti-akinetic properties in rats with 6-hydroxydopamine induced hypothalamic lesions. It decreased dopamine metabolism in striatum and cortex, but did not modify noradrenaline and serotonin metabolism in the rat brain. 201-678 counteracted the in vivo increase of tyrosine hydroxylase activity induced by ..gamma..-butyrolactone. In vitro it stimulated DA-sensitive adenylate cyclase and inhibited acetylcholine release from rat striatal slices. This compound had high affinity for /sup 3/H-dopamine and /sup 3/H-clonidine binding sites. These results indicate that 201-678 is a potent, orally active dopamine agonist with a long duration of action. Furthermore it appears more selective than other dopaminergic drugs. 29 references, 5 figures, 3 tables.

  17. Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus, Prevents Platelet Activation in Human Platelets

    Directory of Open Access Journals (Sweden)

    Ye-Ming Lee

    2012-01-01

    Full Text Available Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulus L.. Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]i mobilization, thromboxane A2 formation, hydroxyl radical (OH● formation, and phospholipase C (PLCγ2, protein kinase C (PKC, mitogen-activated protein kinase (MAPK, and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2 formation, thereby leading to inhibition of [Ca2+]i and finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

  18. Comparative analysis of plant lycopene cyclases.

    Science.gov (United States)

    Koc, Ibrahim; Filiz, Ertugrul; Tombuloglu, Huseyin

    2015-10-01

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

  19. Regulation of hormone-receptor coupling to adenylyl cyclase. Effects of GTP and GDP.

    Science.gov (United States)

    Iyengar, R; Abramowitz, J; Bordelon-Riser, M; Blume, A J; Birnbaumer, L

    1980-11-10

    GDP and GTP regulation of receptor-mediated stimulation of adenylyl cyclases in membranes of S49 murine lymphoma cells (S49), NS-20 murine neuroblastoma cells (NS-20), rabbit corpora lutea (CL), and turkey erythrocytes were studied under assay conditions which minimized conversion of added GTP to GDP and of added GDP to GTP. Hormonal stimulation in all systems required guanine nucleotide addition. In the presence of GTP, adenylyl cyclase activity in S49, NS-20, and CL was stimulated respectively by isoproterenol and prostaglandin E1 (PGE1), by PGE1 and the adenosine analog, phenylisopropyladenosine, and by PGE1 and isoproterenol, with the first of the listed stimulants eliciting higher activities than the second. Activity in turkey erythrocyte membranes was stimulated by isoproterenol. GDP was partially effective in promoting hormonal stimulation, being able to sustain stimulation by isoproterenol and PGE1 in S49 cell membranes and by PGE1 in CL membranes. In NS-20 membranes, both GDP and guanosine-5'-O-(2-thiodiphosphate) (GDP beta S) were inhibitory on basal activity, yet promoted limited but significant stimulation by PGE1. In turkey erythrocytes, stimulation by isoproterenol could not be elicited with GDP or GDP beta S. Thus, although less effective than GTP in promoting hormonal stimulation of several adenylyl cyclase systems, GDP was clearly not inactive. Concentration effect curves for active hormone in the presence of GDP had higher apparent Ka values than in the presence of GTP. In spite of differences between the effects of GTP and GDP on hormonal stimulation of adenylyl cyclase activities, GTP and GDP affected equally well isoproterenol binding, regardless of whether or not its receptor could be shown to stimulate adenylyl cyclase in the presence of GDP. Determination of transphosphorylation of GDP to GTP showed that at saturating concentrations, the proportion of GDP converted to GTP is negligible and unaffected by hormonal stimulation. Concentrations

  20. 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...

  1. Evolution-guided adaptation of an adenylation domain substrate specificity to an unusual amino acid.

    Directory of Open Access Journals (Sweden)

    Simon Vobruba

    Full Text Available Adenylation domains CcbC and LmbC control the specific incorporation of amino acid precursors in the biosynthesis of lincosamide antibiotics celesticetin and lincomycin. Both proteins originate from a common L-proline-specific ancestor, but LmbC was evolutionary adapted to use an unusual substrate, (2S,4R-4-propyl-proline (PPL. Using site-directed mutagenesis of the LmbC substrate binding pocket and an ATP-[32P]PPi exchange assay, three residues, G308, A207 and L246, were identified as crucial for the PPL activation, presumably forming together a channel of a proper size, shape and hydrophobicity to accommodate the propyl side chain of PPL. Subsequently, we experimentally simulated the molecular evolution leading from L-proline-specific substrate binding pocket to the PPL-specific LmbC. The mere change of three amino acid residues in originally strictly L-proline-specific CcbC switched its substrate specificity to prefer PPL and even synthetic alkyl-L-proline derivatives with prolonged side chain. This is the first time that such a comparative study provided an evidence of the evolutionary relevant adaptation of the adenylation domain substrate binding pocket to a new sterically different substrate by a few point mutations. The herein experimentally simulated rearrangement of the substrate binding pocket seems to be the general principle of the de novo genesis of adenylation domains' unusual substrate specificities. However, to keep the overall natural catalytic efficiency of the enzyme, a more comprehensive rearrangement of the whole protein would probably be employed within natural evolution process.

  2. 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

  3. Cholera toxin, a potent inducer of epidermal hyperplasia but with no tumor promoting activity in mouse skin carcinogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Kuroki, T.; Chida, K.; Munakata, K.; Murakami, Y.

    1986-05-29

    Intracutaneous injection of cholera toxin into mice induced epidermal hyperplasia to a greater extent than 12-O-tetra-decanoylphorbol-13-acetate. It also induced adenylate cyclase and through weakly, ornithine decarboxylase of the epidermis. Cholera toxin, however, showed no tumor promoting activity in mouse skin carcinogenesis. In the single stage promotion, cholera toxin (50 ng) was injected once a week for 10 weeks into the skin of SENCAR mice initiated with 25 ..mu..g 7,12-dimethyl-benz(a)anthracene, but no tumors developed. In the two-stage promotion test, cholera toxin (10-100 ng) was injected for one or two weeks into the initiated skin and then mezerein (4 ..mu..g) was applied twice a week for 18 weeks, but the toxin did not increase incidence or numbers of papillomas.

  4. The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    Full Text Available In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA, Adenylate cyclase and Pth11 (a non-canonical GPCR in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

  5. The effect of adenyl compounds on the rat heart.

    OpenAIRE

    Burnstock, G.; Meghji, P.

    1983-01-01

    1 The effects of adenyl compounds were examined on the rat atrium and ventricle. 2 Adenosine, adenosine 5'-monophosphate, adenosine 5'-diphosphate, adenosine 5'-triphosphate (ATP) and beta, gamma-methylene ATP (APPCP) produced negative inotropic effects on the rat atrium. These inhibitory effects were antagonized by 8-phenyltheophylline (8-PT), a P1-purinoceptor antagonist, and potentiated by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), an adenosine deaminase inhibitor, but were not affected...

  6. 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

  7. Bradykinin-activated transmembrane signals are coupled via N/sub o/ or N/sub i/ to production of inositol 1,4,5-trisphosphate, a second messenger in NG108-15 neuroblastoma-glioma hybrid cells

    International Nuclear Information System (INIS)

    Higashida, H.; Streaty, R.A.; Klee, W.; Nirenberg, M.

    1986-01-01

    The addition of bradykinin to NG108-15 cells results in a transient hyperpolarization followed by prolonged cell depolarization. Injection of inositol 1,4,5-trisphosphate or Ca 2+ into the cytoplasm of NG108-15 cells also elicits cell hyperpolarization followed by depolarization. Tetraethylammonium ions inhibit the hyperpolarizing response of cells to bradykinin or inositol 1,4,5-trisphosphate. Thus, the hyperpolarizing phase of the cell response may be due to inositol 1,4,5-trisphosphate-dependent release of stored 45 Ca-labelled Ca 2+ into the cytoplasm, which activates Ca 2+ -dependent K + channels. The depolarizing phase of the cell response to bradykinin is due largely to inhibition of M channels, thereby decreasing the rate of K + efflux from cells and, to a lesser extent, to activation of Ca 2+ -dependent ion channels and Ca 2+ channels. In contrast, injection of inositol 1,4,5-trisphosphate or Ca 2+ into the cytosol did not alter M channel activity. Incubation of NG108-15 cells with pertussis toxin inhibits bradykinin-dependent cell hyperpolarization and depolarization. Bradykinin stimulates low K/sub m/ GTPase activity and inhibits adenylate cyclase in NG108-15 membrane preparations but not in membranes prepared from cells treated with pertussis toxin. These results show that [bradykinin-receptor] complexes interact with N/sub o/ or N/sub i/ and suggest that N/sub o/ and/or N/sub i/ mediate the transduction of signals from bradykinin receptors to phospholipase C and adenylate cyclase

  8. Neuromodulatory effect of Gαs- or Gαq-coupled G-protein-coupled receptor on NMDA receptor selectively activates the NMDA receptor/Ca2+/calcineurin/cAMP response element-binding protein-regulated transcriptional coactivator 1 pathway to effectively induce brain-derived neurotrophic factor expression in neurons.

    Science.gov (United States)

    Fukuchi, Mamoru; Tabuchi, Akiko; Kuwana, Yuki; Watanabe, Shinjiro; Inoue, Minami; Takasaki, Ichiro; Izumi, Hironori; Tanaka, Ayumi; Inoue, Ran; Mori, Hisashi; Komatsu, Hidetoshi; Takemori, Hiroshi; Okuno, Hiroyuki; Bito, Haruhiko; Tsuda, Masaaki

    2015-04-08

    Although coordinated molecular signaling through excitatory and modulatory neurotransmissions is critical for the induction of immediate early genes (IEGs), which lead to effective changes in synaptic plasticity, the intracellular mechanisms responsible remain obscure. Here we measured the expression of IEGs and used bioluminescence imaging to visualize the expression of Bdnf when GPCRs, major neuromodulator receptors, were stimulated. Stimulation of pituitary adenylate cyclase-activating polypeptide (PACAP)-specific receptor (PAC1), a Gαs/q-protein-coupled GPCR, with PACAP selectively activated the calcineurin (CN) pathway that is controlled by calcium signals evoked via NMDAR. This signaling pathway then induced the expression of Bdnf and CN-dependent IEGs through the nuclear translocation of CREB-regulated transcriptional coactivator 1 (CRTC1). Intracerebroventricular injection of PACAP and intraperitoneal administration of MK801 in mice demonstrated that functional interactions between PAC1 and NMDAR induced the expression of Bdnf in the brain. Coactivation of NMDAR and PAC1 synergistically induced the expression of Bdnf attributable to selective activation of the CN pathway. This CN pathway-controlled expression of Bdnf was also induced by stimulating other Gαs- or Gαq-coupled GPCRs, such as dopamine D1, adrenaline β, CRF, and neurotensin receptors, either with their cognate agonists or by direct stimulation of the protein kinase A (PKA)/PKC pathway with chemical activators. Thus, the GPCR-induced expression of IEGs in coordination with NMDAR might occur via the selective activation of the CN/CRTC1/CREB pathway under simultaneous excitatory and modulatory synaptic transmissions in neurons if either the Gαs/adenylate cyclase/PKA or Gαq/PLC/PKC-mediated pathway is activated. Copyright © 2015 the authors 0270-6474/15/355606-19$15.00/0.

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

    International Nuclear Information System (INIS)

    Kolenko, Petr; Koch, Birgit; Rahfeld, Jens-Ulrich; Schilling, Stephan; Demuth, Hans-Ulrich; Stubbs, Milton T.

    2013-01-01

    The structure of ligand-free glutaminyl cyclase from D. melanogaster has been determined in a novel crystal form belonging to space group I4. The structure of ligand-free glutaminyl cyclase (QC) from Drosophila melanogaster (DmQC) has been determined in a novel crystal form. The protein crystallized in space group I4, with unit-cell parameters a = b = 122.3, c = 72.7 Å. The crystal diffracted to a resolution of 2 Å at the home source. The structure was solved by molecular replacement and was refined to an R factor of 0.169. DmQC exhibits a typical α/β-hydrolase fold. The electron density of three monosaccharides could be localized. The accessibility of the active site will facilitate structural studies of novel inhibitor-binding modes

  10. Substrate specificity determinants of class III nucleotidyl cyclases.

    Science.gov (United States)

    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.

  11. 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

  12. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    Science.gov (United States)

    Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

    2013-01-01

    Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

  13. Energetics and Structural Characterization of the large-scale Functional Motion of Adenylate Kinase

    Science.gov (United States)

    Formoso, Elena; Limongelli, Vittorio; Parrinello, Michele

    2015-02-01

    Adenylate Kinase (AK) is a signal transducing protein that regulates cellular energy homeostasis balancing between different conformations. An alteration of its activity can lead to severe pathologies such as heart failure, cancer and neurodegenerative diseases. A comprehensive elucidation of the large-scale conformational motions that rule the functional mechanism of this enzyme is of great value to guide rationally the development of new medications. Here using a metadynamics-based computational protocol we elucidate the thermodynamics and structural properties underlying the AK functional transitions. The free energy estimation of the conformational motions of the enzyme allows characterizing the sequence of events that regulate its action. We reveal the atomistic details of the most relevant enzyme states, identifying residues such as Arg119 and Lys13, which play a key role during the conformational transitions and represent druggable spots to design enzyme inhibitors. Our study offers tools that open new areas of investigation on large-scale motion in proteins.

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

    Science.gov (United States)

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

    2014-10-01

    RNA 3'-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3'-phosphate to form a 2',3'-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA-AMP and RNA(3')pp(5')A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3'-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3'-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3'-phosphate is poised for in-line attack on the P-N bond that links the phosphorous atom of AMP to N(ε) of His307. Thus, we provide the first insights into RNA 3'-phosphate termini recognition and the mechanism of 3'-phosphate activation by an Rtc enzyme. © 2014 Desai et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  15. 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.

  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. Pereskia aculeata Muller (Cactaceae Leaves: Chemical Composition and Biological Activities

    Directory of Open Access Journals (Sweden)

    Lucèia Fàtima Souza

    2016-09-01

    Full Text Available The aims of this work were to study the chemical composition of the essential oil from the leaves of Pereskia aculeata and to evaluate some biological activities of three leaf extracts. The phenolic content, antioxidant activity, and in vitro antimicrobial and antifungal activities were determined. The methanol extract showed antioxidant activity (EC50 7.09 mg/mL and high polyphenols content (15.04 ± 0.31 mg gallic acid equivalents (GAE/g. The petroleum ether extract exhibited potent antibacterial activity against Escherichia coli, whereas the chloroform extract showed inhibitory activity against Bacillus cereus and Staphylococcus aureus. The petroleum ether and methanol extracts were more effective in inhibiting the growth of Aspergillus versicolor. The possible cytotoxicity of extracts on neuroblastoma SH-SY5Y cancer cell line and the influence on adenylate cyclase (ADCY expression was also studied. P. aculeata chloroform extract showed antiproliferative activity with an IC50 value of 262.83 µg/mL. Treatments of SH-SY5Y neuroblastoma cells with 100 µg/mL of methanol extract significantly reduced ADCY1 expression.

  18. Soluble guanylate cyclase : a potential therapeutic target for heart failure

    NARCIS (Netherlands)

    Gheorghiade, Mihai; Marti, Catherine N.; Sabbah, Hani N.; Roessig, Lothar; Greene, Stephen J.; Boehm, Michael; Burnett, John C.; Campia, Umberto; Cleland, John G. F.; Collins, Sean P.; Fonarow, Gregg C.; Levy, Phillip D.; Metra, Marco; Pitt, Bertram; Ponikowski, Piotr; Sato, Naoki; Voors, Adriaan A.; Stasch, Johannes-Peter; Butler, Javed

    The number of annual hospitalizations for heart failure (HF) and the mortality rates among patients hospitalized for HF remains unacceptably high. The search continues for safe and effective agents that improve outcomes when added to standard therapy. The nitric oxide (NO)-soluble guanylate cyclase

  19. 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

  20. A beta-lysine adenylating enzyme and a beta-lysine binding protein involved in poly beta-lysine chain assembly in nourseothricin synthesis in Streptomyces noursei.

    Science.gov (United States)

    Grammel, Nicolas; Pankevych, Kvitka; Demydchuk, Julia; Lambrecht, Klaus; Saluz, Hans-Peter; Krügel, Hans

    2002-01-01

    Nourseothricins (syn. Streptothricins), a group of nucleoside peptides produced by several streptomycete strains, contain a poly beta-lysine chain of variable length attached in amide linkage to the amino sugar moiety gulosamine of the nucleoside portion. We show that the nourseothricin-producing Streptomyces noursei contains an enzyme (NpsA) of an apparent M(r) 56,000 that specifically activates beta-lysine by adenylation but does not bind to it as a thioester. Cloning and sequencing of npsA from S. noursei including its flanking DNA regions revealed that it is closely linked to the nourseothricin resistance gene nat1 and some other genes on the chromosome possibly involved in nourseothricin biosynthesis. The deduced amino-acid sequence revealed that NpsA is a stand-alone adenylation domain with similarity to the adenylation domains of nonribosomal peptide synthetases (NRPS). Further analysis revealed that S. noursei contains a beta-lysine binding enzyme (NpsB) of about M(r) 64,100 which can be loaded by NpsA with beta-lysine as a thioester. Analysis of the deduced amino-acid sequence from the gene (npsB) of NpsB showed that it consists of two domains. The N-terminal domain of approximately 100 amino-acid residues has high similarity to PCP domains of NRPSs whereas the 450-amino-acid C-terminal domain has a high similarity to epimerization (E)-domains of NRPSs. Remarkably, in this E-domain the conserved H-H-motif is changed to H-Q, which suggests that either the domain is nonfunctional or has a specialized function. The presence of one single adenylating beta-lysine activating enzyme in nourseothricin-producing streptomycete and a separate binding protein suggests an iteratively operating NRPS-module catalyses synthesis of the poly beta-lysine chain.

  1. Optimization of ATP synthase function in mitochondria and chloroplasts via the adenylate kinase equilibrium

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

    Full Text Available The bulk of ATP synthesis in plants is performed by ATP synthase, the main bioenergetics engine of cells, operating both in mitochondria and in chloroplasts. The reaction mechanism of ATP synthase has been studied in detail for over half a century; however, its optimal performance depends also on the steady delivery of ATP synthase substrates and the removal of its products. For mitochondrial ATP synthase, we analyze here the provision of stable conditions for (i the supply of ADP and Mg2+, supported by adenylate kinase (AK equilibrium in the intermembrane space, (ii the supply of phosphate via membrane transporter in symport with H+, and (iii the conditions of outflow of ATP by adenylate transporter carrying out the exchange of free adenylates. We also show that, in chloroplasts, AK equilibrates adenylates and governs Mg2+ contents in the stroma, optimizing ATP synthase and Calvin cycle operation, and affecting the import of inorganic phosphate in exchange with triose phosphates. It is argued that chemiosmosis is not the sole component of ATP synthase performance, which also depends on AK-mediated equilibrium of adenylates and Mg2+, adenylate transport and phosphate release and supply.

  2. Constitutive inhibitory action of muscarinic receptors on adenylyl cyclase in cardiac membranes and its stereospecific suppression by hyoscyamine

    Czech Academy of Sciences Publication Activity Database

    Říčný, Jan; Gualtieri, F.; Tuček, Stanislav

    2002-01-01

    Roč. 51, č. 2 (2002), s. 131-137 ISSN 0862-8408 R&D Projects: GA AV ČR IAA7011910; GA ČR GA309/99/0214 Institutional research plan: CEZ:AV0Z5011922 Keywords : muscarinic receptors * adenylyl cyclase * constitutive activity of receptors Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 0.984, year: 2002

  3. Kinetic and structural characterization of bacterial glutaminyl cyclases from Zymomonas mobilis and Myxococcus xanthus.

    Science.gov (United States)

    Carrillo, David Ruiz; Parthier, Christoph; Jänckel, Nadine; Grandke, Julia; Stelter, Marco; Schilling, Stephan; Boehme, Mathias; Neumann, Piotr; Wolf, Raik; Demuth, Hans-Ulrich; Stubbs, Milton T; Rahfeld, Jens-Ulrich

    2010-12-01

    Although enzymes responsible for the cyclization of amino-terminal glutamine residues are present in both plant and mammal species, none have yet been characterized in bacteria. Based on low sequence homologies to plant glutaminyl cyclases (QCs), we cloned the coding sequences of putative microbial QCs from Zymomonas mobilis (ZmQC) and Myxococcus xanthus (MxQC). The two recombinant enzymes exhibited distinct QC activity, with specificity constants k(cat)/K(m) of 1.47±0.33 mm⁻¹ s⁻¹ (ZmQC) and 142±32.7 mm⁻¹ s⁻¹ (MxQC) towards the fluorescent substrate glutamine-7-amino-4-methyl-coumarine. The measured pH-rate profile of the second order rate constant displayed an interesting deviation towards the acidic limb of the pH chart in the case of ZmQC, whereas MxQC showed maximum activity in the mild alkaline pH range. Analysis of the enzyme variants ZmQCGlu⁴⁶Gln and MxQCGln⁴⁶Glu show that the exchanged residues play a significant role in the pH behaviour of the respective enzymes. In addition, we determined the three dimensional crystal structures of both enzymes. The tertiary structure is defined by a five-bladed β-propeller anchored by a core cation. The structures corroborate the putative location of the active site and confirm the proposed relation between bacterial and plant glutaminyl cyclases.

  4. ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A1

    International Nuclear Information System (INIS)

    Gill, D.M.; Coburn, J.

    1987-01-01

    The authors have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S x GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S x GTPγS), forms over a period of 10-15 min at 37 0 C. Both guanosine 5'-O-(2-thiodiphosphate) and GTP block this quasi-permanent activation. Some S x GTPγS forms in membranes that are exposed to CF alone and then to GTPγS, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S x GTPγS dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45 0 C is decreased by GTPγS. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. They suggest that active S interacts directly with the enzymic A 1 fragments of cholera toxin and not with any toxin substrate. The activation and activity of S are independent of the state, or even the presence, of adenylate cyclase and seem to be involved with the cyclase system only via cholera toxin. S is apparently not related by function to certain other GTP binding proteins, including p21/sup ras/, and appears to be a new GTP binding protein whose physiologic role remains to be identified

  5. Activation of oocyte phosphatidylinositol kinase by polyamines

    International Nuclear Information System (INIS)

    Allende, J.E.; Carrasco, D.; Allende, C.C.

    1987-01-01

    Membrane bound phosphatidylinositol is phosphorylated by a specific membrane enzyme to form phosphatidylinositol 4 phosphate (PIP) which in turn is again phosphorylated to generate phosphatidylinositol 4,5 biphosphate (PIPP). The regulation of phosphatidylinositol phosphorylation and hydrolysis is relevant to the possible role of inositol phosphates as second messengers of hormone action. The membranes of Xenopus laevis oocytes contain a phosphatidylinositol kinase that can generate radioactive PIP after incubation with [ 32 ATP]. The radioactive product is extracted with methanol-chloroform and isolated by thin layer chromatography. The oocyte enzyme has an app Km for ATP of 80 μM and cannot use GTP as a phosphate donor. The formation of PIP is greatly stimulated by the addition of synthetic peptides containing clusters of polylysine at concentrations 0.5 mM. A similar effect is observed with a lysine rich peptide that corresponds to the 14 amino acids of the carboxyl terminus of the Kirstein ras 2 protein and also by polyornithine. Polyarginine and histone H 1 have much lower effects. Peptides containing polylysine clusters have also been found to affect the activity of other key membrane enzymes such as protein kinases and adenylate cyclase

  6. Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2

    Directory of Open Access Journals (Sweden)

    Kabir Hassan Biswas

    2015-04-01

    Full Text Available GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem (GAFab domain. In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.

  7. Enhanced Ex Vivo Stimulation of Mycobacterium tuberculosis-Specific T Cells in Human Immunodeficiency Virus-Infected Persons via Antigen Delivery by the Bordetella pertusis Adenylate Cyclase Vector

    Czech Academy of Sciences Publication Activity Database

    Connell, T. G.; Shey, M. S.; Seldon, R.; Rangaka, M. X.; van Cutsem, G.; Šimšová, Marcela; Marčeková, Zuzana; Šebo, Peter; Curtis, N.; Diwakar, L.; Meintjes, G. A.; Leclerc, C.; Wilkinson, R. J.; Wilkinson, K. A.

    2007-01-01

    Roč. 14, č. 7 (2007), s. 847-854 ISSN 1556-6811 R&D Projects: GA MŠk 2B06161 Institutional research plan: CEZ:AV0Z50200510 Keywords : mycobacterium tuberculosis * bordetella pertusis * human immunodeficiency virus Subject RIV: EE - Microbiology, Virology Impact factor: 1.995, year: 2007

  8. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin

    Czech Academy of Sciences Publication Activity Database

    Mašín, Jiří; Osičková, Adriana; Suková, Anna; Fišer, Radovan; Halada, Petr; Bumba, Ladislav; Linhartová, Irena; Osička, Radim; Šebo, Peter

    2016-01-01

    Roč. 6, Septemeber 1 (2016), s. 29137 ISSN 2045-2322 R&D Projects: GA MŠk(CZ) LO1509; GA MŠk(CZ) LM2015064; GA ČR(CZ) GA16-05919S; GA ČR GA15-09157S; GA ČR(CZ) GA15-11851S; GA ČR(CZ) GA13-14547S Institutional support: RVO:61388971 Keywords : BORDETELLA-PERTUSSIS CYAA * SECONDARY STRUCTURE PREDICTION * ANTIGEN-PRESENTING CELLS Subject RIV: EE - Microbiology, Virology Impact factor: 4.259, year: 2016

  9. Bordetella Adenylate Cyclase Toxin Mobilizes Its beta(2) Integrin Receptor into Lipid Rafts to Accomplish Translocation across Target Cell Membrane in Two Steps

    Czech Academy of Sciences Publication Activity Database

    Bumba, Ladislav; Mašín, Jiří; Fišer, R.; Šebo, Peter

    2010-01-01

    Roč. 6, č. 5 (2010), s. 1-15 ISSN 1553-7366 R&D Projects: GA ČR GP310/07/P115; GA MŠk 1M0506; GA AV ČR IAA500200914; GA MŠk 2B06161; GA ČR GA310/08/0447 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50520701 Keywords : SHEEP ERYTHROCYTES * BACILLUS-ANTHRACIS * ESCHERICHIA-COLI Subject RIV: EE - Microbiology, Virology Impact factor: 9.079, year: 2010

  10. Prime/boost immunotherapy of HPV16-induced tumors with E7 protein delivered by Bordetella adenylate cyclase and modified vaccinia virus Ankara

    Czech Academy of Sciences Publication Activity Database

    Macková, J.; Stasíková, J.; Kutinová, L.; Mašín, Jiří; Hainz, P.; Šimšová, Marcela; Gabriel, P.; Šebo, Peter; Němečková, P.

    2006-01-01

    Roč. 55, - (2006), s. 39-46 ISSN 0340-7004 R&D Projects: GA AV ČR IBS5020311; GA ČR GA310/04/0004; GA MZd NR8004 Grant - others:GA MZd NC6570 Institutional research plan: CEZ:AV0Z50200510 Keywords : vaccine * hpv -e7 * vaccinia virus Subject RIV: EE - Microbiology, Virology Impact factor: 4.313, year: 2006

  11. The cell invasive Bordetella pertussis adenylate cyclase toxin: a model of protein penetration across cell membranes and a new tool for the immunologist

    Czech Academy of Sciences Publication Activity Database

    Osička, Radim; Osičková, Adriana; Basar, T.; Bezoušková, Silvia; Havlíček, Vladimír; Halada, Petr; Loucká, Jiřina; Vojtová, Jana; Kalhous, Miloš; Forman, Stanislav

    2000-01-01

    Roč. 94, č. 8 (2000), s. 599-600 ISSN 0009-2770. [Biochemický sjezd /17./. 07.09.2000-10.09.2000, Prague] R&D Projects: GA ČR GA310/98/0432; GA AV ČR IAA5020907; GA MŠk VS96149; GA MŠk ME 167 Subject RIV: CE - Biochemistry

  12. In vivo induction of CTL responses by recombinant adenylate cyclase of Bordetella pertussis carrying multiple copies of a viral CD8+ T-cell epitope

    Czech Academy of Sciences Publication Activity Database

    Šebo, Peter; Moukrim, Z.; Kalhous, M.; Schaft, N.; Dadaglio, G.; Sheshko, V.; Fayolle, C.; Leclerc, C.

    1999-01-01

    Roč. 26, - (1999), s. 167-173 ISSN 0928-8244 R&D Projects: GA ČR GA310/98/0432; GA MŠk VS96149; GA MŠk ME 167 Grant - others:Barrande(FR) 97054 Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EE - Microbiology , Virology Impact factor: 1.329, year: 1999

  13. Induction of a polarized Th1 response by insertion of multiple copies of a viral T-cell epitope into adenylate cyclase of Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Dadaglio, G.; Moukrim, Z.; Lo-Man, R.; Sheshko, V.; Šebo, Peter; Leclerc, C.

    2000-01-01

    Roč. 68, č. 7 (2000), s. 3867-3872 ISSN 0019-9567 R&D Projects: GA ČR GA310/98/0432; GA MŠk VS96149; GA MŠk ME 167 Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EE - Microbiology, Virology Impact factor: 4.204, year: 2000

  14. Acylation of Lysine 860 Allows Tight Binding and Cytotoxicity of Bordetella Adenylate Cyclase on CD1 1b-Expressing Cells

    Czech Academy of Sciences Publication Activity Database

    Mašín, Jiří; Basler, Marek; Knapp, O.; El-Azami-El-Idrissi, M.; Maier, E.; Konopásek, I.; Benz, R.; Leclerc, C.; Šebo, Peter

    2005-01-01

    Roč. 44, - (2005), s. 12766-12759 ISSN 0006-2960 R&D Projects: GA AV ČR IAA5020406; GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50200510 Keywords : lysine 860 * bordetella Subject RIV: EE - Microbiology, Virology Impact factor: 3.848, year: 2005

  15. Complete protection against P. berghei malaria upon heterologous prime/boost immunization against circumsporozoite protein employing Salmonella type III secretion system and Bordetella adenylate cyclase toxoid

    Czech Academy of Sciences Publication Activity Database

    Tartz, S.; Rüssmann, H.; Kamanová, Jana; Šebo, Peter; Sturm, A.; Heussler, V.; Fleischer, B.; Jacobs, T.

    2008-01-01

    Roč. 26, č. 47 (2008), s. 5935-5943 ISSN 0264-410X R&D Projects: GA MŠk 2B06161 Institutional research plan: CEZ:AV0Z50200510 Keywords : circumsporozoite protein * vaccine * salmonella Subject RIV: EE - Microbiology, Virology Impact factor: 3.298, year: 2008

  16. A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain from Bordetella pertussis Adenylate Cyclase

    Science.gov (United States)

    2009-10-22

    inMaterials andMethods. (C)Rapid kinetics ofC-BR(L)-Y folding (9) andunfolding (b) at the indicated CaCl2 concentrations was measured using FRET as...described inMaterials andMethods. Error bars represent standard errors (Ng 3). Representative fluorescence time course traces are shown inFigure S1 of...aContour length (lc) between the chromophores of the FPs, calculated as described inMaterials andMethods. bAverage end-to-end distance of a theoretical

  17. Mycobacterium tuberculosis Rv3586 (DacA is a diadenylate cyclase that converts ATP or ADP into c-di-AMP.

    Directory of Open Access Journals (Sweden)

    Yinlan Bai

    Full Text Available Cyclic diguanosine monophosphate (c-di-GMP and cyclic diadenosine monophosphate (c-di-AMP are recently identified signaling molecules. c-di-GMP has been shown to play important roles in bacterial pathogenesis, whereas information about c-di-AMP remains very limited. Mycobacterium tuberculosis Rv3586 (DacA, which is an ortholog of Bacillus subtilis DisA, is a putative diadenylate cyclase. In this study, we determined the enzymatic activity of DacA in vitro using high-performance liquid chromatography (HPLC, mass spectrometry (MS and thin layer chromatography (TLC. Our results showed that DacA was mainly a diadenylate cyclase, which resembles DisA. In addition, DacA also exhibited residual ATPase and ADPase in vitro. Among the potential substrates tested, DacA was able to utilize both ATP and ADP, but not AMP, pApA, c-di-AMP or GTP. By using gel filtration and analytical ultracentrifugation, we further demonstrated that DacA existed as an octamer, with the N-terminal domain contributing to tetramerization and the C-terminal domain providing additional dimerization. Both the N-terminal and the C-terminal domains were essential for the DacA's enzymatically active conformation. The diadenylate cyclase activity of DacA was dependent on divalent metal ions such as Mg(2+, Mn(2+ or Co(2+. DacA was more active at a basic pH rather than at an acidic pH. The conserved RHR motif in DacA was essential for interacting with ATP, and mutation of this motif to AAA completely abolished DacA's diadenylate cyclase activity. These results provide the molecular basis for designating DacA as a diadenylate cyclase. Our future studies will explore the biological function of this enzyme in M. tuberculosis.

  18. Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

    Directory of Open Access Journals (Sweden)

    Andre Terzic

    2009-04-01

    Full Text Available Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7 are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network.

  19. Crystal Structure of the PAC1R Extracellular Domain Unifies a Consensus Fold for Hormone Recognition by Class B G-Protein Coupled Receptors

    OpenAIRE

    Kumar, Shiva; Pioszak, Augen; Zhang, Chenghai; Swaminathan, Kunchithapadam; Xu, H. Eric

    2011-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism...

  20. Adenylate kinase I does not affect cellular growth characteristics under normal and metabolic stress conditions.

    Science.gov (United States)

    de Bruin, Wieke; Oerlemans, Frank; Wieringa, Bé

    2004-07-01

    Adenylate kinase (AK)-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy in cells of fully differentiated tissues with highly variable energy demand, such as muscle and brain. To investigate if AK isoenzymes have a comparable function in the energy-demand management of proliferating cells, AK1 and AK1beta were expressed in mouse neuroblastoma N2a cells and in human colon carcinoma SW480 cells. Glucose deprivation, galactose feeding, and metabolic inhibitor tests revealed a differential energy dependency for these two cell lines. N2a cells showed a faster proliferation rate and strongest coupling to mitochondrial activity, SW480 proliferation was more dependent on glycolysis. Despite these differences, ectopic expression of AK1 or AK1beta did not affect their growth characteristics under normal conditions. Also, no differential effects were seen under metabolic stress upon treatment with mitochondrial and glycolytic inhibitors in in vitro culture or in solid tumors grown in vivo. Although many intimate connections have been revealed between cell death and metabolism, our results suggest that AK1- or AK1beta-mediated high-energy phosphoryl transfer is not a modulating factor in the survival of tumor cells during episodes of metabolic crisis.

  1. Anoctamin 9/TMEM16J is a cation channel activated by cAMP/PKA signal.

    Science.gov (United States)

    Kim, Hyungsup; Kim, Hyesu; Lee, Jesun; Lee, Byeongjun; Kim, Hee-Ryang; Jung, Jooyoung; Lee, Mi-Ock; Oh, Uhtaek

    2018-05-01

    Anoctamins (ANOs) are multifunctional membrane proteins that consist of 10 homologs. ANO1 (TMEM16A) and ANO2 (TMEM16B) are anion channels activated by intracellular calcium that meditate numerous physiological functions. ANO6 is a scramblase that redistributes phospholipids across the cell membrane. The other homologs are not well characterized. We found ANO9/TMEM16J is a cation channel activated by a cAMP-dependent protein kinase A (PKA). Intracellular cAMP-activated robust currents in whole cells expressing ANO9, which were inhibited by a PKA blocker. A cholera toxin that persistently stimulated adenylate cyclase activated ANO9 as did the application of PKA. The cAMP-induced ANO9 currents were permeable to cations. The cAMP-dependent ANO9 currents were augmented by intracellular Ca 2+ . Ano9 transcripts were predominant in the intestines. Human intestinal SW480 cells expressed high levels of Ano9 transcripts and showed PKA inhibitor-reversible cAMP-dependent currents. We conclude that ANO9 is a cation channel activated by a cAMP/PKA pathway and could play a role in intestine function. Copyright © 2017. Published by Elsevier Ltd.

  2. Vasodilatory activity and antihypertensive profile mediated by inhibition of phosphodiesterase type 1 induced by a novel sulfonamide compound.

    Science.gov (United States)

    Pontes, Luana Braga; Antunes, Fernanda; Sudo, Roberto Takashi; Raimundo, Juliana Montani; Lima, Lidia Moreira; Barreiro, Eliezer Jesus; Zapata-Sudo, Gisele

    2012-12-01

    LASSBio-985 is a sulfonamide compound designed as a simplified structure of a nonselective phosphodiesterase type 4 (PDE-4) inhibitor that promotes vasodilatory activity in vitro. PDE are enzymes responsible for the hydrolysis of cyclic adenosine 3',5'- monophosphate and cyclic guanosine 3',5'-monophosphate. Five different isozymes of PDE are found in vascular smooth muscle (PDE1-PDE5). Aortic rings, with or without endothelium, from male normotensive and spontaneously hypertensive rats (SHR) were prepared for isometric tension recording. Blood pressure was measured in Wistar Kyoto (WKY) rats and SHR during intravenous infusion of LASSBio-985 (10 mg/kg/min) during 15 min. LASSBio-985 induced a concentration-dependent vasodilation in aortic rings from normotensive and SHR, which was almost completely inhibited in endothelium-denuded vessels. Vasodilatory activity was also reduced in endothelium-intact aortic rings that had been pretreated with N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase inhibitor and 1H-[1,2,4]oxadiazolod[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor. LASSBio-985-induced vasodilation was also inhibited by sildenafil (100 μm) and SQ 22536, a PDE5 inhibitor and adenylate cyclase inhibitor, respectively. To evaluate the involvement of some endothelial receptors, atropine, diphenhydramine, HOE 140, naloxone, propranolol, indomethacin, and wortmannin were tested, but none inhibited the effects of LASSBio-985. The residual effect observed on endothelium-denuded aortic rings was abolished by nicardipine, a voltage-sensitive-Ca(2+)-channel blocker. Intravenous infusion of LASSBio-985 (10 mg/kg/min) significantly reduced systolic and diastolic pressures in both WKY and SHR. LASSBio-985 is a compound with vasodilatory activity, which could be consequent to PDE1 inhibition and voltage-sensitive-Ca(2+)-channel blockade. © 2011 The Authors Fundamental and Clinical Pharmacology © 2011 Société Fran

  3. Cyclic nucleotides and mitogen-activated protein kinases: regulation of simvastatin in platelet activation

    Directory of Open Access Journals (Sweden)

    Hou Ssu-Yu

    2010-06-01

    Full Text Available Abstract Background 3-Hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA reductase inhibitors (statins have been widely used to reduce cardiovascular risk. These statins (i.e., simvastatin may exert other effects besides from their cholesterol-lowering actions, including inhibition of platelet activation. Platelet activation is relevant to a variety of coronary heart diseases. Although the inhibitory effect of simvastatin in platelet activation has been studied; the detailed signal transductions by which simvastatin inhibit platelet activation has not yet been completely resolved. Methods The aim of this study was to systematically examine the detailed mechanisms of simvastatin in preventing platelet activation. Platelet aggregation, flow cytometric analysis, immunoblotting, and electron spin resonance studies were used to assess the antiplatelet activity of simvastatin. Results Simvastatin (20-50 μM exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen than other agonists (i.e., thrombin. Simvastatin inhibited collagen-stimulated platelet activation accompanied by [Ca2+]i mobilization, thromboxane A2 (TxA2 formation, and phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (i.e., p38 MAPK, JNKs phosphorylation in washed platelets. Simvastatin obviously increased both cyclic AMP and cyclic GMP levels. Simvastatin markedly increased NO release, vasodilator-stimulated phosphoprotein (VASP phosphorylation, and endothelial nitric oxide synthase (eNOS expression. SQ22536, an inhibitor of adenylate cyclase, markedly reversed the simvastatin-mediated inhibitory effects on platelet aggregation, PLCγ2 and p38 MAPK phosphorylation, and simvastatin-mediated stimulatory effects on VASP and eNOS phosphorylation. Conclusion The most important findings of this study demonstrate for the first time that inhibitory effect of simvastatin in platelet activation may involve activation of the cyclic AMP

  4. Membrane Guanylyl Cyclase Complexes Shape the Photoresponses of Retinal Rods and Cones

    Directory of Open Access Journals (Sweden)

    Xiao-Hong eWen

    2014-06-01

    Full Text Available In vertebrate rods and cones, photon capture by rhodopsin leads to the destruction of cyclic GMP (cGMP and the subsequent closure of cyclic nucleotide gated (CNG ion channels in the outer segment plasma membrane. Replenishment of cGMP and reopening of the channels limit the growth of the photon response and are requisite for its recovery. In different vertebrate retinas, there may be as many as four types of membrane guanylyl cyclases (GCs for cGMP synthesis. Ten neuronal Ca2+ sensor proteins could potentially modulate their activities. The mouse is proving to be an effective model for characterizing the roles of individual components because its relative simplicity can be reduced further by genetic engineering. There are two types of guanylyl cyclase activating proteins (GCAPs and two types of GCs in mouse rods, whereas cones express one type of GCAP and one type of GC. Mutant mouse rods and cones bereft of both GCAPs have large, long lasting photon responses. Thus, GCAPs normally mediate negative feedback tied to the light-induced decline in intracellular Ca2+ that accelerates GC activity to curtail the growth and duration of the photon response. Rods from other mutant mice that express a single GCAP type reveal how the two GCAPs normally work together as a team. Because of its lower Ca2+ affinity, GCAP1 is the first responder that senses the initial decrease in Ca2+ following photon absorption and acts to limit response amplitude. GCAP2, with a higher Ca2+ affinity, is recruited later during the course of the photon response as Ca2+ levels continue to decline further. The main role of GCAP2 is to provide for a timely response recovery and it is particularly important after exposure to very bright light. The multiplicity of GC isozymes and GCAP homologs in the retinas of other vertebrates confers greater flexibility in shaping the photon responses in order to tune visual sensitivity, dynamic range and frequency response.

  5. Enzymatic synthesis of c-di-GMP using inclusion bodies of Thermotoga maritima full-length diguanylate cyclase.

    Science.gov (United States)

    Korovashkina, A S; Rymko, A N; Kvach, S V; Zinchenko, A I

    2012-12-15

    Recombinant full-length diguanylate cyclases (DGCs) of Thermotoga maritima with native and mutant allosteric sites were overexpressed in Escherichia coli cells and characterized. It has been shown that target enzymes are produced substantially in the form of active inclusion bodies. Introduction of the mutation in allosteric site resulted in 7-fold increase of the T. maritima DGC activity. Possibility of applying full-length DGC of T. maritima in the form of inclusion bodies for synthesis of c-di-GMP was originally demonstrated. Copyright © 2012 Elsevier B.V. All rights reserved.

  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. Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration

    Science.gov (United States)

    Watson, Richard L.; Buck, Jochen; Levin, Lonny R.; Winger, Ryan C.; Wang, Jing; Arase, Hisashi

    2015-01-01

    CD99 is a critical regulator of leukocyte transendothelial migration (TEM). How CD99 signals during this process remains unknown. We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC). PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium. Pharmacologic or genetic inhibition of EC sAC or PKA, like CD99 blockade, arrests neutrophils and monocytes partway through EC junctions, in vitro and in vivo, without affecting leukocyte adhesion or the expression of relevant cellular adhesion molecules. This is the first description of the CD99 signaling pathway in TEM as well as the first demonstration of a role for sAC in leukocyte TEM. PMID:26101266

  8. Synergism between soluble guanylate cyclase signaling and neuropeptides extends lifespan in the nematode Caenorhabditis elegans.

    Science.gov (United States)

    Abergel, Rachel; Livshits, Leonid; Shaked, Maayan; Chatterjee, Arijit Kumar; Gross, Einav

    2017-04-01

    Oxygen (O 2 ) homeostasis is important for all aerobic animals. However, the manner by which O 2 sensing and homeostasis contribute to lifespan regulation is poorly understood. Here, we use the nematode Caenorhabditis elegans to address this question. We demonstrate that a loss-of-function mutation in the neuropeptide receptor gene npr-1 and a deletion mutation in the atypical soluble guanylate cyclase gcy-35 O 2 sensor interact synergistically to extend worm lifespan. The function of npr-1 and gcy-35 in the O 2 -sensing neurons AQR, PQR, and URX shortens the lifespan of the worm. By contrast, the activity of the atypical soluble guanylate cyclase O 2 sensor gcy-33 in these neurons is crucial for lifespan extension. In addition to AQR, PQR, and URX, we show that the O 2 -sensing neuron BAG and the interneuron RIA are also important for the lifespan lengthening. Neuropeptide processing by the proprotein convertase EGL-3 is essential for lifespan extension, suggesting that the synergistic effect of joint loss of function of gcy-35 and npr-1 is mediated through neuropeptide signal transduction. The extended lifespan is regulated by hypoxia and insulin signaling pathways, mediated by the transcription factors HIF-1 and DAF-16. Moreover, reactive oxygen species (ROS) appear to play an important function in lifespan lengthening. As HIF-1 and DAF-16 activities are modulated by ROS, we speculate that joint loss of function of gcy-35 and npr-1 extends lifespan through ROS signaling. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

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

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Hashimoto, Naohiro

    2008-01-01

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

  10. 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

  11. Adenylate kinase I does not affect cellular growth characteristics under normal and metabolic stress conditions.

    NARCIS (Netherlands)

    Bruin, W.C.C. de; Oerlemans, F.T.J.J.; Wieringa, B.

    2004-01-01

    Adenylate kinase (AK)-catalyzed phosphotransfer is essential in the maintenance of cellular energetic economy in cells of fully differentiated tissues with highly variable energy demand, such as muscle and brain. To investigate if AK isoenzymes have a comparable function in the energy-demand

  12. 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.

  13. Investigating the Potential Signaling Pathways That Regulate Activation of the Novel PKC Downstream of Serotonin in Aplysia.

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    Carole A Farah

    Full Text Available Activation of the novel PKC Apl II in sensory neurons by serotonin (5HT underlies the ability of 5HT to reverse synaptic depression, but the pathway from 5HT to PKC Apl II activation remains unclear. Here we find no evidence for the Aplysia-specific B receptors, or for adenylate cyclase activation, to translocate fluorescently-tagged PKC Apl II. Using an anti-PKC Apl II antibody, we monitor translocation of endogenous PKC Apl II and determine the dose response for PKC Apl II translocation, both in isolated sensory neurons and sensory neurons coupled with motor neurons. Using this assay, we confirm an important role for tyrosine kinase activation in 5HT mediated PKC Apl II translocation, but rule out roles for intracellular tyrosine kinases, epidermal growth factor (EGF receptors and Trk kinases in this response. A partial inhibition of translocation by a fibroblast growth factor (FGF-receptor inhibitor led us to clone the Aplysia FGF receptor. Since a number of related receptors have been recently characterized, we use bioinformatics to define the relationship between these receptors and find a single FGF receptor orthologue in Aplysia. However, expression of the FGF receptor did not affect translocation or allow it in motor neurons where 5HT does not normally cause PKC Apl II translocation. These results suggest that additional receptor tyrosine kinases (RTKs or other molecules must also be involved in translocation of PKC Apl II.

  14. G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase.

    Science.gov (United States)

    Lavine, Natalie; Ethier, Nathalie; Oak, James N; Pei, Lin; Liu, Fang; Trieu, Phan; Rebois, R Victor; Bouvier, Michel; Hebert, Terence E; Van Tol, Hubert H M

    2002-11-29

    A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.

  15. Coriandrum sativum and Lavandula angustifolia Essential Oils: Chemical Composition and Activity on Central Nervous System.

    Science.gov (United States)

    Caputo, Lucia; Souza, Lucéia Fátima; Alloisio, Susanna; Cornara, Laura; De Feo, Vincenzo

    2016-11-30

    The aims of this study are to determine the chemical composition of Lavandula angustifolia Mill. and Coriandrum sativum L. essential oils, to evaluate their cytotoxic effects in SH-SY5Y human neuroblastoma cells, to investigate whether an alteration of adenylate cyclase 1 (ADCY1) and of extracellular signal-regulated kinase (ERK) expression can take part in the molecular mechanisms of the essential oils, and to study their possible neuronal electrophysiological effects. The essential oils were obtained by hydrodistillation, and studied by GC and GC-MS. In the oils from L. angustifolia and C. sativum , linalool was the main component (33.1% and 67.8%, respectively). SH-SY5Y cells were incubated with different concentrations of essential oils and of linalool. Cell viability and effects on ADCY1 and ERK expression were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT and Western blotting, respectively. Variation in cellular electrophysiology was studied in primary cultures of rat cortical neurons with a multi-electrode array (MEA)-based approach. The essential oils and linalool revealed different cytotoxic activities. Linalool inhibited ADCY1 and ERK expression. Neuronal networks subjected to L. angustifolia and C. sativum essential oils showed a concentration-dependent inhibition of spontaneous electrical activity.

  16. Influence of bacterial toxins on the GTPase activity of transducin from bovine retinal rod outer segments

    International Nuclear Information System (INIS)

    Rybin, V.O.; Gureeva, A.A.

    1986-01-01

    The action of cholera toxin, capable of ADP-ribosylation of the activator N/sub s/ protein, and pertussis toxin, capable of ADP-ribosylation of the inhibitor N/sub i/ protein of the adenylate cyclase complex, on transducin, the GTP-binding protein of the rod outer segments of the retina, was investigated. It was shown that under the action of pertussis and cholera toxins, the GTPase activity of transducin is inhibited. Pertussin toxin inhibits the GTPase of native retinal rod outer segments by 30-40%, while GTPase of homogeneous transducin produces a 70-80% inhibition. The action of toxins on transducin depends on the presence and nature of the guanylic nucleotide with which incubation is performed. On the basis of the data obtained it is suggested that pertussis toxin interacts with pretransducin and with the transducin-GDP complex, while cholera toxin ADP-ribosylates the transducin-GTP complex and does not act on transducin lacking GTP

  17. Ca²+/calmodulin-dependent protein kinase II (CaMKII activity and sinoatrial nodal pacemaker cell energetics.

    Directory of Open Access Journals (Sweden)

    Yael Yaniv

    Full Text Available : Ca(2+-activated basal adenylate cyclase (AC in rabbit sinoatrial node cells (SANC guarantees, via basal cAMP/PKA-calmodulin/CaMKII-dependent protein phosphorylation, the occurrence of rhythmic, sarcoplasmic-reticulum generated, sub-membrane Ca(2+ releases that prompt rhythmic, spontaneous action potentials (APs. This high-throughput signaling consumes ATP.We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca(2+ indirectly, regulate mitochondrial ATP production. While, clearly, Ca(2+-calmodulin-CaMKII activity regulates ATP consumption, whether it has a role in the control of ATP production is unknown.We superfused single, isolated rabbit SANC at 37°C with physiological saline containing CaMKII inhibitors, (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP, or a calmodulin inhibitor (W-7 and measured cytosolic Ca(2+, flavoprotein fluorescence and spontaneous AP firing rate. We measured cAMP, ATP and O2 consumption in cell suspensions. Graded reductions in basal CaMKII activity by KN-93 (0.5-3 µmol/L or AIP (2-10 µmol/L markedly slow the kinetics of intracellular Ca(2+ cycling, decrease the spontaneous AP firing rate, decrease cAMP, and reduce O2 consumption and flavoprotein fluorescence. In this context of graded reductions in ATP demand, however, ATP also becomes depleted, indicating reduced ATP production.CaMKII signaling, a crucial element of normal automaticity in rabbit SANC, is also involved in SANC bioenergetics.

  18. 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.

  19. The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase

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    Kenyon Colin P

    2012-08-01

    Full Text Available Abstract Background It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP as a molecular probe with site directed mutagenesis (SDM of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings. Results We have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK and adenylate kinase 1 (AK1, are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP. Conclusions The data outlined serves to demonstrate the “push” mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It

  20. Germacrene C synthase from Lycopersicon esculentum cv. VFNT cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase.

    Science.gov (United States)

    Colby, S M; Crock, J; Dowdle-Rizzo, B; Lemaux, P G; Croteau, R

    1998-03-03

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, beta-caryophyllene, alpha-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton delta-cadinene synthase (50% identity).

  1. Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity.

    Science.gov (United States)

    Brandt, D R; Asano, T; Pedersen, S E; Ross, E M

    1983-09-13

    beta-Adrenergic receptors were partially purified from turkey erythrocyte membranes by alprenolol-agarose chromatography to 0.25-2 nmol/mg of protein, and the stimulatory guanosine 5'-triphosphate (GTP) binding protein of adenylate cyclase (Gs) was purified from rabbit liver. These proteins were reconstituted into phospholipid vesicles by addition of phospholipids and removal of detergent by gel filtration. This preparation hydrolyzes GTP to guanosine 5'-diphosphate (GDP) plus inorganic phosphate (Pi) in response to beta-adrenergic agonists. The initial rate of isoproterenol-stimulated hydrolysis is approximately 1 mol of GTP hydrolyzed min-1 X mol-1 of Gs. This low rate may be limited by the hormone-stimulated binding of substrate, since it is roughly equal to the rate of binding of the GTP analogue guanosine 5'-O-(3-[35S] thiotriphosphate) [( 35S]GTP gamma S) to Gs in the vesicles. Activity in the absence of agonist, or in the presence of agonist plus a beta-adrenergic antagonist, is 8-25% of the hormone-stimulated activity. Guanosinetriphosphatase (GTPase) is not saturated at 10 microM GTP, and the response to GTP is formally consistent either with the existence of multiple Km's or of a separate stimulatory site for GTP. The GTPase activity of Gs in vesicles is also stimulated by 50 mM MgCl2 in the presence or absence of receptor. Significant GTPase activity is not observed with Lubrol-solubilized Gs, although [35S]-GTP gamma S binding is increased by Lubrol solubilization.

  2. 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

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

    Science.gov (United States)

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

    2018-01-01

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

  4. The arsonomethyl analogue of adenosine 5'-phosphate. An uncoupler of adenylate kinase.

    Science.gov (United States)

    Adams, S R; Sparkes, M J; Dixon, H B

    1984-01-01

    Adenosine was converted into the arsonomethyl analogue of AMP. The reactions used provide a general route for converting an alcohol, R-CH2-OH, into the arsonomethyl analogue, R-CH2-CH2-AsO3H2, of its phosphate, R-CH2-O-PO3H2. The analogue of AMP proves to be a substrate for rabbit adenylate kinase, which shows a limiting velocity with it of 1/17 that with AMP, a Michaelis constant raised 70-fold to about 10 mM, and hence a specificity constant lowered about 1200-fold. The product of transfer of a phospho group from ATP to the analogue is, like all anhydrides of arsonic acids, unstable to hydrolysis, and so breaks down to yield orthophosphate and regenerate the analogue. Hence adenylate kinase is converted into an ATPase by the presence of the analogue. PMID:6089748

  5. Crystal structure of a trimeric archaeal adenylate kinase from the mesophile Methanococcus maripaludis with an unusually broad functional range and thermal stability.

    Science.gov (United States)

    Davlieva, Milya; Shamoo, Yousif

    2010-02-01

    The structure of the trimeric adenylate kinase from the Archaebacteria Methanococcus mariplaludis (AK(MAR)) has been solved to 2.5-A resolution and the temperature dependent stability and kinetics of the enzyme measured. The K(M) and V(max) of AK(MAR) exhibit only modest temperature dependence from 30 degrees -60 degrees C. Although M. mariplaludis is a mesophile with a maximum growth temperature of 43 degrees C, AK(MAR) has a very broad functional range and stability (T(m) = 74.0 degrees C) that are more consistent with a thermophilic enzyme with high thermostability and exceptional activity over a wide range of temperatures, suggesting that this microbe may have only recently invaded a mesophilic niche and has yet to fully adapt. A comparison of the Local Structural Entropy (LSE) for AK(MAR) to the related adenylate kinases from the mesophile Methanococcus voltae and thermophile Methanococcus thermolithotrophicus show that changes in LSE are able to fully account for the intermediate stability of AK(MAR) and highlights a general mechanism for protein adaptation in this class of enzymes. (c) 2009 Wiley-Liss, Inc.

  6. A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness

    OpenAIRE

    Burroughs, A. Maxwell; Ando, Yoshinari; de Hoon, Michiel J.L.; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O.

    2010-01-01

    Animal microRNA sequences are subject to 3′ nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3′ adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a...

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

    Directory of Open Access Journals (Sweden)

    Emmanuel S Buys

    2014-05-01

    Full Text Available Glaucoma is a progressive optic neuropathy characterized by visual field defects that ultimately lead to irreversible blindness (1, 2. By the year 2020, an estimated 80 million people will have glaucoma, 11 million of which will be bilaterally blind. Primary open-angle glaucoma (POAG is the most common type of glaucoma. Elevated intraocular pressure (IOP is currently the only risk factor amenable to treatment. How IOP is regulated and can be modulated remains a topic of active investigation. Available therapies, mostly geared towards lowering IOP, offer incomplete protection, and POAG often goes undetected until irreparable damage has been done, highlighting the need for novel therapeutic approaches, drug targets, and biomarkers (3, 4. In this review, the role of soluble (nitric oxide (NO-activated and membrane-bound, natriuretic peptide (NP-activated guanylate cyclases that generate the secondary signaling molecule cyclic guanosine monophosphate (cGMP in the regulation of IOP and in the pathophysiology of POAG will be discussed.

  8. The prelimbic cortex muscarinic M₃ receptor-nitric oxide-guanylyl cyclase pathway modulates cardiovascular responses in rats.

    Science.gov (United States)

    Fassini, Aline; Antero, Leandro S; Corrêa, Fernando M A; Joca, Sâmia R; Resstel, Leonardo B M

    2015-05-01

    The prelimbic cortex (PL), a limbic structure, sends projections to areas involved in the control of cardiovascular responses. Stimulation of the PL with acetylcholine (ACh) evokes depressor and tachycardiac responses mediated by local PL muscarinic receptors. Early studies demonstrated that stimulation of muscarinic receptors induced nitric oxide (NO) synthesis and cyclic guanosine cyclic monophosphate (cGMP) formation. Hence, this study investigates which PL muscarinic receptor subtype is involved in the cardiovascular response induced by ACh and tests the hypothesis that cardiovascular responses caused by muscarinic receptor stimulation in the PL are mediated by local NO and cGMP formation. PL pretreatment with J104129 (an M3 receptor antagonist) blocked the depressor and tachycardiac response evoked by injection of ACh into the PL. Pretreatment with either pirenzepine (an M1 receptor antagonist) or AF-DX 116 (an M2 and M4 receptor antagonist) did not affect cardiovascular responses evoked by ACh. Moreover, similarly to the antagonism of PL M3 receptors, pretreatment with N(ω)-propyl-L-arginine (an inhibitor of neuronal NO synthase), carboxy-PTIO(S)-3-carboxy-4-hydroxyphenylglicine (an NO scavenger), or 1H-[1,2,4]oxadiazolol-[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) blocked both the depressor and the tachycardiac response evoked by ACh. The current results demonstrate that cardiovascular responses evoked by microinjection of ACh into the PL are mediated by local activation of the M3 receptor-NO-guanylate cyclase pathway. © 2015 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

    2016-06-01

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

  10. Guanylate cyclase-C/cGMP: an emerging pathway in the regulation of visceral pain

    Directory of Open Access Journals (Sweden)

    Gerhard eHannig

    2014-04-01

    Full Text Available Activation of guanylate cyclase-C (GC-C expressed predominantly on intestinal epithelial cells by guanylin, uroguanylin or the closely related GC-C agonist peptide, linaclotide, stimulates generation and release of cyclic guanosine-3’,5’-monophosphate (cGMP. Evidence that the visceral analgesic effects of linaclotide are mediated by a novel, GC-C-dependent peripheral sensory mechanism was first demonstrated in animal models of visceral pain. Subsequent studies with uroguanylin or linaclotide have confirmed the activation of a GC-C/cGMP pathway leading to increased submucosal cGMP mediated by cGMP efflux pumps, which modulates intestinal nociceptor function resulting in peripheral analgesia. These effects can be reproduced by the addition of exogenous cGMP and support a role for GC-C/cGMP signaling in the regulation of visceral sensation, a physiological function that has not previously been linked to the GC-C/cGMP pathway. Notably, targeting the GC-C/cGMP pathway for treatment of gastrointestinal pain and abdominal sensory symptoms has now been validated in the clinic. In 2012, linaclotide was approved in the United States and European Union for the treatment of adult patients with irritable bowel syndrome with constipation.

  11. Adenylyl cyclase-mediated effects contribute to increased Isoprenaline-induced cardiac contractility in TRPM4-deficient mice.

    Science.gov (United States)

    Uhl, Sebastian; Mathar, Ilka; Vennekens, Rudi; Freichel, Marc

    2014-09-01

    TRPM4 and TRPM5 proteins belong to the Transient Receptor Potential (TRP) ion channel family and form Ca(2+)-activated nonselective cation channels. Recently we showed a significant increase of Isoprenaline-induced inotropy in TRPM4-deficient (Trpm4(-/-)) mice. This is caused by increased Ca(2+) entry via L-type calcium channels due to faster action potential repolarization in Trpm4(-/-) ventricular myocytes [Mathar et al., 2013]. Here, we investigated the contribution of various steps of the β-adrenergic signalling cascade to the augmented positive inotropic response in the absence of TRPM4, and whether the closely related TRPM5 additively contributes to this process using TRPM4/TRPM5-double deficient (Trpm4/Trpm5((-/-)2)) mice. We performed contractility measurements on isolated papillary muscles from wild type, Trpm4(-/-) and Trpm4/Trpm5((-/-)2) mice. As shown in Trpm4(-/-) mice, Isoprenaline-induced inotropy in Trpm4/Trpm5((-/-)2) papillary muscles was significantly increased compared to wild type, whereas basal, frequency- and Ca(2+)-dependent contractility was unaltered. Equivalent to Isoprenaline, activation of adenylyl cyclase using Forskolin led to a significantly increased twitch force in Trpm4(-/-) heart preparations whereas the Isoprenaline-mediated increase in cAMP level was comparable to wild type mice. Notably, the positive inotropic response evoked by phosphodiesterase inhibition with 3-isobutyl-1-methylxanthine (IBMX) was unchanged between both genotypes. Furthermore, experiments performed with increasing concentrations of IBMX after prestimulation with Forskolin and vice versa did not provide evidence that the increased β-adrenergic positive inotropic response in TRPM4-deficient papillary muscles is due to differences in accumulation of cAMP. Compared to inhibition of phosphodiesterase, the rise of intracellular cAMP by activating adenylyl cyclase is accompanied by ATP breakdown. To test the relevance of TRPM4 during forced ATP consumption we

  12. Cloning, expression, and characterization of epi-cedrol synthase, a sesquiterpene cyclase from Artemisia annua L.

    Science.gov (United States)

    Mercke, P; Crock, J; Croteau, R; Brodelius, P E

    1999-09-15

    Sesquiterpene cyclases (synthases) catalyze the conversion of the isoprenoid intermediate farnesyl diphosphate to various sesquiterpene structural types. In plants, many sesquiterpenes are produced as defensive chemicals (phytoalexins) or mediators of chemical communication (i.e., pollinator attractants). A number of sesquiterpene synthases are present in Artemisia annua L. (annual wormwood). We have isolated a cDNA clone encoding one of these, epi-cedrol synthase. This clone contains a 1641-bp open reading frame coding for 547 amino acids (63.5 kDa), a 38-bp 5'-untranslated end, and a 272-bp 3'-untranslated sequence. The deduced amino acid sequence was 32 to 43% identical with the sequences of other known sesquiterpene cyclases from angiosperms. When expressed in Escherichia coli, the recombinant enzyme catalyzed the formation of both olefinic (3%) and oxygenated (97%) sesquiterpenes from farnesyl diphosphate. GC-MS analysis identified the olefins as alpha-cedrene (57% of the olefins), beta-cedrene (13%), (E)-beta-farnesene (5%), alpha-acoradiene (1%), (E)-alpha-bisabolene (8%), and three unknown olefins (16%) and the oxygenated sesquiterpenes (97% of total sesquiterpene generated, exclusive of farnesol and nerolidol) as cedrol (4%) and epi-cedrol (96%). epi-Cedrol synthase was not active with geranylgeranyl diphosphate as substrate, whereas geranyl diphosphate was converted to monoterpenes by the recombinant enzyme at a rate of about 15% of that observed with farnesyl diphosphate as substrate. The monoterpene olefin products are limonene (45%), terpinolene (42%), gamma-terpinene (8%), myrcene (5%), and alpha-terpinene (2%); a small amount of the monoterpene alcohol terpinen-4-ol is also produced. The pH optimum for the recombinant enzyme is 8.5-9.0 (with farnesyl diphosphate as substrate) and the K(m) values for farnesyl diphosphate are 0.4 and 1.3 microM at pH 7. 0 and 9.0, respectively. The K(m) for Mg(2+) is 80 microM at pH 7.0 and 9.0.

  13. The effect of alcohol on recombinant proteins derived from mammalian adenylyl cyclase

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    Emily Qualls-Creekmore

    2017-07-01

    Full Text Available The cyclic AMP (cAMP signaling pathway is implicated in the development of alcohol use disorder. Previous studies have demonstrated that ethanol enhances the activity of adenylyl cyclase (AC in an isoform specific manner; AC7 is most enhanced by ethanol, and regions responsible for enhancement by ethanol are located in the cytoplasmic domains of the AC7 protein. We hypothesize that ethanol modulates AC activity by directly interacting with the protein and that ethanol effects on AC can be studied using recombinant AC in vitro. AC recombinant proteins containing only the C1a or C2 domains of AC7 and AC9 individually were expressed in bacteria, and purified. The purified recombinant AC proteins retained enzymatic activity and isoform specific alcohol responsiveness. The combination of the C1a or C2 domains of AC7 maintained the same alcohol cutoff point as full-length AC7. We also find that the recombinant AC7 responds to alcohol differently in the presence of different combinations of activators including MnCl2, forskolin, and Gsα. Through a series of concentration-response experiments and curve fitting, the values for maximum activities, Hill coefficients, and EC50 were determined in the absence and presence of butanol as a surrogate of ethanol. The results suggest that alcohol modulates AC activity by directly interacting with the AC protein and that the alcohol interaction with the AC protein occurs at multiple sites with positive cooperativity. This study indicates that the recombinant AC proteins expressed in bacteria can provide a useful model system to investigate the mechanism of alcohol action on their activity.

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

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    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.

  15. Molecular cloning, subcellular localization and characterization of two adenylate kinases from cassava, Manihot esculenta Crantz cv. KU50.

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    Boonrueng, Channarong; Tangpranomkorn, Surachat; Yazhisai, Uthaman; Sirikantaramas, Supaart

    2016-10-01

    Adenylate kinase (ADK) is a phosphotransferase that plays an important role in cellular energy homeostasis. Many isozymes located in different subcellular compartments have been reported. In this study, we focus on the characterization of cassava (Manihot esculenta) ADKs. We found 15 ADKs that are publicly available in the African cassava genome database. We cloned two ADKs, namely MeADK1 and MeADK2, which are phylogenetically grouped together with the plastidial ADK in potato. Both MeADK1 and MeADK2 showed 66% identity in the amino acid sequences with plastidial ADK in potato. However, we demonstrated that they are localized to mitochondria using GFP fusions of MeADK1 and MeADK2. The Escherichia coli-produced recombinant MeADK1 and MeADK2 preferred forward reactions that produce ATP. They exhibited similar specific activities. The semi-quantitative RT-PCR analysis showed that MeADK1 and MeADK2 in 2-month-old leaves have similar expression patterns under a diurnal light-dark cycle. However, MeADK2 transcripts were expressed at much higher levels than MeADK1 in 5-month-old leaves and roots. Thus, we conclude that MeADK2 might play a vital role in energy homeostasis in cassava mitochondria. Copyright © 2016 Elsevier GmbH. All rights reserved.

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

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    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. Glutaminyl Cyclases as Novel Targets for the Treatment of Septic Arthritis

    Science.gov (United States)

    Hellvard, Annelie; Maresz, Katarzyna; Schilling, Stephan; Graubner, Sigrid; Heiser, Ulrich; Jonsson, Roland; Cynis, Holger; Demuth, Hans-Ulrich; Potempa, Jan; Mydel, Piotr

    2013-01-01

    Background. Septic arthritis is a severe and rapidly debilitating disease mainly caused by Staphylococcus aureus. Here, we assess the antiarthritic efficiency of glutaminyl cyclase (QC) inhibitors. Methods. Mice were inoculated with an arthritogenic amount of S. aureus intravenously or by local administration into the knee joint. Animals were treated with QC inhibitors (PBD155 and PQ529) via chow during the experiment. QC and isoQC knockout mice were also analyzed for arthritis symptoms after local administration of bacteria. Results. Both QC inhibitors significantly delayed the onset of clinical signs of arthritis, and inhibitors significantly decreased weight loss in treated animals. Following intraarticular injection of S. aureus, PBD155-treated mice had lower levels of synovitis and bone erosion, as well as less myeloperoxidase in synovial tissue. Fluorescence-activated cell sorter analysis revealed that PBD155 treatment affected the expression pattern of adhesion molecules, preventing the upregulation of cells expressing CD11b/CD18. Conclusion. The compounds investigated here represent a novel class of small molecular antiarthritic inhibitors. In our studies, they exerted strong antiinflammatory actions, and therefore they might be suited for disease-modifying treatment of infectious arthritis. PMID:23204180

  18. 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.

  19. Functional analysis of the sporulation-specific diadenylate cyclase CdaS in Bacillus thuringiensis

    Science.gov (United States)

    Zheng, Cao; Ma, Yang; Wang, Xun; Xie, Yuqun; Ali, Maria K.; He, Jin

    2015-01-01

    Cyclic di-AMP (c-di-AMP) is a recently discovered bacterial secondary messenger molecule, which is associated with various physiological functions. In the genus Bacillus, the intracellular level and turnover of c-di-AMP are mainly regulated by three diadenylate cyclases (DACs), including DisA, CdaA and CdaS, and two c-di-AMP-specific phosphodiesterases (GdpP and PgpH). In this study, we demonstrated that CdaS protein from B. thuringiensis is a hexameric DAC protein that can convert ATP or ADP to c-di-AMP in vitro and the N-terminal YojJ domain is essential for the DAC activity. Based on the markerless gene knock-out method, we demonstrated that the transcription of cdaS was initiated by the sporulation-specific sigma factor σH and the deletion of cdaS significantly delayed sporulation and parasporal crystal formation. These findings contrast with similar experiments conducted using B. subtilis, wherein transcription of its cdaS was initiated by the sigma factor σG. Deletion of all the three DAC genes from a single strain was unsuccessful, suggesting that c-di-AMP is an indispensable molecule in B. thuringiensis. Phylogenetic analysis indicated increased diversity of CdaS in the B. cereus and B. subtilis Bacillus subgroups. In summary, this study identifies important aspects in the regulation of c-di-AMP in the genus Bacillus. PMID:26441857

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

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    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.

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

    Science.gov (United States)

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

    2001-01-01

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

  2. 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

  3. Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

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    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

  4. A novel role of sesamol in inhibiting NF-κB-mediated signaling in platelet activation

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    Chang Chao-Chien

    2011-12-01

    Full Text Available Abstract Background Platelet activation is relevant to a variety of coronary heart diseases. Our previous studies revealed that sesamol possesses potent antiplatelet activity through increasing cyclic AMP formation. Although platelets are anucleated cells, they also express the transcription factor, NF-κB, that may exert non-genomic functions in platelet activation. Therefore, we further investigated the inhibitory roles of sesamol in NF-κB-mediated platelet function. Methods Platelet aggregation, Fura 2-AM fluorescence, and immunoblotting analysis were used in this study. Results NF-κB signaling events, including IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation, were markedly activated by collagen (1 μg/ml in washed human platelets, and these signaling events were attenuated by sesamol (2.5~25 μM. Furthermore, SQ22536 and ODQ, inhibitors of adenylate cyclase and guanylate cyclase, respectively, strongly reversed the sesamol (25 μM-mediated inhibitory effects of IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation stimulated by collagen. The protein kinase A (PKA inhibitor, H89, also reversed sesamol-mediated inhibition of IκBα degradation. Moreover, BAY11-7082, an NF-κB inhibitor, abolished IκBα degradation, phospholipase C (PLCγ2 phosphorylation, protein kinase C (PKC activation, [Ca2+]i mobilization, and platelet aggregation stimulated by collagen. Preincubation of platelets with the inhibitors, SQ22536 and H89, both strongly reversed sesamol-mediated inhibition of platelet aggregation and [Ca2+]i mobilization. Conclusions Sesamol activates cAMP-PKA signaling, followed by inhibition of the NF-κB-PLC-PKC cascade, thereby leading to inhibition of [Ca2+]i mobilization and platelet aggregation. Because platelet activation is not only linked to hemostasis, but also has a relevant role in inflammation and metastasis, our data demonstrating that inhibition of NF-κB interferes with platelet function may

  5. Crosstalk between adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism under red blood cell microrheological changes.

    Science.gov (United States)

    Muravyov, Alexei V; Tikhomirova, Irina A; Maimistova, Alla A; Bulaeva, Svetlana V; Zamishlayev, Andrey V; Batalova, Ekaterina A

    2010-01-01

    There are evidences that red blood cell (RBC) deformation and aggregation change under their incubation with catecholamines and it is connected with activation of intracellular signaling pathways. The present study was designed to explore the adenylyl cyclase signaling pathway and Ca2+ regulatory mechanism of RBCs together with their microrheological changes. The washed RBCs were resuspended in PBS. In each of the three research sessions RBC suspensions were divided into two aliquots: 1) control (without drug) and 2) with an appropriate drug. After cell incubation RBC deformability (RBCD) and aggregation (RBCA) were estimated. RBC incubation with catecholamines resulted in RBCD changes by 18-30%. RBCs incubation with forskolin facilitated an increase of RBCD by 17% (p RBCA; whereas red cell deformability was changed only slightly. On the other hand, Ca2+ entry blocking into the cells by verapamil has led to significant RBCA decrease and RBCD rise. The obtained results make us believe that RBCD change was closely associated with Ca2+ control mechanisms. An effect of Ca2+ concentration increase on RBC microrheology was removed, if it was preliminary added to incubation medium EGTA as Ca2+ chelator. It was found that all four PDE inhibitors: IBMX, vinpocetine, rolipram, pentoxifylline decreased RBCA significantly and, quite the contrary, they increased red cell deformability. Our data have shown that Ca2+ entry increase was accompanied by red cell aggregation rise, while adenylyl cyclase-cAMP system stimulation led to red cell deformability increase and its aggregation lowered. The crosstalk between two intracellular signaling systems is probably connected with phosphodiesterase activity.

  6. Multiple lineage specific expansions within the guanylyl cyclase gene family

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    O'Halloran Damien M

    2006-03-01

    Full Text Available Abstract Background Guanylyl cyclases (GCs are responsible for the production of the secondary messenger cyclic guanosine monophosphate, which plays important roles in a variety of physiological responses such as vision, olfaction, muscle contraction, homeostatic regulation, cardiovascular and nervous function. There are two types of GCs in animals, soluble (sGCs which are found ubiquitously in cell cytoplasm, and receptor (rGC forms which span cell membranes. The complete genomes of several vertebrate and invertebrate species are now available. These data provide a platform to investigate the evolution of GCs across a diverse range of animal phyla. Results In this analysis we located GC genes from a broad spectrum of vertebrate and invertebrate animals and reconstructed molecular phylogenies for both sGC and rGC proteins. The most notable features of the resulting phylogenies are the number of lineage specific rGC and sGC expansions that have occurred during metazoan evolution. Among these expansions is a large nematode specific rGC clade comprising 21 genes in C. elegans alone; a vertebrate specific expansion in the natriuretic receptors GC-A and GC-B; a vertebrate specific expansion in the guanylyl GC-C receptors, an echinoderm specific expansion in the sperm rGC genes and a nematode specific sGC clade. Our phylogenetic reconstruction also shows the existence of a basal group of nitric oxide (NO insensitive insect and nematode sGCs which are regulated by O2. This suggests that the primordial eukaryotes probably utilized sGC as an O2 sensor, with the ligand specificity of sGC later switching to NO which provides a very effective local cell-to-cell signalling system. Phylogenetic analysis of the sGC and bacterial heme nitric oxide/oxygen binding protein domain supports the hypothesis that this domain originated from a cyanobacterial source. Conclusion The most salient feature of our phylogenies is the number of lineage specific expansions

  7. Mechanism of adenylate kinase: Site-directed mutagenesis versus x-ray and NMR

    International Nuclear Information System (INIS)

    Tsai, Mingdaw; Yan, Honggao

    1991-01-01

    Controversy is an integral part of scientific research and is often a precursor to the truth. However, this lesson has been learned in a very hard way in the case of the structure-function relationship of adenylate kinase (AK), which catalyzes the interconversion between MgATP+AMP and MgADP+ADP. While this small kinase has been considered a model kinase and the enzyme-substrate interaction of AK was among the first investigated by X-ray crystallography and NMR the substrate binding sites deduced from the early studies by these two powerful techniques (termed the X-ray model and the NMR model, respectively) were dramatically different. Ironically, both models have had substantial impact on researchers in related fields. The problems have finally been dealt with since 1987 by the interplay between site-directed mutagenesis, X-ray, and NMR. The purpose of this review is not only to summarize the current knowledge in the structure-function relationship of adenylate kinase but also to accurately document and critically analyze historical developments in the hope that history will not be repeated

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

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    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

  9. Inhibition of Heat-Stable Toxin-Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors.

    Science.gov (United States)

    Bijvelds, Marcel J C; Loos, Michaela; Bronsveld, Inez; Hellemans, Ann; Bongartz, Jean-Pierre; Ver Donck, Luc; Cox, Eric; de Jonge, Hugo R; Schuurkes, Jan A J; De Maeyer, Joris H

    2015-12-01

    Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Germacrene C synthase from Lycopersicon esculentum cv. VFNT Cherry tomato: cDNA isolation, characterization, and bacterial expression of the multiple product sesquiterpene cyclase

    Science.gov (United States)

    Colby, Sheila M.; Crock, John; Dowdle-Rizzo, Barbara; Lemaux, Peggy G.; Croteau, Rodney

    1998-01-01

    Germacrene C was found by GC-MS and NMR analysis to be the most abundant sesquiterpene in the leaf oil of Lycopersicon esculentum cv. VFNT Cherry, with lesser amounts of germacrene A, guaia-6,9-diene, germacrene B, β-caryophyllene, α-humulene, and germacrene D. Soluble enzyme preparations from leaves catalyzed the divalent metal ion-dependent cyclization of [1-3H]farnesyl diphosphate to these same sesquiterpene olefins, as determined by radio-GC. To obtain a germacrene synthase cDNA, a set of degenerate primers was constructed based on conserved amino acid sequences of related terpenoid cyclases. With cDNA prepared from leaf epidermis-enriched mRNA, these primers amplified a 767-bp fragment that was used as a hybridization probe to screen the cDNA library. Thirty-one clones were evaluated for functional expression of terpenoid cyclase activity in Escherichia coli by using labeled geranyl, farnesyl, and geranylgeranyl diphosphates as substrates. Nine cDNA isolates expressed sesquiterpene synthase activity, and GC-MS analysis of the products identified germacrene C with smaller amounts of germacrene A, B, and D. None of the expressed proteins was active with geranylgeranyl diphosphate; however, one truncated protein converted geranyl diphosphate to the monoterpene limonene. The cDNA inserts specify a deduced polypeptide of 548 amino acids (Mr = 64,114), and sequence comparison with other plant sesquiterpene cyclases indicates that germacrene C synthase most closely resembles cotton δ-cadinene synthase (50% identity). PMID:9482865

  11. Nitroxyl (HNO stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation.

    Directory of Open Access Journals (Sweden)

    Eliane Q Lin

    Full Text Available New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP however has not been investigated.Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II in the presence and absence of the HNO donor Angeli's salt (sodium trioxodinitrate or B-type natriuretic peptide, BNP (all 1 µmol/L. Hypertrophic responses and its triggers, as well as cGMP signaling, were determined.We now demonstrate that Angeli's salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli's salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation, as well as p38 mitogen-activated protein kinase (p38MAPK. The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli's salt were mimicked by BNP. We also demonstrate that the effects of Angeli's salt are specifically mediated by HNO (with no role for NO• or nitrite, with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP.Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.

  12. cAMP-dependent kinase does not modulate the Slack sodium-activated potassium channel.

    Science.gov (United States)

    Nuwer, Megan O; Picchione, Kelly E; Bhattacharjee, Arin

    2009-09-01

    The Slack gene encodes a Na(+)-activated K(+) channel and is expressed in many different types of neurons. Like the prokaryotic Ca(2+)-gated K(+) channel MthK, Slack contains two 'regulator of K(+) conductance' (RCK) domains within its carboxy terminal, domains likely involved in Na(+) binding and channel gating. It also contains multiple consensus protein kinase C (PKC) and protein kinase A (PKA) phosphorylation sites and although regulated by protein kinase C (PKC) phosphorylation, modulation by PKA has not been determined. To test if PKA directly regulates Slack, nystatin-perforated patch whole-cell currents were recorded from a human embryonic kidney (HEK-293) cell line stably expressing Slack. Bath application of forskolin, an adenylate cyclase activator, caused a rapid and complete inhibition of Slack currents however, the inactive homolog of forskolin, 1,9-dideoxyforskolin caused a similar effect. In contrast, bath application of 8-bromo-cAMP did not affect the amplitude nor the activation kinetics of Slack currents. In excised inside-out patch recordings, direct application of the PKA catalytic subunit to patches did not affect the open probability of Slack channels nor was open probability affected by direct application of protein phosphatase 2B. Preincubation of cells with the protein kinase A inhibitor KT5720 also did not change current density. Finally, mutating the consensus phosphorylation site located between RCK domain 1 and domain 2 from serine to glutamate did not affect current activation kinetics. We conclude that unlike PKC, phosphorylation by PKA does not acutely modulate the function and gating activation kinetics of Slack channels.

  13. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    Science.gov (United States)

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. 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 : embryonic heart * embryotoxicity * adenylyl cyclase * G protein * beta-blocking agents Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 1.579, year: 2014

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

    Science.gov (United States)

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

    2016-04-01

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

  16. 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

  17. ADP-ribosylation by cholera toxin: functional analysis of a cellular system that stimulates the enzymic activity of cholera toxin fragment A/sub 1/

    Energy Technology Data Exchange (ETDEWEB)

    Gill, D.M.; Coburn, J.

    1987-10-06

    The authors have clarified relationships between cholera toxin, cholera toxin substrates, a membrane protein S that is required for toxin activity, and a soluble protein CF that is needed for the function of S. The toxin has little intrinsic ability to catalyze ADP-ribosylations unless it encounters the active form of the S protein, which is S liganded to GTP or to a GTP analogue. In the presence of CF, S x GTP forms readily, though reversibly, but a more permanent active species, S-guanosine 5'-O-(3-thiotriphosphate) (S x GTP..gamma..S), forms over a period of 10-15 min at 37/sup 0/C. Both guanosine 5'-O-(2-thiodiphosphate) and GTP block this quasi-permanent activation. Some S x GTP..gamma..S forms in membranes that are exposed to CF alone and then to GTP..gamma..S, with a wash in between, and it is possible that CF facilitates a G nucleotide exchange. S x GTP..gamma..S dissolved by nonionic detergents persists in solution and can be used to support the ADP-ribosylation of nucleotide-free substrates. In this circumstance, added guanyl nucleotides have no further effect. This active form of S is unstable, especially when heated, but the thermal inactivation above 45/sup 0/C is decreased by GTP..gamma..S. Active S is required equally for the ADP-ribosylation of all of cholera toxin's protein substrates, regardless of whether they bind GTP or not. They suggest that active S interacts directly with the enzymic A/sub 1/ fragments of cholera toxin and not with any toxin substrate. The activation and activity of S are independent of the state, or even the presence, of adenylate cyclase and seem to be involved with the cyclase system only via cholera toxin. S is apparently not related by function to certain other GTP binding proteins, including p21/sup ras/, and appears to be a new GTP binding protein whose physiologic role remains to be identified.

  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. Cloning and Functional Characterization of Three Branch Point Oxidosqualene Cyclases from Withania somnifera (L.) Dunal*

    Science.gov (United States)

    Dhar, Niha; Rana, Satiander; Razdan, Sumeer; Bhat, Wajid Waheed; Hussain, Aashiq; Dhar, Rekha S.; Vaishnavi, Samantha; Hamid, Abid; Vishwakarma, Ram; Lattoo, Surrinder K.

    2014-01-01

    Oxidosqualene cyclases (OSCs) positioned at a key metabolic subdividing junction execute indispensable enzymatic cyclization of 2,3-oxidosqualene for varied triterpenoid biosynthesis. Such branch points present favorable gene targets for redirecting metabolic flux toward specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for the desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full-length OSC cDNAs, β-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS), and cycloartenol synthase (WsOSC/CS), having open reading frames of 2289, 2268, and 2277 bp, were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses, and kinetic studies confirmed their monofunctionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript profiling under methyl jasmonate, gibberellic acid, and yeast extract elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by Western blotting. Negative regulation by yeast extract resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool toward WsOSC/CS for increased withanolide production. PMID:24770414

  20. Decreased Soluble Guanylate Cyclase Contributes to Cardiac Dysfunction Induced by Chronic Doxorubicin Treatment in Mice.

    Science.gov (United States)

    Vandenwijngaert, Sara; Swinnen, Melissa; Walravens, Ann-Sophie; Beerens, Manu; Gillijns, Hilde; Caluwé, Ellen; Tainsh, Robert E; Nathan, Daniel I; Allen, Kaitlin; Brouckaert, Peter; Bartunek, Jozef; Scherrer-Crosbie, Marielle; Bloch, Kenneth D; Bloch, Donald B; Janssens, Stefan P; Buys, Emmanuel S

    2017-02-01

    The use of doxorubicin, a potent chemotherapeutic agent, is limited by cardiotoxicity. We tested the hypothesis that decreased soluble guanylate cyclase (sGC) enzyme activity contributes to the development of doxorubicin-induced cardiotoxicity. Doxorubicin administration (20 mg/kg, intraperitoneally [IP]) reduced cardiac sGC activity in wild-type (WT) mice. To investigate whether decreased sGC activity contributes to doxorubicin-induced cardiotoxicity, we studied mice with cardiomyocyte-specific deficiency of the sGC α1-subunit (mice with cardiomyocyte-specific deletion of exon 6 of the sGCα1 allele [sGCα1 -/-CM ]). After 12 weeks of doxorubicin administration (2 mg/kg/week IP), left ventricular (LV) systolic dysfunction was greater in sGCα1 -/-CM than WT mice. To further assess whether reduced sGC activity plays a pathogenic role in doxorubicin-induced cardiotoxicity, we studied a mouse model in which decreased cardiac sGC activity was induced by cardiomyocyte-specific expression of a dominant negative sGCα1 mutant (DNsGCα1) upon doxycycline removal (Tet-off). After 8 weeks of doxorubicin administration, DNsGCα1 tg/+ , but not WT, mice displayed LV systolic dysfunction and dilatation. The difference in cardiac function and remodeling between DNsGCα1 tg/+ and WT mice was even more pronounced after 12 weeks of treatment. Further impairment of cardiac function was attenuated when DNsGCα1 gene expression was inhibited (beginning at 8 weeks of doxorubicin treatment) by administering doxycycline. Furthermore, doxorubicin-associated reactive oxygen species generation was higher in sGCα1-deficient than WT hearts. Innovation and Conclusion: These data demonstrate that a reduction in cardiac sGC activity worsens doxorubicin-induced cardiotoxicity in mice and identify sGC as a potential therapeutic target. Various pharmacological sGC agonists are in clinical development or use and may represent a promising approach to limit doxorubicin-associated cardiotoxicity

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

    Science.gov (United States)

    Sharma, Rameshwar K; Duda, Teresa

    2014-01-01

    A sequel to these authors' earlier comprehensive reviews which covered the field of mammalian membrane guanylate cyclase (MGC) from its origin to the year 2010, this article contains 13 sections. The first is historical and covers MGC from the year 1963-1987, summarizing its colorful developmental stages from its passionate pursuit to its consolidation. The second deals with the establishment of its biochemical identity. MGC becomes the transducer of a hormonal signal and founder of the peptide hormone receptor family, and creates the notion that hormone signal transduction is its sole physiological function. The third defines its expansion. The discovery of ROS-GC subfamily is made and it links ROS-GC with the physiology of phototransduction. Sections ROS-GC, a Ca(2+)-Modulated Two Component Transduction System to Migration Patterns and Translations of the GCAP Signals Into Production of Cyclic GMP are Different cover its biochemistry and physiology. The noteworthy events are that augmented by GCAPs, ROS-GC proves to be a transducer of the free Ca(2+) signals generated within neurons; ROS-GC becomes a two-component transduction system and establishes itself as a source of cyclic GMP, the second messenger of phototransduction. Section ROS-GC1 Gene Linked Retinal Dystrophies demonstrates how this knowledge begins to be translated into the diagnosis and providing the molecular definition of retinal dystrophies. Section Controlled By Low and High Levels of [Ca(2+)]i, ROS-GC1 is a Bimodal Transduction Switch discusses a striking property of ROS-GC where it becomes a "[Ca(2+)]i bimodal switch" and transcends its signaling role in other neural processes. In this course, discovery of the first CD-GCAP (Ca(2+)-dependent guanylate cyclase activator), the S100B protein, is made. It extends the role of the ROS-GC transduction system beyond the phototransduction to the signaling processes in the synapse region between photoreceptor and cone ON-bipolar cells; in section Ca(2

  2. Development of novel biosensors to study receptor-mediated activation of the G-protein α subunits Gsand Golf.

    Science.gov (United States)

    Yano, Hideaki; Provasi, Davide; Cai, Ning Sheng; Filizola, Marta; Ferré, Sergi; Javitch, Jonathan A

    2017-12-08

    Gα s (G s ) and Gα olf (G olf ) are highly homologous G-protein α subunits that activate adenylate cyclase, thereby serving as crucial mediators of intracellular signaling. Because of their dramatically different brain expression patterns, we studied similarities and differences between their activation processes with the aim of comparing their receptor coupling mechanisms. We engineered novel luciferase- and Venus-fused Gα constructs that can be used in bioluminescence resonance energy transfer assays. In conjunction with molecular simulations, these novel biosensors were used to determine receptor activation-induced changes in conformation. Relative movements in G s were consistent with the crystal structure of β 2 adrenergic receptor in complex with G s Conformational changes in G olf activation are shown to be similar to those in G s Overall the current study reveals general similarities between G s and G olf activation at the molecular level and provides a novel set of tools to search for G s - and G olf -specific receptor pharmacology. In view of the wide functional and pharmacological roles of G s - and G olf -coupled dopamine D 1 receptor and adenosine A 2A receptor in the brain and other organs, elucidating their differential structure-function relationships with G s and G olf might provide new approaches for the treatment of a variety of neuropsychiatric disorders. In particular, these novel biosensors can be used to reveal potentially therapeutic dopamine D 1 receptor and adenosine A 2A receptor ligands with functionally selective properties between G s and G olf signaling.

  3. The Subcellular Dynamics of the Gs-Linked Receptor GPR3 Contribute to the Local Activation of PKA in Cerebellar Granular Neurons.

    Science.gov (United States)

    Miyagi, Tatsuhiro; Tanaka, Shigeru; Hide, Izumi; Shirafuji, Toshihiko; Sakai, Norio

    2016-01-01

    G-protein-coupled receptor (GPR) 3 is a member of the GPR family that constitutively activates adenylate cyclase. We have reported that the expression of GPR3 in cerebellar granular neurons (CGNs) contributes to neurite outgrowth and modulates neuronal proliferation and survival. To further identify its role, we have analyzed the precise distribution and local functions of GPR3 in neurons. The fluorescently tagged GPR3 protein was distributed in the plasma membrane, the Golgi body, and the endosomes. In addition, we have revealed that the plasma membrane expression of GPR3 functionally up-regulated the levels of PKA, as measured by a PKA FRET indicator. Next, we asked if the PKA activity was modulated by the expression of GPR3 in CGNs. PKA activity was highly modulated at the neurite tips compared to the soma. In addition, the PKA activity at the neurite tips was up-regulated when GPR3 was transfected into the cells. However, local PKA activity was decreased when endogenous GPR3 was suppressed by a GPR3 siRNA. Finally, we determined the local dynamics of GPR3 in CGNs using time-lapse analysis. Surprisingly, the fluorescent GPR3 puncta were transported along the neurite in both directions over time. In addition, the anterograde movements of the GPR3 puncta in the neurite were significantly inhibited by actin or microtubule polymerization inhibitors and were also disturbed by the Myosin II inhibitor blebbistatin. Moreover, the PKA activity at the tips of the neurites was decreased when blebbistatin was administered. These results suggested that GPR3 was transported along the neurite and contributed to the local activation of PKA in CGN development. The local dynamics of GPR3 in CGNs may affect local neuronal functions, including neuronal differentiation and maturation.

  4. Selective splitting of 3'-adenylated dinucleoside polyphosphates by specific enzymes degrading dinucleoside polyphosphates.

    Science.gov (United States)

    Guranowski, Andrzej; Sillero, Antonio; Günther Sillero, María Antonia

    2003-01-01

    Several 3'-[(32)P]adenylated dinucleoside polyphosphates (Np(n)N'p*As) were synthesized by the use of poly(A) polymerase (Sillero MAG et al., 2001, Eur J Biochem.; 268: 3605-11) and three of them, ApppA[(32)P]A or ApppAp*A, AppppAp*A and GppppGp*A, were tested as potential substrates of different dinucleoside polyphosphate degrading enzymes. Human (asymmetrical) dinucleoside tetraphosphatase (EC 3.6.1.17) acted almost randomly on both AppppAp*A, yielding approximately equal amounts of pppA + pAp*A and pA + pppAp*A, and GppppGp*, yielding pppG + pGp*A and pG + pppGp*A. Narrow-leafed lupin (Lupinus angustifolius) tetraphosphatase acted preferentially on the dinucleotide unmodified end of both AppppAp*A (yielding 90% of pppA + pAp*A and 10 % of pA + pppAp*A) and GppppGp*A (yielding 89% pppG + pGp*A and 11% of pG + pppGp*A). (Symmetrical) dinucleoside tetraphosphatase (EC 3.6.1.41) from Escherichia coli hydrolyzed AppppAp*A and GppppGp*A producing equal amounts of ppA + ppAp*A and ppG + ppGp*A, respectively, and, to a lesser extent, ApppAp*A producing pA + ppAp*A. Two dinucleoside triphosphatases (EC 3.6.1.29) (the human Fhit protein and the enzyme from yellow lupin (Lupinus luteus)) and dinucleoside tetraphosphate phosphorylase (EC 2.7.7.53) from Saccharomyces cerevisiae did not degrade the three 3'-adenylated dinucleoside polyphosphates tested.

  5. 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.

  6. 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.

  7. 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.

  8. A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

    Directory of Open Access Journals (Sweden)

    Chen Yun-Ru

    2012-09-01

    Full Text Available Abstract Background Deep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available. Results We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum using the T4 RNA ligase 1 adenylated adapter. Conclusion We provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.

  9. Characterization of the Human ?1?1 Soluble Guanylyl Cyclase Promoter

    OpenAIRE

    Marro, Mart?n L.; Peir?, Concepci?n; Panayiotou, Catherine M.; Baliga, Reshma S.; Meurer, Sabine; Schmidt, Harald H. H. W.; Hobbs, Adrian J.

    2008-01-01

    Soluble guanylyl cyclase (sGC) is the principal receptor for NO and plays a ubiquitous role in regulating cellular function. This is exemplified in the cardiovascular system where sGC governs smooth muscle tone and growth, vascular permeability, leukocyte flux, and platelet aggregation. As a consequence, aberrant NO-sGC signaling has been linked to diseases including hypertension, atherosclerosis, and stroke. Despite these key (patho)physiological roles, little is known about the expressional...

  10. Antiarrhythmic effect of prolonged morphine exposure is accompanied by altered myocardial adenylyl cyclase signaling in rats

    Czech Academy of Sciences Publication Activity Database

    Škrabalová, J.; Neckář, Jan; Hejnová, L.; Bartoňová, I.; Kolář, František; Novotný, J.

    2012-01-01

    Roč. 64, č. 2 (2012), s. 351-359 ISSN 1734-1140 R&D Projects: GA AV ČR(CZ) IAA501110901 Grant - others:Univerzita Karlova(CZ) 429511 Institutional research plan: CEZ:AV0Z50110509 Keywords : rat myocardium * morphine * adenylyl cyclase * G-proteins * arrhythmias Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 1.965, year: 2012

  11. Chemical shift assignments of polyketide cyclase_like protein CGL2373 from Corynebacterium glutamicum.

    Science.gov (United States)

    Liang, Chunjie; Hu, Rui; Ramelot, Theresa A; Kennedy, Michael A; Li, Xuegang; Yang, Yunhuang; Zhu, Jiang; Liu, Maili

    2017-10-01

    Protein CGL2373 from Corynebacterium glutamicum, which is 155 amino acids long and 17.7 kDa, is a member of the polyketide_cyc2 family. As a potential polyketide cyclase, it may play an important role in the biosynthesis of aromatic polyketides that are the source of many bioactive molecules. Here we report the complete 1 H, 13 C and 15 N chemical shift assignments of CGL2373, which lays a foundation for further structural and functional research.

  12. 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

  13. A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

    Directory of Open Access Journals (Sweden)

    Lauren Forbes

    Full Text Available Mycobacterium tuberculosis (Mtb is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.

  14. Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

    Energy Technology Data Exchange (ETDEWEB)

    Azarkan, Mohamed [Laboratoire de Chimie Générale I, Faculté de Médecine-ULB CP609, 808 Route de Lennik, B-1070 Brussels (Belgium); Clantin, Bernard; Bompard, Coralie [CNRS-UMR 8525, Institut de Biologie de Lille, BP 477, 1 Rue du Professeur Calmette, F-59021 Lille (France); Belrhali, Hassan [EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP 181, F-38042 Grenoble CEDEX 9 (France); Baeyens-Volant, Danielle [Laboratoire de Chimie Générale I, Faculté de Médecine-ULB CP609, 808 Route de Lennik, B-1070 Brussels (Belgium); Looze, Yvan [Laboratoire de Chimie Générale, Institut de Pharmacie-ULB CP206/04, Boulevard du Triomphe, B-1050 Brussels (Belgium); Villeret, Vincent, E-mail: vincent.villeret@ibl.fr [CNRS-UMR 8525, Institut de Biologie de Lille, BP 477, 1 Rue du Professeur Calmette, F-59021 Lille (France); Wintjens, René, E-mail: vincent.villeret@ibl.fr [Laboratoire de Chimie Générale, Institut de Pharmacie-ULB CP206/04, Boulevard du Triomphe, B-1050 Brussels (Belgium); Laboratoire de Chimie Générale I, Faculté de Médecine-ULB CP609, 808 Route de Lennik, B-1070 Brussels (Belgium)

    2005-01-01

    The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution. In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å.

  15. Linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with TEV protease and autonomous N-terminal cyclization with glutaminyl cyclase in vivo.

    Directory of Open Access Journals (Sweden)

    Yan-Ping Shih

    Full Text Available Overproduction of N-terminal pyroglutamate (pGlu-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I, and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.

  16. Membrane Guanylate Cyclase, A Multimodal Transduction Machine: History, Present and Future Directions

    Directory of Open Access Journals (Sweden)

    Rameshwar K Sharma

    2014-07-01

    Full Text Available A sequel to these authors’ earlier comprehensive reviews which covered the field of mammalian membrane guanylate cyclase (MGC from its origin to the year 2010, this article contains 13 parts. The first is HISTORICAL and covers MGC from the year 1963-1987, summarizing its colorful developmental stages from its passionate pursuit to its consolidation. The second deals with the establishment of its BIOCHEMICAL IDENTITY. MGC becomes the transducer of a hormonal signal and founder of the peptide hormone receptor family, and creates the notion that hormone signal transduction is its sole physiological function. The third defines its EXPANSION. The discovery of ROS-GC subfamily is made and it links ROS-GC with the physiology of PHOTOTRANSDUCTION. Parts 4 to 7 cover its BIOCHEMISTRY and PHYSIOLOGY. The noteworthy events are that augmented by GCAPs, ROS-GC proves to be a transducer of the free Ca2+ signals generated within neurons; ROS-GC becomes a two-component transduction system and establishes itself as a source of cyclic GMP, the second messenger of phototransduction. Part 8 demonstrates how this knowledge begins to be TRANSLATED into the diagnosis and providing the molecular definition of retinal dystrophies. Part 9 discusses a striking property of ROS-GC where it becomes a [Ca2+]i bimodal switch and transcends its signaling role in other neural transduction processes. In this course, discovery of the first CD-GCAP (Ca2+-dependent guanylate cycles activator, the S100B protein, is made. It extends the role of ROS-GC transduction system beyond the photoreceptor cells to the signaling processes in the synapse region between photoreceptor and cone ON-bipolar cells; in Part 10, discovery of ANOTHER CD-GCAP, NC, is made and its linkage with signaling of the inner plexiform layer neurons is established. Part 11 discusses linkage of the ROS-GC transduction system with other sensory transduction processes: Pineal gland, Olfaction and Gustation. In the

  17. Carotenoid analysis of sweetpotato Ipomoea batatas and functional identification of its lycopene β- and ε-cyclase genes.

    Science.gov (United States)

    Khan, Muhammad Zubair; Takemura, Miho; Maoka, Takashi; Otani, Motoyasu; Misawa, Norihiko

    2016-09-01

    Sweetpotato Ipomoea batatas is known as a hexaploid species. Here, we analyzed carotenoids contained in the leaves and tubers of sweetpotato cultivars 'White Star' (WS) and W71. These cultivars were found to contain several carotenoids unique to sweetpotato tubers such as β-carotene-5,6,5',8'-diepoxide and β-carotene-5,8-epoxide. Next, we isolated two kinds of carotene cyclase genes that encode lycopene β- and ε-cyclases from the WS and W71 leaves, by RT-PCR and subsequent RACE. Two and three lycopene β-cyclase gene sequences were, respectively, isolated from WS, named IbLCYb1, 2, and from W71, IbLCYb3, 4, 5. Meanwhile, only a single lycopene ε-cyclase gene sequence, designated IbLCYe, was isolated from both WS and W71. These genes were separately introduced into a lycopene-synthesizing Escherichia coli transformed with the Pantoea ananatis crtE, crtB and crtI genes, followed by HPLC analysis. β-Carotene was detected in E. coli cells that carried IbLCYb1-4, indicating that the IbLCYb1-4 genes encode lycopene β-cyclase. Meanwhile, the introduction of IbLCYe into the lycopene-synthesizing E. coli led to efficient production of δ-carotene with a monocyclic ε-ring, providing evidence that the IbLCYe gene codes for lycopene ε-(mono)cyclase. Expression of the β- and ε-cyclase genes was analyzed as well.

  18. Combinatorial effects of genistein and sex-steroids on the level of cystic fibrosis transmembrane regulator (CFTR), adenylate cyclase (AC) and cAMP in the cervix of ovariectomised rats.

    Science.gov (United States)

    Salleh, Naguib; Ismail, Nurain; Muniandy, Sekaran; Korla, Praveen Kumar; Giribabu, Nelli

    2015-12-01

    The combinatorial effects of genistein and estrogen (E) or estrogen plus progesterone (E+P) on CFTR, AC and cAMP levels in cervix were investigated. Ovariectomised adult female rats received 50 or 100mg/kg/day genistein with E or E followed by E+P [E+(E+P)] for seven consecutive days. Cervixes were harvested and analyzed for CFTR mRNA levels by Real-time PCR. Distribution of AC and CFTR proteins in endocervix were observed by immunohistochemistry. Levels of cAMP were measured by enzyme-immunoassay. Molecular docking predicted interaction between genistein and AC. Our results indicate that levels of CFTR, AC and cAMP in cervix of rats receiving genistein plus E were higher than E-only treatment (pcervix of E and E+(E+P)-treated rats by genistein could affect the cervical secretory function which could influence the female reproductive processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Immunization with a Circumsporozoite Epitope Fused to Bordetella pertussis Adenylate Cyclase in Conjunction with Cytotoxic T-Lymphocyte-Associated Antigen 4 Blockade Confers Protection against Plasmodium berghei Liver-Stage Malaria

    Czech Academy of Sciences Publication Activity Database

    Tartz, S.; Kamanová, Jana; Šimšová, Marcela; Šebo, Peter; Bolte, S.; Heussler, V.; Fleischer, B.; Jacobs, T.

    2006-01-01

    Roč. 74, č. 4 (2006), s. 2277-2285 ISSN 0019-9567 R&D Projects: GA AV ČR IBS5020311 Institutional research plan: CEZ:AV0Z50200510 Keywords : plasmodium berghei * immunity * malaria Subject RIV: EE - Microbiology, Virology Impact factor: 4.004, year: 2006

  20. Delivery of a MalE CD4+-T-Cell Epitope into the Major Histocompatibility Complex Class II Antigen Presentation Pathway by Bordetella pertussis Adenylate Cyclase ral NPKSupply

    Czech Academy of Sciences Publication Activity Database

    Loucká, Jiřina; Schlecht, G.; Vojtová, Jana; Leclerc, C.; Šebo, Peter

    2002-01-01

    Roč. 70, č. 2 (2002), s. 1002-1005 ISSN 0019-9567 R&D Projects: GA ČR GA310/01/0934; GA AV ČR IAA5020907; GA MŠk ME 167 Grant - others:QLK2-CT(US) 00556 Institutional research plan: CEZ:AV0Z5020903 Keywords : delivery * epitope * complex Subject RIV: EE - Microbiology, Virology Impact factor: 4.039, year: 2002

  1. Delivery of CD8+ T-cell epitopes into major histocompatibility complex class I antigen presentation pathway by Bordetella pertussis adenylate cyclase:delineation of cell invasive structures and permissive insertion sites

    Czech Academy of Sciences Publication Activity Database

    Osička, Radim; Osičková, Adriana; Basar, T.; Guermonprez, P.; Rojas, M.; Leclerc, C.; Šebo, Peter

    2000-01-01

    Roč. 68, č. 1 (2000), s. 247-256 ISSN 0019-9567 R&D Projects: GA ČR GA310/98/0432; GA AV ČR IAA5020907; GA MŠk VS96149; GA MŠk ME 167 Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EE - Microbiology, Virology Impact factor: 4.204, year: 2000

  2. Recombinant adenylate kinase 3 from liver fluke Clonorchis sinensis for histochemical analysis and serodiagnosis of clonorchiasis.

    Science.gov (United States)

    Kwon, Soon Bin; Kim, Paul; Woo, Hae Sun; Kim, Tae Yun; Kim, Ju Yeong; Lee, Hye Min; Jang, Yun Soo; Kim, Eun-Min; Yong, Tai-Soon; Seong, Baik Lin

    2018-03-27

    Due to the lack of an effective prophylactic intervention and diagnosis, human liver fluke Clonorchis sinensis continues to afflict a large human population, causing a chronic inflammatory bile duct disease. With an aim to identify target antigens for sensitive serodiagnosis, adenylate kinase 3 of C. sinensis (CsAK3) was successfully expressed in soluble form in Escherichia coli by fusion to an RNA-interacting domain derived from human Lys-tRNA synthetase and purified by Ni2+-affinity chromatography. Anti-CsAK3 serum was raised by immunization of mice, and Western blotting confirmed that CsAK3 was expressed in adult-stage C. sinensis. Histochemical analysis showed that CsAK3 was localized to the subtegumental tissue of C. sinensis and was excreted into the bile duct of the host. When tested against sera from various parasite-infected patients by enzyme-linked immunosorbent assay, the recombinant CsAK3 elicited a specific response to C. sinensis-infected sera. The results suggest that CsAK3, either alone or in combination with other antigens, could be used for improving the clinical diagnosis of clonorchiasis.

  3. [Effect of Qishen Yiqi droplet on adenylate contents in myocardium of cor pulmonale rats].

    Science.gov (United States)

    Geng, Shi-Han; Shan, Qiu-Yue; Xu, Meng-Xi; Zhang, Yi-Dan; Song, Li-Li; Hou, Ning; Zhang, Wen; Zhang, Mi-Xia; Zhuang, Peng-Wei; Zhang, Yan-Jun

    2017-01-01

    Cor pulmonale rat models were induced by a single intraperitoneal injection of monocrotaline(MCT), and the sham group received a single intraperitioneal injection of normal saline. After the model rats received intragastric administration of Qishen Yiqi droplet(QS) for 6 weeks, the contents of adenylate(ATP, ADP and AMP) in right myocardial tissues were measured by HPLC, and then the metabolism changes in myocardium of cor pulmonale rats with QS were investigated. The results showed that ATP, ADP, and AMP were well separated, with a good linearity within a certain range of concentration; and the recovery rates were within the range of 90%-108%. As compared with model group, the level of ATP was significantly elevated in high-dose treatment group; ADP contents showed an increasing trend and AMP contents showed a decreasing trend, indicating that QS could significantly improve energy metabolism system in myocardium. By using the HPLC, a qualitative and quantitative analysis method was given for the determination of ATP, ADP and AMP contents in myocardium, providing a method for energy metabolism measurement in biological samples. Copyright© by the Chinese Pharmaceutical Association.

  4. Deciphering hierarchical features in the energy landscape of adenylate kinase folding/unfolding

    Science.gov (United States)

    Taylor, J. Nicholas; Pirchi, Menahem; Haran, Gilad; Komatsuzaki, Tamiki

    2018-03-01

    Hierarchical features of the energy landscape of the folding/unfolding behavior of adenylate kinase, including its dependence on denaturant concentration, are elucidated in terms of single-molecule fluorescence resonance energy transfer (smFRET) measurements in which the proteins are encapsulated in a lipid vesicle. The core in constructing the energy landscape from single-molecule time-series across different denaturant concentrations is the application of rate-distortion theory (RDT), which naturally considers the effects of measurement noise and sampling error, in combination with change-point detection and the quantification of the FRET efficiency-dependent photobleaching behavior. Energy landscapes are constructed as a function of observation time scale, revealing multiple partially folded conformations at small time scales that are situated in a superbasin. As the time scale increases, these denatured states merge into a single basin, demonstrating the coarse-graining of the energy landscape as observation time increases. Because the photobleaching time scale is dependent on the conformational state of the protein, possible nonequilibrium features are discussed, and a statistical test for violation of the detailed balance condition is developed based on the state sequences arising from the RDT framework.

  5. In vitro activation of CMV-specific human CD8ţ T cells by adenylate

    Czech Academy of Sciences Publication Activity Database

    Jelínek, J.; Adkins, Irena; Mikulková, Z.; Jagosová, J.; Pacasová, R.; Michličková, S.; Šebo, Peter; Michálek, J.

    2012-01-01

    Roč. 47, č. 2 (2012), s. 243-250 ISSN 0268-3369 R&D Projects: GA MŠk 2B06161; GA ČR GP310/09/P582 Institutional research plan: CEZ:AV0Z50200510 Keywords : T cells * antigenic peptide epitopes * CyaA toxoid Subject RIV: EE - Microbiology, Virology Impact factor: 3.541, year: 2012

  6. The soluble guanylate cyclase CYG12 is required for the acclimation to hypoxia and trophic regimes in Chlamydomonas reinhardtii.

    Science.gov (United States)

    Düner, Melis; Lambertz, Jan; Mügge, Carolin; Hemschemeier, Anja

    2018-01-01

    Oxygenic phototrophs frequently encounter environmental conditions that result in intracellular energy crises. Growth of the unicellular green alga Chlamydomonas reinhardtii in hypoxia in the light depends on acclimatory responses of which the induction of photosynthetic cyclic electron flow is essential. The microalga cannot grow in the absence of molecular oxygen (O 2 ) in the dark, although it possesses an elaborate fermentation metabolism. Not much is known about how the microalga senses and signals the lack of O 2 or about its survival strategies during energy crises. Recently, nitric oxide (NO) has emerged to be required for the acclimation of C. reinhardtii to hypoxia. In this study, we show that the soluble guanylate cyclase (sGC) CYG12, a homologue of animal NO sensors, is also involved in this response. CYG12 is an active sGC, and post-transcriptional down-regulation of the CYG12 gene impairs hypoxic growth and gene expression in C. reinhardtii. However, it also results in a disturbed photosynthetic apparatus under standard growth conditions and the inability to grow heterotrophically. Transcriptome profiles indicate that the mis-expression of CYG12 results in a perturbation of responses that, in the wild-type, maintain the cellular energy budget. We suggest that CYG12 is required for the proper operation of the photosynthetic apparatus which, in turn, is essential for survival in hypoxia and darkness. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  7. Synthesis of Triazole-Linked Analogues of c-di-GMP and Their Interactions with Diguanylate Cyclase.

    Science.gov (United States)

    Fernicola, Silvia; Torquati, Ilaria; Paiardini, Alessandro; Giardina, Giorgio; Rampioni, Giordano; Messina, Marco; Leoni, Livia; Del Bello, Fabio; Petrelli, Riccardo; Rinaldo, Serena; Cappellacci, Loredana; Cutruzzolà, Francesca

    2015-10-22

    Cyclic di-GMP (c-di-GMP) is a widespread second messenger that plays a key role in bacterial biofilm formation. The compound's ability to assume multiple conformations allows it to interact with a diverse set of target macromolecules. Here, we analyzed the binding mode of c-di-GMP to the allosteric inhibitory site (I-site) of diguanylate cyclases (DGCs) and compared it to the conformation adopted in the catalytic site of the EAL phosphodiesterases (PDEs). An array of novel molecules has been designed and synthesized by simplifying the native c-di-GMP structure and replacing the charged phosphodiester backbone with an isosteric nonhydrolyzable 1,2,3-triazole moiety. We developed the first neutral small molecule able to selectively target DGCs discriminating between the I-site of DGCs and the active site of PDEs; this molecule represents a novel tool for mechanistic studies, particularly on those proteins bearing both DGC and PDE modules, and for future optimization studies to target DGCs in vivo.

  8. Insights into the role of RD3 in guanylate cyclase trafficking, photoreceptor degeneration and Leber Congenital Amaurosis

    Directory of Open Access Journals (Sweden)

    Robert S. Molday

    2014-05-01

    Full Text Available RD3 is an evolutionarily conserved 23 kDa protein expressed in rod and cone photoreceptor cells. Mutations in the gene encoding RD3 resulting in unstable non-functional C-terminal truncated proteins are responsible for early onset photoreceptor degeneration in Leber Congenital Amaurosis 12 (LCA12 patients, the rd3 mice, and the rcd2 collies. Recent studies have shown that RD3 interacts with guanylate cyclases GC1 and GC2 in retinal cell extracts and HEK293 cells co-expressing GC and RD3. This interaction inhibits GC catalytic activity and promotes the exit of GC1 and GC2 from the endoplasmic reticulum and their trafficking to photoreceptor outer segments. Adeno-associated viral vector delivery of the normal RD3 gene to photoreceptors of the Rd3 mouse restores GC1 and GC2 expression and outer segment localization and leads to the long-term recovery of visual function and photoreceptor cell survival. This review focuses on the genetic and biochemical studies that have provided insight into the role of RD3 in photoreceptor function and survival.

  9. 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.

  10. The receptor guanylyl cyclase type D (GC-D) ligand uroguanylin promotes the acquisition of food preferences in mice.

    Science.gov (United States)

    Arakawa, Hiroyuki; Kelliher, Kevin R; Zufall, Frank; Munger, Steven D

    2013-06-01

    Rodents rely on olfactory stimuli to communicate information between conspecifics that is critical for health and survival. For example, rodents that detect a food odor simultaneously with the social odor carbon disulfide (CS(2)) will acquire a preference for that food. Disruption of the chemosensory transduction cascade in CS(2-)sensitive olfactory sensory neurons (OSNs) that express the receptor guanylyl cyclase type D (GC-D; GC-D+ OSNs) will prevent mice from acquiring these preferences. GC-D+ OSNs also respond to the natriuretic peptide uroguanylin, which is excreted into urine and feces. We analyzed if uroguanylin could also act as a social stimulus to promote the acquisition of food preferences. We found that feces of mice that had eaten odored food, but not unodored food, promoted a strong preference for that food in mice exposed to the feces. Olfactory exploration of uroguanylin presented with a food odor similarly produced a preference that was absent when mice were exposed to the food odor alone. Finally, the acquisition of this preference was dependent on GC-D+ OSNs, as mice lacking GC-D (Gucy2d(-)(/-) mice) showed no preference for the demonstrated food. Together with our previous findings, these results demonstrate that the diverse activators of GC-D+ OSNs elicit a common behavioral result and suggest that this specialized olfactory subsystem acts as a labeled line for a type of associative olfactory learning.

  11. Natural Products from Microalgae with Potential against Alzheimer’s Disease: Sulfolipids Are Potent Glutaminyl Cyclase Inhibitors

    Directory of Open Access Journals (Sweden)

    Stephanie Hielscher-Michael

    2016-11-01

    Full Text Available In recent years, many new enzymes, like glutaminyl cyclase (QC, could be associated with pathophysiological processes and represent targets for many diseases, so that enzyme-inhibiting properties of natural substances are becoming increasingly important. In different studies, the pathophysiology connection of QC to various diseases including Alzheimer’s disease (AD was described. Algae are known for the ability to synthesize complex and highly-diverse compounds with specific enzyme inhibition properties. Therefore, we screened different algae species for the presence of QC inhibiting metabolites using a new “Reverse Metabolomics” technique including an Activity-correlation Analysis (AcorA, which is based on the correlation of bioactivities to mass spectral data with the aid of mathematic informatics deconvolution. Thus, three QC inhibiting compounds from microalgae belonging to the family of sulfolipids were identified. The compounds showed a QC inhibition of 81% and 76% at concentrations of 0.25 mg/mL and 0.025 mg/mL, respectively. Thus, for the first time, sulfolipids are identified as QC inhibiting compounds and possess substructures with the required pharmacophore qualities. They represent a new lead structure for QC inhibitors.

  12. Genetic Ablation of Type III Adenylyl Cyclase Exerts Region-Specific Effects on Cilia Architecture in the Mouse Nose.

    Directory of Open Access Journals (Sweden)

    Rosemary C Challis

    Full Text Available We recently reported that olfactory sensory neurons in the dorsal zone of the mouse olfactory epithelium exhibit drastic location-dependent differences in cilia length. Furthermore, genetic ablation of type III adenylyl cyclase (ACIII, a key olfactory signaling protein and ubiquitous marker for primary cilia, disrupts the cilia length pattern and results in considerably shorter cilia, independent of odor-induced activity. Given the significant impact of ACIII on cilia length in the dorsal zone, we sought to further investigate the relationship between cilia length and ACIII level in various regions throughout the mouse olfactory epithelium. We employed whole-mount immunohistochemical staining to examine olfactory cilia morphology in phosphodiesterase (PDE 1C-/-;PDE4A-/- (simplified as PDEs-/- hereafter and ACIII-/- mice in which ACIII levels are reduced and ablated, respectively. As expected, PDEs-/- animals exhibit dramatically shorter cilia in the dorsal zone (i.e., where the cilia pattern is found, similar to our previous observation in ACIII-/- mice. Remarkably, in a region not included in our previous study, ACIII-/- animals (but not PDEs-/- mice have dramatically elongated, comet-shaped cilia, as opposed to characteristic star-shaped olfactory cilia. Here, we reveal that genetic ablation of ACIII has drastic, location-dependent effects on cilia architecture in the mouse nose. These results add a new dimension to our current understanding of olfactory cilia structure and regional organization of the olfactory epithelium. Together, these findings have significant implications for both cilia and sensory biology.

  13. Genetic Ablation of Type III Adenylyl Cyclase Exerts Region-Specific Effects on Cilia Architecture in the Mouse Nose.

    Science.gov (United States)

    Challis, Rosemary C; Tian, Huikai; Yin, Wenbin; Ma, Minghong

    2016-01-01

    We recently reported that olfactory sensory neurons in the dorsal zone of the mouse olfactory epithelium exhibit drastic location-dependent differences in cilia length. Furthermore, genetic ablation of type III adenylyl cyclase (ACIII), a key olfactory signaling protein and ubiquitous marker for primary cilia, disrupts the cilia length pattern and results in considerably shorter cilia, independent of odor-induced activity. Given the significant impact of ACIII on cilia length in the dorsal zone, we sought to further investigate the relationship between cilia length and ACIII level in various regions throughout the mouse olfactory epithelium. We employed whole-mount immunohistochemical staining to examine olfactory cilia morphology in phosphodiesterase (PDE) 1C-/-;PDE4A-/- (simplified as PDEs-/- hereafter) and ACIII-/- mice in which ACIII levels are reduced and ablated, respectively. As expected, PDEs-/- animals exhibit dramatically shorter cilia in the dorsal zone (i.e., where the cilia pattern is found), similar to our previous observation in ACIII-/- mice. Remarkably, in a region not included in our previous study, ACIII-/- animals (but not PDEs-/- mice) have dramatically elongated, comet-shaped cilia, as opposed to characteristic star-shaped olfactory cilia. Here, we reveal that genetic ablation of ACIII has drastic, location-dependent effects on cilia architecture in the mouse nose. These results add a new dimension to our current understanding of olfactory cilia structure and regional organization of the olfactory epithelium. Together, these findings have significant implications for both cilia and sensory biology.

  14. A comprehensive survey of 3' animal miRNA modification events and a possible role for 3' adenylation in modulating miRNA targeting effectiveness.

    Science.gov (United States)

    Burroughs, A Maxwell; Ando, Yoshinari; de Hoon, Michiel J L; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O

    2010-10-01

    Animal microRNA sequences are subject to 3' nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3' adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1-EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3' addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3' adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake.

  15. A comprehensive survey of 3′ animal miRNA modification events and a possible role for 3′ adenylation in modulating miRNA targeting effectiveness

    Science.gov (United States)

    Burroughs, A. Maxwell; Ando, Yoshinari; de Hoon, Michiel J.L.; Tomaru, Yasuhiro; Nishibu, Takahiro; Ukekawa, Ryo; Funakoshi, Taku; Kurokawa, Tsutomu; Suzuki, Harukazu; Hayashizaki, Yoshihide; Daub, Carsten O.

    2010-01-01

    Animal microRNA sequences are subject to 3′ nucleotide addition. Through detailed analysis of deep-sequenced short RNA data sets, we show adenylation and uridylation of miRNA is globally present and conserved across Drosophila and vertebrates. To better understand 3′ adenylation function, we deep-sequenced RNA after knockdown of nucleotidyltransferase enzymes. The PAPD4 nucleotidyltransferase adenylates a wide range of miRNA loci, but adenylation does not appear to affect miRNA stability on a genome-wide scale. Adenine addition appears to reduce effectiveness of miRNA targeting of mRNA transcripts while deep-sequencing of RNA bound to immunoprecipitated Argonaute (AGO) subfamily proteins EIF2C1–EIF2C3 revealed substantial reduction of adenine addition in miRNA associated with EIF2C2 and EIF2C3. Our findings show 3′ addition events are widespread and conserved across animals, PAPD4 is a primary miRNA adenylating enzyme, and suggest a role for 3′ adenine addition in modulating miRNA effectiveness, possibly through interfering with incorporation into the RNA-induced silencing complex (RISC), a regulatory role that would complement the role of miRNA uridylation in blocking DICER1 uptake. PMID:20719920

  16. Identification of a specific assembly of the G protein Golf as a critical and regulated module of dopamine and adenosine-activated cAMP pathways in the striatum

    Directory of Open Access Journals (Sweden)

    Denis eHervé

    2011-08-01

    Full Text Available In the principal neurons of striatum (medium spiny neurons, MSNs, cAMP pathway is primarily activated through the stimulation of dopamine D1 and adenosine A2A receptors, these receptors being mainly expressed in striatonigral and striatopallidal MSNs, respectively. Since cAMP signaling pathway could be altered in various physiological and pathological situations, including drug addiction and Parkinson’s disease, it is of crucial importance to identify the molecular components involved in the activation of this pathway. In MSNs, cAMP pathway activation is not dependent on the classical Gs GTP-binding protein but requires a specific G protein subunit heterotrimer containing Galpha-olf/beta2/gamma7 in particular association with adenylate cyclase type 5. This assembly forms an authentic functional signaling unit since loss of one of its members leads to defects of cAMP pathway activation in response to D1 or A2A receptor stimulation, inducing dramatic impairments of behavioral responses dependent on these receptors. Interestingly, D1 receptor-dependent cAMP signaling is modulated by the neuronal levels of Galpha-olf, indicating that Galpha-olf represents the rate-limiting step in this signaling cascade and could constitute a critical element for regulation of D1 receptor responses. In both Parkinsonian patients and several animal models of Parkinson’s disease, the lesion of dopamine neurons produces a prolonged elevation of Galpha-olf levels. This observation gives an explanation for the cAMP pathway hypersensitivity to D1 stimulation, occurring despite an unaltered D1 receptor density. In conclusion, alterations in the highly specialized assembly of Galpha-olf/beta2/gamma7 subunits can happen in pathological conditions, such as Parkinson’s disease, and it could have important functional consequences in relation to changes in D1 receptor signaling in the striatum.

  17. A signaling pathway involving the diguanylate cyclase CelR and the response regulator DivK controls cellulose synthesis in Agrobacterium tumefaciens.

    Science.gov (United States)

    Barnhart, D Michael; Su, Shengchang; Farrand, Stephen K

    2014-03-01

    The production of cellulose fibrils is involved in the attachment of Agrobacterium tumefaciens to its plant host. Consistent with previous studies, we reported recently that a putative diguanylate cyclase, celR, is required for synthesis of this polymer in A. tumefaciens. In this study, the effects of celR and other components of the regulatory pathway of cellulose production were explored. Mutational analysis of celR demonstrated that the cyclase requires the catalytic GGEEF motif, as well as the conserved aspartate residue of a CheY-like receiver domain, for stimulating cellulose production. Moreover, a site-directed mutation within the PilZ domain of CelA, the catalytic subunit of the cellulose synthase complex, greatly reduced cellulose production. In addition, deletion of divK, the first gene of the divK-celR operon, also reduced cellulose production. This requirement for divK was alleviated by expression of a constitutively active form of CelR, suggesting that DivK acts upstream of CelR activation. Based on bacterial two-hybrid assays, CelR homodimerizes but does not interact with DivK. The mutation in divK additionally affected cell morphology, and this effect was complementable by a wild-type copy of the gene, but not by the constitutively active allele of celR. These results support the hypothesis that CelR is a bona fide c-di-GMP synthase and that the nucleotide signal produced by this enzyme activates CelA via the PilZ domain. Our studies also suggest that the DivK/CelR signaling pathway in Agrobacterium regulates cellulose production independent of cell cycle checkpoint systems that are controlled by divK.

  18. Butanol tolerance in microorganisms

    Energy Technology Data Exchange (ETDEWEB)

    Bramucci, Michael G.; Nagarajan, Vasantha

    2016-03-01

    Provided herein are recombinant yeast host cells and methods for their use for production of fermentation products from a pyruvate utilizing pathway. Yeast host cells provided herein comprise reduced pyruvate decarboxylase activity and modified adenylate cyclase activity. In embodiments, yeast host cells provided herein comprise resistance to butanol and increased biomass production.

  19. cAMP Signaling Compartmentation: Adenylyl Cyclases as Anchors of Dynamic Signaling Complexes.

    Science.gov (United States)

    Johnstone, Timothy B; Agarwal, Shailesh R; Harvey, Robert D; Ostrom, Rennolds S

    2018-04-01

    It is widely accepted that cAMP signaling is compartmentalized within cells. However, our knowledge of how receptors, cAMP signaling enzymes, effectors, and other key proteins form specific signaling complexes to regulate specific cell responses is limited. The multicomponent nature of these systems and the spatiotemporal dynamics involved as proteins interact and move within a cell make cAMP responses highly complex. Adenylyl cyclases, the enzymatic source of cAMP production, are key starting points for understanding cAMP compartments and defining the functional signaling complexes. Three basic elements are required to form a signaling compartment. First, a localized signal is generated by a G protein-coupled receptor paired to one or more of the nine different transmembrane adenylyl cyclase isoforms that generate the cAMP signal in the cytosol. The diffusion of cAMP is subsequently limited by several factors, including expression of any number of phosphodiesterases (of which there are 24 genes plus spice variants). Finally, signal response elements are differentially localized to respond to cAMP produced within each locale. A-kinase-anchoring proteins, of which there are 43 different isoforms, facilitate this by targeting protein kinase A to specific substrates. Thousands of potential combinations of these three elements are possible in any given cell type, making the characterization of cAMP signaling compartments daunting. This review will focus on what is known about how cells organize cAMP signaling components as well as identify the unknowns. We make an argument for adenylyl cyclases being central to the formation and maintenance of these signaling complexes. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  20. ePAT: A simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications

    Science.gov (United States)

    Jänicke, Amrei; Vancuylenberg, John; Boag, Peter R.; Traven, Ana; Beilharz, Traude H.

    2012-01-01

    The addition of a poly(A)-tail to the 3′ termini of RNA molecules influences stability, nuclear export, and efficiency of translation. In the cytoplasm, dynamic changes in the length of the poly(A)-tail have long been recognized as reflective of the switch between translational silence and activation. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. Here, we describe a simple new method to 3′-tag adenylated RNA in total RNA samples using the intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template. This tag can serve as an anchor for cDNA synthesis and subsequent gene-specific PCR to assess poly(A)-tail length. We call this method extension Poly(A) Test (ePAT). The ePAT approach is as efficient as traditional Ligation-Mediated Poly(A) Test (LM-PAT) assays, avoids problems of internal priming associated with oligo-dT-based methods, and allows for the accurate analysis of both the poly(A)-tail length and alternate 3′ UTR usage in 3′ RACE applications. PMID:22543866

  1. cAMP Regulation of the lactose operon.

    Science.gov (United States)

    Szeberenyi, Jozsef

    2004-05-01

    Terms to be familiar with before you start to solve the test: lactose operon, adenylate cyclase, cAMP, catabolite activator protein (CAP), expression plasmid, lac operator, lac repressor, lactose, glucose, promoter, cis- and trans-acting factors. Copyright © 2004 International Union of Biochemistry and Molecular Biology, Inc.

  2. Headache and prolonged dilatation of the middle meningeal artery by PACAP38 in healthy volunteers

    DEFF Research Database (Denmark)

    Amin, Faisal Mohammad; Asghar, Mohammad Sohail; Guo, Song

    2012-01-01

    To explore a possible relationship between vasodilatation and delayed headache we examined the effect of pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) on the middle meningeal artery (MMA) and middle cerebral artery (MCA) using high resolution magnetic resonance angiography (MRA)....

  3. VIP and PACAP

    DEFF Research Database (Denmark)

    Fahrenkrug, Jan

    2010-01-01

    amounts. Carboxyamidation of VIP and PHI is not critical and glycine-extended forms of both peptides have been demonstrated. Pituitary adenylate cyclase activating polypeptide (PACAP) is derived from a 170 amino acid long precursor, which gives rise to PACAP 38, PACAP 27 and PACAP related peptide (PRP...

  4. The Electrophysiologic Mechanisms of Halogenated Alkane Arrhythmogenesis.

    Science.gov (United States)

    1983-03-01

    signal was also recorded. The tissue was carefully removed from the bath, placed in 1.0 ml of cold 5% trichloroacetic acid (TCA), and homogenized using...and Daly, J.W.: Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc. Natl. Acad. Sci. USA, 76:3363

  5. Vascular effects and cyclic AMP production produced by VIP, PHM, PHV, PACAP-27, PACAP-38, and NPY on rabbit ovarian artery

    DEFF Research Database (Denmark)

    Yao, W; Sheikh, S P; Ottesen, B

    1996-01-01

    The relationship between vessel tone and cAMP production induced by vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), peptide histidine valine (PHV), pituitary adenylate cyclase activating polypeptide (PACAP-27 and PACAP-38), and neuropeptide Y (NPY) was investigated in...

  6. Tritium labelling of PACAP-38 using a synthetic diiodinated precursor peptide

    DEFF Research Database (Denmark)

    Pedersen, Martin Holst Friborg; Baun, Michael

    2012-01-01

    In the interest of developing efficient methods for tritium labelling peptides, we here demonstrate the successful labelling of PACAP-38 (pituitary adenylate cyclase-activating polypeptide), a 38-mer peptide, using a synthetic diiodinated PACAP-38 precursor. In this example, we employ standard...... hydrogenation chemistry with the use of a heterogeneous palladium catalyst and carrier-free tritium gas on a tritium manifold system....

  7. Prostaglandins stimulate renin secretion and renin mRNA in mouse renal juxtaglomerular cells

    DEFF Research Database (Denmark)

    Jensen, B L; Schmid, C; Kurtz, A

    1996-01-01

    identified PGI2 and PGE2 as stimulators of renin secretion; the effects were dose and time dependent. PGE2 also increased renin mRNA accumulation time and dose dependent. PGE2 and PGI2 activated adenylate cyclase concentration dependent in granular cells. PGE2 stimulations of renin secretion and renin m...

  8. Plasma Bile Acids Are Associated with Energy Expenditure and Thyroid Function in Humans

    NARCIS (Netherlands)

    Ockenga, Johann; Valentini, Luzia; Schuetz, Tatjana; Wohlgemuth, Franziska; Glaeser, Silja; Omar, Ajmal; Kasim, Esmatollah; duPlessis, Daniel; Featherstone, Karen; Davis, Julian R.; Tietge, Uwe J. F.; Kroencke, Thomas; Biebermann, Heike; Koehrle, Josef; Brabant, Georg

    Background/Aims: Animal studies implicate a role of bile acids (BA) in thyroid-regulated energy expenditure (EE) via activation of the TGR-5/adenylate cyclase/deiodinase type 2 pathway. Here we investigated these possible associations in humans. Methods: EE, BA, and thyroid hormone status were

  9. Long-acting β2-agonists increase fluticasone propionate-induced mitogen-activated protein kinase phosphatase 1 (MKP-1 in airway smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Melanie Manetsch

    Full Text Available Mitogen-activated protein kinase phosphatase 1 (MKP-1 represses MAPK-driven signalling and plays an important anti-inflammatory role in asthma and airway remodelling. Although MKP-1 is corticosteroid-responsive and increased by cAMP-mediated signalling, the upregulation of this critical anti-inflammatory protein by long-acting β2-agonists and clinically-used corticosteroids has been incompletely examined to date. To address this, we investigated MKP-1 gene expression and protein upregulation induced by two long-acting β2-agonists (salmeterol and formoterol, alone or in combination with the corticosteroid fluticasone propionate (abbreviated as fluticasone in primary human airway smooth muscle (ASM cells in vitro. β2-agonists increased MKP-1 protein in a rapid but transient manner, while fluticasone induced sustained upregulation. Together, long-acting β2-agonists increased fluticasone-induced MKP-1 and modulated ASM synthetic function (measured by interleukin 6 (IL-6 and interleukin 8 (IL-8 secretion. As IL-6 expression (like MKP-1 is cAMP/adenylate cyclase-mediated, the long-acting β2-agonist formoterol increased IL-6 mRNA expression and secretion. Nevertheless, when added in combination with fluticasone, β2-agonists significantly repressed IL-6 secretion induced by tumour necrosis factor α (TNFα. Conversely, as IL-8 is not cAMP-responsive, β2-agonists significantly inhibited TNFα-induced IL-8 in combination with fluticasone, where fluticasone alone was without repressive effect. In summary, long-acting β2-agonists increase fluticasone-induced MKP-1 in ASM cells and repress synthetic function of this immunomodulatory airway cell type.

  10. 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.

  11. Type 7 adenylyl cyclase is involved in the ethanol and CRF sensitivity of GABAergic synapses in mouse central amygdala

    Directory of Open Access Journals (Sweden)

    Maureen T. Cruz

    2011-01-01

    Full Text Available AbstractThe GABAergic system in the central amygdala (CeA plays a major role in ethanol dependence and in the anxiogenic response to ethanol withdrawal. Previously, we found that both ethanol and corticotropin releasing factor (CRF increase GABAergic transmission in mouse and rat CeA neurons, in part by enhancing the release of GABA via activation of presynaptic CRF1 receptors. CRF1 receptors are coupled to the enzyme adenylyl cyclase (AC, which produces the second messenger cyclic AMP. There are nine isoforms of AC, but we recently found that CRF1 receptors in the pituitary were coupled to the Type 7 AC (AC7. Therefore, using an in vitro electrophysiological approach in brain slices, here we have investigated a possible role of the AC7 signaling pathway in ethanol and CRF effects on CeA GABAergic synapses of genetically modified mice with diminished brain Adcy7 activity (HET compared to their littermate male wild type (WT mice. We found no significant differences in basal membrane properties, mean baseline amplitude of evoked GABAA receptor-mediated inhibitory postsynaptic potentials (IPSPs, or paired-pulse facilitation (PPF of GABAA-IPSPs between HET and WT mice. In CeA neurons of WT mice, ethanol superfusion significantly augmented (by 39% GABAA-IPSPs and decreased PPF (by 25%, suggesting increased presynaptic GABA release. However, these effects were absent in HET mice. CRF superfusion also significantly augmented IPSPs (by 38% and decreased PPF (by 23% in WT CeA neurons, and still elicited a significant but smaller (by 13% increase of IPSP amplitude, but no effect on PPF, in HET mice. These electrophysiological data suggest that AC7 plays an important role in ethanol and CRF modulation of presynaptic GABA release in CeA and thus may underlie ethanol-related behaviors such as anxiety and dependence.

  12. A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis.

    Science.gov (United States)

    Huang, Kai-Fa; Wang, Yu-Ruei; Chang, En-Cheng; Chou, Tsung-Lin; Wang, Andrew H-J

    2008-04-01

    QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid arthritis and melanoma. The crystal structure of human QC revealed an unusual H-bond (hydrogen-bond) network in the active site, formed by several highly conserved residues (Ser(160), Glu(201), Asp(248), Asp(305) and His(319)), within which Glu(201) and Asp(248) were found to bind to substrate. In the present study we combined steady-state enzyme kinetic and X-ray structural analyses of 11 single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu(201)...Asp(305) and Asp(248)...Asp(305), reduced the steady-state catalysis dramatically. The roles of these two COOH...COOH bonds on catalysis could be partly replaced by COOH...water bonds, but not by COOH...CONH(2) bonds, reminiscent of the low-barrier Asp...Asp H-bond in the active site of pepsin-like aspartic peptidases. Mutations on Asp(305), a residue located at the centre of the H-bond network, raised the K(m) value of the enzyme by 4.4-19-fold, but decreased the k(cat) value by 79-2842-fold, indicating that Asp(305) primarily plays a catalytic role. In addition, results from mutational studies on Ser(160) and His(319) suggest that these two residues might help to stabilize the conformations of Asp(248) and Asp(305) respectively. These data allow us to propose an essential proton transfer between Glu(201), Asp(305) and Asp(248) during the catalysis by animal QCs.

  13. Impairment of F1F0-ATPase, adenine nucleotide translocator and adenylate kinase causes mitochondrial energy deficit in human skin fibroblasts with chromosome 21 trisomy.

    Science.gov (United States)

    Valenti, Daniela; Tullo, Apollonia; Caratozzolo, Mariano F; Merafina, Riccardo S; Scartezzini, Paolo; Marra, Ersilia; Vacca, Rosa A

    2010-10-15

    A central role for mitochondrial dysfunction has been proposed in the pathogenesis of DS (Down's syndrome), a multifactorial disorder caused by trisomy of human chromosome 21. To explore whether and how abnormalities in mitochondrial energy metabolism are involved in DS pathogenesis, we investigated the catalytic properties, gene expression and protein levels of certain proteins involved in mitochondrial ATP synthesis, such as F1F0-ATPase, ANT (adenine nucleotide translocator) and AK (adenylate kinase), in DS-HSF (human skin fibroblasts with trisomic karyotype), comparing them with euploid fibroblasts. In DS-HSF, we found a strong impairment of mitochondrial ATP synthesis due to a reduction in the catalytic efficiency of each of the investigated proteins. This impairment occurred in spite of unchanged gene expression and an increase in ANT and AK protein content, whereas the amount of ATPase subunits was selectively reduced. Interestingly, exposure of DS-HSF to dibutyryl-cAMP, a permanent derivative of cAMP, stimulated ANT, AK and ATPase activities, whereas H89, a specific PKA (protein kinase A) inhibitor, suppressed this cAMPdependent activation, indicating an involvement of the cAMP/PKA-mediated signalling pathway in the ATPase, ANT and AK deficit. Consistently, DS-HSF showed decreased basal levels of cAMP and reduced PKA activity. Despite the impairment of mitochondrial energy apparatus, no changes in cellular energy status, but increased basal levels of L-lactate, were found in DS-HSF, which partially offset for the mitochondrial energy deficit by increasing glycolysis and mitochondrial mass.These results provide new insight into the molecular basis for mitochondrial dysfunction in DS and might provide a molecular explanation for some clinical features of the syndrome.

  14. Characterisation of the ArmA adenylation domain implies a more diverse secondary metabolism in the genus Armillaria.

    Science.gov (United States)

    Misiek, Mathias; Braesel, Jana; Hoffmeister, Dirk

    2011-08-01

    The armA-gene, encoding a tridomain enzyme reminiscent of nonribosomal peptide synthetases, was identified in the genome of the basidiomycete Armillaria mellea. Heterologously expressed enzyme and the ATP-pyrophosphate exchange assay were used for the in vitro biochemical characterisation of the ArmA adenylation domain. l-leucine was the preferred substrate, while l-threonine, l-valine, l-alanine, and l-isoleucine were turned over at lower rates (83 %, 62 %, 56 %, and 44 %, respectively). Other proteinogenic amino acids, 2-oxo acids, and benzoic acid derivatives were not accepted. As the substrate range of ArmA is incompatible with the secondary metabolites known from the genus Armillaria, our results imply greater natural product diversity in this genus. This is the first biochemical characterisation of a basidiomycete amino acid-adenylating domain, and our results may help refine computer algorithms to predict substrate specificities for basidiomycete nonribosomal peptide synthetases whose genes are discovered through genome sequencing. Copyright © 2011 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  15. Scutellarin Suppresses NLRP3 Inflammasome Activation in Macrophages and Protects Mice against Bacterial Sepsis.

    Science.gov (United States)

    Liu, Yi; Jing, Yan-Yun; Zeng, Chen-Ying; Li, Chen-Guang; Xu, Li-Hui; Yan, Liang; Bai, Wen-Jing; Zha, Qing-Bing; Ouyang, Dong-Yun; He, Xian-Hui

    2017-01-01

    The NLRP3 inflammasome plays a critical role in mediating the innate immune defense against pathogenic infections, but aberrant activation of NLRP3 inflammasome has been linked to a variety of inflammatory diseases. Thus targeting the NLRP3 inflammasome represents a promising therapeutic for the treatment of such diseases. Scutellarin is a flavonoid isolated from Erigeron breviscapus (Vant.) Hand.-Mazz. and has been reported to exhibit potent anti-inflammatory activities, but the underlying mechanism is only partly understood. In this study, we aimed to investigate whether scutellarin could affect the activation of NLRP3 inflammasome in macrophages. The results showed that scutellarin dose-dependently reduced caspase-1 activation and decreased mature interleukin-1β (IL-1β) release in lipopolysaccharide (LPS)-primed macrophages upon ATP or nigericin stimulation, indicating that scutellarin inhibited NLRP3 inflammasome activation in macrophages. Consistent with this, scutellarin also suppressed pyroptotic cell death in LPS-primed macrophages treated with ATP or nigericin. ATP or nigericin-induced ASC speck formation and its oligomerization were blocked by scutellarin pre-treatment. Intriguingly, scutellarin augmented PKA-specific phosphorylation of NLRP3 in LPS-primed macrophages, which was completely blocked by selective PKA inhibitor H89, suggesting that PKA signaling had been involved in the action of scutellarin to suppress NLRP3 inflammasome activation. Supporting this, the inhibitory effect of scutellarin on NLRP3 inflammasome activation was completely counteracted by H89 or adenyl cyclase inhibitor MDL12330A. As NLRP3-dependent release of IL-1β has a critical role in sepsis, the in vivo activity of scutellarin was assayed in a mouse model of bacterial sepsis, which was established by intraperitoneally injection of a lethal dose of viable Escherichia coli . Oral administration of scutellarin significantly improved the survival of mice with bacterial sepsis

  16. Scutellarin Suppresses NLRP3 Inflammasome Activation in Macrophages and Protects Mice against Bacterial Sepsis

    Directory of Open Access Journals (Sweden)

    Yi Liu

    2018-01-01

    Full Text Available The NLRP3 inflammasome plays a critical role in mediating the innate immune defense against pathogenic infections, but aberrant activation of NLRP3 inflammasome has been linked to a variety of inflammatory diseases. Thus targeting the NLRP3 inflammasome represents a promising therapeutic for the treatment of such diseases. Scutellarin is a flavonoid isolated from Erigeron breviscapus (Vant. Hand.-Mazz. and has been reported to exhibit potent anti-inflammatory activities, but the underlying mechanism is only partly understood. In this study, we aimed to investigate whether scutellarin could affect the activation of NLRP3 inflammasome in macrophages. The results showed that scutellarin dose-dependently reduced caspase-1 activation and decreased mature interleukin-1β (IL-1β release in lipopolysaccharide (LPS-primed macrophages upon ATP or nigericin stimulation, indicating that scutellarin inhibited NLRP3 inflammasome activation in macrophages. Consistent with this, scutellarin also suppressed pyroptotic cell death in LPS-primed macrophages treated with ATP or nigericin. ATP or nigericin-induced ASC speck formation and its oligomerization were blocked by scutellarin pre-treatment. Intriguingly, scutellarin augmented PKA-specific phosphorylation of NLRP3 in LPS-primed macrophages, which was completely blocked by selective PKA inhibitor H89, suggesting that PKA signaling had been involved in the action of scutellarin to suppress NLRP3 inflammasome activation. Supporting this, the inhibitory effect of scutellarin on NLRP3 inflammasome activation was completely counteracted by H89 or adenyl cyclase inhibitor MDL12330A. As NLRP3-dependent release of IL-1β has a critical role in sepsis, the in vivo activity of scutellarin was assayed in a mouse model of bacterial sepsis, which was established by intraperitoneally injection of a lethal dose of viable Escherichia coli. Oral administration of scutellarin significantly improved the survival of mice with

  17. 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.

  18. β3GnT2 maintains adenylyl cyclase-3 signaling and axon guidance molecule expression in the olfactory epithelium.

    Science.gov (United States)

    Henion, Timothy R; Faden, Ashley A; Knott, Thomas K; Schwarting, Gerald A

    2011-04-27

    In the olfactory epithelium (OE), odorant receptor stimulation generates cAMP signals that function in both odor detection and the regulation of axon guidance molecule expression. The enzyme that synthesizes cAMP, adenylyl cyclase 3 (AC3), is coexpressed in olfactory sensory neurons (OSNs) with poly-N-acetyllactosamine (PLN) oligosaccharides determined by the glycosyltransferase β3GnT2. The loss of either enzyme results in similar defects in olfactory bulb (OB) innervation and OSN survival, suggesting that glycosylation may be important for AC3 function. We show here that AC3 is extensively modified with N-linked PLN, which is essential for AC3 activity and localization. On Western blots, AC3 from the wild-type OE migrates diffusely as a heavily glycosylated 200 kDa band that interacts with the PLN-binding lectin LEA. AC3 from the β3GnT2(-/-) OE loses these PLN modifications, migrating instead as a 140 kDa glycoprotein. Furthermore, basal and forskolin-stimulated cAMP production is reduced 80-90% in the β3GnT2(-/-) OE. Although AC3 traffics normally to null OSN cilia, it is absent from axon projections that aberrantly target the OB. The cAMP-dependent guidance receptor neuropilin-1 is also lost from β3GnT2(-/-) OSNs and axons, while semaphorin-3A ligand expression is upregulated. In addition, kirrel2, a mosaically expressed adhesion molecule that functions in axon sorting, is absent from β3GnT2(-/-) OB projections. These results demonstrate that PLN glycans are essential in OSNs for proper AC3 localization and function. We propose that the loss of cAMP-dependent guidance cues is also a critical factor in the severe axon guidance defects observed in β3GnT2(-/-) mice.

  19. Putative protein VC0395_0300 from Vibrio cholerae is a diguanylate cyclase with a role in biofilm formation.

    Science.gov (United States)

    Bandekar, Divya; Chouhan, Om Prakash; Mohapatra, Swati; Hazra, Mousumi; Hazra, Saugata; Biswas, Sumit

    2017-09-01

    The hallmark of the lifecycle of Vibrio cholerae is its ability to switch between two lifestyles - the sessile, non-pathogenic form and the motile, infectious form in human hosts. One of these changes is in the formation of surface biofilms, when in sessile aquatic habitats. The cell-cell interactions within a V. cholerae biofilm are stabilized by the production of an exopolysachharide (EPS) matrix, which in turn is regulated by the ubiquitous secondary messenger, cyclic di-GMP (c-di-GMP), synthesized by proteins containing GGD(/E)EF domains in all prokaryotic systems. Here, we report the functional role of the VC0395_0300 protein (Sebox3) encoded by the chromosome I of V. cholerae, with a GGEEF signature sequence, in the formation of surface biofilms. In our study, we have shown that Escherichia coli containing the full-length Sebox3 displays enhanced biofilm forming ability with cellulose production as quantified and visualized by multiple assays, most notably using FEG-SEM. This has also been corroborated with the lack of motility of host containing Sebox3 in semi-solid media. Searching for the reasons for this biofilm formation, we have demonstrated in vitro that Sebox3 can synthesize c-di-GMP from GTP. The homology derived model of Sebox3 displayed significant conservation of the GGD(/E)EF architecture as well. Hence, we propose that the putative protein VC0395_0300 from V. cholerae is a diguanylate cyclase which has an active role in biofilm formation. Copyright © 2017 Elsevier GmbH. All rights reserved.

  20. Comparative analysis of oligonucleotide primers for high-throughput screening of genes encoding adenylation domains of nonribosomal peptide synthetases in actinomycetes

    Czech Academy of Sciences Publication Activity Database

    Bakal, Tomáš; Goo, K.-S.; Najmanová, Lucie; Plháčková, Kamila; Kadlčík, Stanislav; Ulanová, Dana

    2015-01-01

    Roč. 108, č. 5 (2015), s. 1267-1274 ISSN 0003-6072 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Nonribosomal peptide synthetase * Adenylation domain * Actinomycetes Subject RIV: EE - Microbiology, Virology Impact factor: 1.944, year: 2015

  1. Crystal structures at 2.5 Angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate

    DEFF Research Database (Denmark)

    Belrhali, H.; Yaremchuk, A.; Tukalo, M.

    1994-01-01

    in the crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions...

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

    DEFF Research Database (Denmark)

    Chen, Yicai; Yuan, Mingjun; Mohanty, Anee

    2015-01-01

    intrinsic feature of c-di-GMP signalling is the abundance of DGCs and PDEs encoded by many bacterial species. It is unclear whether the different DGCs or PDEs coordinately establish the c-di-GMP regulation or function independently of each other. Here, we provide evidence that multiple DGCs are involved......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...... in regulation of c-di-GMP on synthesis of the major iron siderophore pyoverdine in Pseudomonas aeruginosa. Constitutive expression of the WspG or YedQ DGC in P.aeruginosa is able to induce its pyoverdine synthesis. Induction of pyoverdine synthesis by high intracellular c-di-GMP depends on the synthesis...

  3. 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.

  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. 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.

  6. Enzymatic Profiles of Activated Sludge from a Wastewater Treatment ...

    African Journals Online (AJOL)

    Activated sludge samples collected from a treatment plant, with foaming experience in the month of July, was characterized enzymatically. Hexokinase, Glyceraldehyde-3-phosphate dehydrogenase and Adenylate kinase activity assays were conducted before, during and after the foaming episode. The spectrum of enzyme ...

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

    Directory of Open Access Journals (Sweden)

    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

  8. Peroxisome proliferator-activated receptors, estrogenic responses and biotransformation system in the liver of salmon exposed to tributyltin and second messenger activator

    International Nuclear Information System (INIS)

    Pavlikova, Nela; Kortner, Trond M.; Arukwe, Augustine

    2010-01-01

    The mechanisms by which organotin compounds produce modulations of the endocrine systems and other biological responses are not fully understood. In this study, juvenile salmon were force-fed diet containing TBT (0: solvent control, 0.1, 1 and 10 mg/kg fish) for 72 h. Subsequently, fish exposed to solvent control and 10 mg TBT were exposed to waterborne concentration (200 μg/l) of the adenylate cyclase (AC) stimulator, forskolin for 2 and 4 h. The overall aim of the study was to explore whether TBT endocrine disruptive effects involve second messenger activation. Liver was sampled from individual fish (n = 8) at the end of the exposures. The transcription patterns of peroxisome proliferator-activated receptor (PPAR) isotype and acyl-coenzyme A oxidase 1 (ACOX1), aromatase isoform, estrogen receptor-α (ERα), pregnane X receptor (PXR), CYP3A and glutathione S-transferase (GST) genes were measured by quantitative polymerase chain reaction (qPCR). Our data showed a consistent increase in PPARα, PPARβ and PPARγ mRNA and protein expression after TBT exposure that were inversely correlated with ACOX1 mRNA levels. Forskolin produced PPAR isotype-specific mRNA and protein effects that were modulated by TBT. ACOX1 expression was decreased (at 2 h) and increased (at 4 h) by forskolin and the presence of TBT potentiated these effects. TBT apparently increased mRNA and protein levels of cyp19a, compared to the solvent control, whereas cyp19b mRNA levels were unaffected by TBT treatment. Combined TBT and forskolin exposure produced respective decrease and increase of mRNA levels of cyp19a and cyp19b, compared with control. TBT decreased ERα mRNA at low dose (1 mg/kg) and forskolin exposure alone produced a consistent decrease of ERα mRNA levels that were not affected by the presence of TBT. Interestingly, PXR and CYP3A mRNA levels were differentially affected, either decreased or increased, after exposure to TBT and forskolin, singly and also in combination. GST mRNA was

  9. cAMP-dependent protein kinase inhibits α7 nicotinic receptor activity in layer 1 cortical interneurons through activation of D1/D5 dopamine receptors.

    Science.gov (United States)

    Komal, Pragya; Estakhr, Jasem; Kamran, Melad; Renda, Anthony; Nashmi, Raad

    2015-08-15

    Protein kinases can modify the function of many proteins including ion channels. However, the role of protein kinase A in modifying nicotinic receptors in the CNS has never been investigated. We showed through whole-cell recordings of layer 1 prefrontal cortical interneurons that α7 nicotinic responses are negatively modulated by protein kinase A. Furthermore, we show that stimulation of dopamine receptors can similarly attenuate α7 nicotinic responses through the activation of protein kinase A. These results suggest how the interaction of the cholinergic and dopaminergic systems may influence neuronal excitability in the brain. Phosphorylation of ion channels, including nicotinic acetylcholine receptors (nAChRs), by protein kinases plays a key role in the modification of synaptic transmission and neuronal excitability. α7 nAChRs are the second most prevalent nAChR subtype in the CNS following α4β2. Serine 365 in the M3-M4 cytoplasmic loop of the α7 nAChR is a phosphorylation site for protein kinase A (PKA). D1/D5 dopamine receptors signal through the adenylate cyclase-PKA pathway and play a key role in working memory and attention in the prefrontal cortex. Thus, we examined whether the dopaminergic system, mediated through PKA, functionally interacts with the α7-dependent cholinergic neurotransmission. In layer 1 interneurons of mouse prefrontal cortex, α7 nicotinic currents were decreased upon stimulation with 8-Br-cAMP, a PKA activator. In HEK 293T cells, dominant negative PKA abolished 8-Br-cAMP's effect of diminishing α7 nicotinic currents, while a constitutively active PKA catalytic subunit decreased α7 currents. In brain slices, the PKA inhibitor KT-5720 nullified 8-Br-cAMP's effect of attenuating α7 nicotinic responses, while applying a PKA catalytic subunit in the pipette solution decreased α7 currents. 8-Br-cAMP stimulation reduced surface expression of α7 nAChRs, but there was no change in single-channel conductance. The D1/D5 dopamine

  10. Differentiation of cGMP-dependent and -independent nitric oxide effects on platelet apoptosis and reactive oxygen species production using platelets lacking soluble guanylyl cyclase.

    Science.gov (United States)

    Rukoyatkina, N; Walter, U; Friebe, A; Gambaryan, S

    2011-11-01

    Platelet activation is an irreversible process resulting in platelet apoptosis and necrosis, and circulating platelets contain many components of the apoptotic machinery. Cyclic guanosine monophosphate (cGMP) generated by nitric oxide (NO) activated soluble guanylyl cyclase (sGC) plays a crucial role in preventing platelet activation. However, in addition to activation of sGC, cGMP-independent NO effects in platelets have been described. To differentiate between cGMP-dependent and -independent NO effects on platelet apoptosis and reactive oxygen species (ROS) production, we generated platelet-specific sGC-deficient mice (PS-GCKO). Platelet apoptosis was induced by a combination of thrombin/convulxin (Thr/Cvx) and assessed by phosphatidylserine (PS) surface exposure, and loss of the mitochondrial membrane potential. NO-induced inhibition of PS externalisation was mediated only by cGMP-dependent mechanisms. Inhibition of the mitochondrial membrane potential decrease at low NO concentration was also cGMP-dependent but became cGMP-independent at high NO concentrations. In contrast, inhibition of ROS formation at any NO concentration was mediated by cGMP-independent mechanisms, very likely due to direct radical scavenging. NO inhibits platelet apoptosis by cGMP-dependent mechanisms and ROS production by cGMP-independent mechanisms. The PS-GCKO mouse model is an important tool for the differentiation of cGMP-dependent and -independent NO effects on platelets.

  11. NMR structural characterization of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Mavoungou, Chrystelle [Max Planck Institute for Biochemistry (Germany); Israel, Lars [Ludwig Maximilians-University, Adolf Butenandt Institute, Cell Biology (Germany); Rehm, Till; Ksiazek, Dorota; Krajewski, Marcin; Popowicz, Grzegorz [Max Planck Institute for Biochemistry (Germany); Noegel, Angelika A. [University of Cologne, Institute for Biochemistry (Germany); Schleicher, Michael [Ludwig Maximilians-University, Adolf Butenandt Institute, Cell Biology (Germany); Holak, Tad A. [Max Planck Institute for Biochemistry (Germany)

    2004-05-15

    Cyclase-associated proteins (CAPs) are highly conserved, ubiquitous actin binding proteins that are involved in microfilament reorganization. The N-termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C-termini bind to G-actin. We report here the NMR characterization of the amino-terminal domain of CAP from Dictyostelium discoideum (CAP(1-226)). NMR data, including the steady state {sup 1}H-{sup 15}N heteronuclear NOE experiments, indicate that the first 50 N-terminal residues are unstructured and that this highly flexible serine-rich fragment is followed by a stable, folded core starting at Ser 51. The NMR structure of the folded core is an {alpha}-helix bundle composed of six antiparallel helices, in a stark contrast to the recently determined CAP C-terminal domain structure, which is solely built by {beta}-strands.

  12. The Receptor Guanylyl Cyclase Type D (GC-D) Ligand Uroguanylin Promotes the Acquisition of Food Preferences in Mice

    OpenAIRE

    Arakawa, Hiroyuki; Kelliher, Kevin R.; Zufall, Frank; Munger, Steven D.

    2013-01-01

    Rodents rely on olfactory stimuli to communicate information between conspecifics that is critical for health and survival. For example, rodents that detect a food odor simultaneously with the social odor carbon disulfide (CS2) will acquire a preference for that food. Disruption of the chemosensory transduction cascade in CS2-sensitive olfactory sensory neurons (OSNs) that express the receptor guanylyl cyclase type D (GC-D; GC-D+ OSNs) will prevent mice from acquiring these preferences. GC-D+...

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

    Directory of Open Access Journals (Sweden)

    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.

  14. 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.

  15. Elevated Adenylyl Cyclase 9 Expression Is a Potential Prognostic Biomarker for Patients with Colon Cancer.

    Science.gov (United States)

    Yi, Hua; Wang, Kun; Jin, Jun-Feng; Jin, He; Yang, Lihua; Zou, Yidan; Du, Biaoyan; Liu, Xiaodong

    2018-01-02

    BACKGROUND Adenylyl cyclase 9 (ADCY9) is an enzyme that modulates signal transduction by producing the second messenger, cyclic adenosine monophosphate (cAMP). The aim of the present study was to investigate the association of ADCY9 expression with clinicopathological features and disease-free survival of colon cancer patients. MATERIAL AND METHODS Immunohistochemistry staining with ADCY9 antibody was performed on a tissue microarray. Immunoreactivity scores (IRS) were recorded and applied for association analysis. ADCY9 mRNA expression and clinicopathogical information were also extracted from TCGA colon cancer dataset and analyzed using univariate and multivariate Cox proportional hazards models.  RESULTS ADCY9 IRS was significantly higher (P=0.002) in tumor tissues (6.40±1.26, n=200) than in adjacent normal samples (4.13±0.83, n=8). The IRS and mRNA expression of ADCY9 were correlated to colon cancer TNM staging. Longer disease-free survival was observed in patients with lower ADCY9 expression (P=0.001). In the multivariate models, ADCY9 expression level (hazard ratio [HR] 5.495, 95% confidence interval [CI] 1.753-17.227, P=0.003), and distant metastasis (HR 4.329, 95% CI 1.374-13.636, P=0.012) were still associated with disease-free survival. CONCLUSIONS High ADCY9 expression is a poor prognostic factor for disease-free survival in colon cancer.

  16. Photomanipulation of antibiotic susceptibility and biofilm formation of Escherichia coli heterologously expressing photoactivated adenylyl cyclase.

    Science.gov (United States)

    Yasukawa, Hiro; Konno, Noriko; Haneda, Yukari; Yamamori, Baku; Iseki, Mineo; Shibusawa, Mami; Ono, Yasushi; Kodaira, Ken-ichi; Funada, Hisashi; Watanabe, Masakatsu

    2012-01-01

    A cyaA-deficient Escherichia coli strain was transformed by a plasmid carrying the gene for BsPAC, a photoactivated adenylyl cyclase identified from a Beggiatoa sp., and was subjected to an antibiotic susceptibility assay and biofilm formation assay under a light or dark condition. Cells expressing BsPAC that were incubated under blue light (470 nm) were more susceptible to fosfomycin, nalidixic acid and streptomycin than were cells incubated in the dark. Cells expressing BsPAC formed more biofilms when incubated under the light than did cells cultured in the dark. We concluded from these observations that it is possible to determine the importance of cAMP in antibiotic susceptibility and biofilm formation of E. coli by photomanipulating the cellular cAMP level by the use of BsPAC. A site-directed mutant of BsPAC in which Tyr7 was replaced by Phe functioned even in the dark, indicating that Tyr7 plays an important role in photoactivation of BsPAC. Results of mutational analysis of BsPAC should contribute to an understanding of the molecular basis for photoactivation of the protein.

  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. 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.

  19. Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi.

    Directory of Open Access Journals (Sweden)

    Jorge García-Martínez

    Full Text Available The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. Here we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments, reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, ΔacyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress (H(2O(2, but no change in heavy metal resistance or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group.

  20. Bioinformatics analysis of the oxidosqualene cyclase gene and the amino acid sequence in mangrove plants

    Science.gov (United States)

    Basyuni, M.; Wati, R.

    2017-01-01

    This study described the bioinformatics methods to analyze seven oxidosqualene cyclase (OSC) genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, similarity, subcellular localization and phylogenetic. The physical and chemical properties of seven mangrove OSC showed variation among the genes. The percentage of the secondary structure of seven mangrove OSC genes followed the order of a helix > random coil > extended chain structure. The values of chloroplast or signal peptide were too low, indicated that no chloroplast transit peptide or signal peptide of secretion pathway in mangrove OSC genes. The target peptide value of mitochondria varied from 0.163 to 0.430, indicated it was possible to exist. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove OSC genes. To clarify the relationship among the mangrove OSC gene, a phylogenetic tree was constructed. The phylogenetic tree shows that there are three clusters, Kandelia KcMS join with Bruguiera BgLUS, Rhizophora RsM1 was close to Bruguiera BgbAS, and Rhizophora RcCAS join with Kandelia KcCAS. The present study, therefore, supported the previous results that plant OSC genes form distinct clusters in the tree.

  1. Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii.

    OpenAIRE

    Konisky, J; Michels, P C; Clark, D S

    1995-01-01

    The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens. Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D. J. Hei and D. S. Clark, Appl. Environ. Microbiol. 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles.

  2. Similarly potent inhibition of adenylyl cyclase by P-site inhibitors in hearts from wild type and AC5 knockout mice.

    Directory of Open Access Journals (Sweden)

    Joerg H Braeunig

    Full Text Available Adenylyl cyclase type 5 (AC5 was described as major cardiac AC isoform. The knockout of AC5 (AC5KO exerted cardioprotective effects in heart failure. Our study explored the impact of AC5KO on mouse heart AC activities and evaluated putative AC5-selective inhibitors. In cardiac membranes from AC5KO mice, basal AC activity was decreased, while AC stimulation was intact. The putative AC5-selective P-site inhibitors SQ22,536 [9-(tetra-hydro-2-furanyl-9H-purin-6-amine], vidarabine (9-β-D-arabinosyladenine and NKY80 [2-amino-7-(2-furanyl-7,8-dihydro-5(6H-quinazolinone] inhibited recombinant AC5 more potently than AC2 and AC1, but selectivity was only modest (∼4-40-fold. These compounds inhibited cardiac AC from WT and AC5KO mice with similar potencies. In conclusion, AC regulation in AC5KO hearts was unimpaired, questioning the supposed dominant role of AC5 in the heart. Moreover, the AC inhibitors SQ22,536, NKY80 and vidarabine lack adequate selectivity for AC5 and, therefore, do not present suitable tools to study AC5-specific functions.

  3. Ca2+-stimulated adenylyl cyclase isoform AC1 is preferentially expressed in guinea-pig sino-atrial node cells and modulates the I(f) pacemaker current.

    Science.gov (United States)

    Mattick, Paul; Parrington, John; Odia, Efe; Simpson, Alasdair; Collins, Thomas; Terrar, Derek

    2007-08-01

    Ca(2+)-stimulated adenylyl cyclases (AC) are known to play important roles in neurons but have not previously been reported in the heart. Here we present the first evidence for selective expression of Ca(2+)-stimulated AC in the sino-atrial node (SAN) but not in ventricular muscle of the guinea-pig heart. The AC1 isoform of Ca(2+)-stimulated AC was shown to be present in SAN, both as mRNA using RT-PCR and as protein using immuno-blotting with a specific antibody. Confocal immuno-fluorescence studies detected membrane localization of AC1 in SAN cells, but no AC1 in ventricular muscle. Ca(2+)-stimulated AC8 may also be present in SAN. The functional importance of AC activity was investigated by monitoring activation of I(f) (gated by hyperpolarization and regulated by cAMP, which shifts activation to more depolarized voltages). Basal activity of AC in isolated SAN myocytes was demonstrated by the observations that an inhibitor of AC activity (MDL 12330A, 10 microm) shifted activation in the hyperpolarizing direction, while inhibition of phosphodiesterases (IBMX, 100 microm) shifted I(f) activation in the depolarizing direction. Buffering cytosolic Ca(2+) with the Ca(2+) chelator BAPTA (by exposure to BAPTA-AM) shifted activation of I(f) in the hyperpolarizing direction, and under these conditions the AC inhibitor MDL had little or no further effect. The actions of BAPTA were overcome by exposure to forskolin (10 microm), a direct stimulator of all AC isoforms, to restore cAMP levels. These effects are consistent with the functional importance of Ca(2+)-stimulated AC, which is expected to be fundamental to initiation and regulation of the heartbeat.

  4. A New Type of Metal-Binding Site in Cobalt- And Zinc-Containing Adenylate Kinases Isolated From Sulfate-Reducers D. Gigas And D. Desulfuricans ATCC 27774

    Energy Technology Data Exchange (ETDEWEB)

    Gavel, O.Y.; Bursakov, S.A.; Rocco, G.Di; Trincao, J.; Pickering, I.J.; George, G.N.; Calvete, J.J.; Brondino, C.; Pereira, A.S.; Lampreia, J.; Tavares, P.; Moura, J.J.G.; Moura, I.

    2009-05-18

    Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterized in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the 'LID' domain. The sequence {sup 129}Cys-X{sub 5}-His-X{sub 15}-Cys-X{sub 2}-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

  5. A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774.

    Science.gov (United States)

    Gavel, Olga Yu; Bursakov, Sergey A; Di Rocco, Giulia; Trincão, José; Pickering, Ingrid J; George, Graham N; Calvete, Juan J; Shnyrov, Valery L; Brondino, Carlos D; Pereira, Alice S; Lampreia, Jorge; Tavares, Pedro; Moura, José J G; Moura, Isabel

    2008-01-01

    Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

  6. Altered Circadian Food Anticipatory Activity Rhythms in PACAP Receptor 1 (PAC1 Deficient Mice.

    Directory of Open Access Journals (Sweden)

    Jens Hannibal

    Full Text Available Light signals from intrinsically photosensitive retinal ganglion cells (ipRGCs entrain the circadian clock and regulate negative masking. Two neurotransmitters, glutamate and Pituitary Adenylate Cyclase Activating Polypeptide (PACAP, found in the ipRGCs transmit light signals to the brain via glutamate receptors and the specific PACAP type 1 (PAC1 receptor. Light entrainment occurs during the twilight zones and has little effect on clock phase during daytime. When nocturnal animals have access to food only for a few hours during the resting phase at daytime, they adapt behavior to the restricted feeding (RF paradigm and show food anticipatory activity (FAA. A recent study in mice and rats demonstrating that light regulates FAA prompted us to investigate the role of PACAP/PAC1 signaling in the light mediated regulation of FAA. PAC1 receptor knock out (PAC1-/- and wild type (PAC1+/+ mice placed in running wheels were examined in a full photoperiod (FPP of 12:12 h light/dark (LD and a skeleton photoperiod (SPP 1:11:1:11 h L:DD:L:DD at 300 and 10 lux light intensity. Both PAC1-/- mice and PAC1+/+ littermates entrained to FPP and SPP at both light intensities. However, when placed in RF with access to food for 4-5 h during the subjective day, a significant change in behavior was observed in PAC1-/- mice compared to PAC1+/+ mice. While PAC1-/- mice showed similar FAA as PAC1+/+ animals in FPP at 300 lux, PAC1-/- mice demonstrated an advanced onset of FAA with a nearly 3-fold increase in amplitude compared to PAC1+/+ mice when placed in SPP at 300 lux. The same pattern of FAA was observed at 10 lux during both FPP and SPP. The present study indicates a role of PACAP/PAC1 signaling during light regulated FAA. Most likely, PACAP found in ipRGCs mediating non-image forming light information to the brain is involved.

  7. 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.

  8. Increased Nicotiana tabacum fitness through positive regulation of carotenoid, gibberellin and chlorophyll pathways promoted by Daucus carota lycopene β-cyclase (Dclcyb1) expression.

    Science.gov (United States)

    Moreno, J C; Cerda, A; Simpson, K; Lopez-Diaz, I; Carrera, E; Handford, M; Stange, C

    2016-04-01

    Carotenoids, chlorophylls and gibberellins are derived from the common precursor geranylgeranyl diphosphate (GGPP). One of the enzymes in carotenoid biosynthesis is lycopene β-cyclase (LCYB) that catalyzes the conversion of lycopene into β-carotene. In carrot, Dclcyb1 is essential for carotenoid synthesis in the whole plant. Here we show that when expressed in tobacco, increments in total carotenoids, β-carotene and chlorophyll levels occur. Furthermore, photosynthetic efficiency is enhanced in transgenic lines. Interestingly, and contrary to previous observations where overexpression of a carotenogenic gene resulted in the inhibition of the synthesis of gibberellins, we found raised levels of active GA4 and the concommitant increases in plant height, leaf size and whole plant biomass, as well as an early flowering phenotype. Moreover, a significant increase in the expression of the key carotenogenic genes, Ntpsy1, Ntpsy2 and Ntlcyb, as well as those involved in the synthesis of chlorophyll (Ntchl), gibberellin (Ntga20ox, Ntcps and Ntks) and isoprenoid precursors (Ntdxs2 and Ntggpps) was observed. These results indicate that the expression of Dclcyb1 induces a positive feedback affecting the expression of isoprenoid gene precursors and genes involved in carotenoid, gibberellin and chlorophyll pathways leading to an enhancement in fitness measured as biomass, photosynthetic efficiency and carotenoid/chlorophyll composition. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  9. 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.

  10. 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.

  11. 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.

  12. A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy

    International Nuclear Information System (INIS)

    Hass, Mathias A. S.; Liu, Wei-Min; Agafonov, Roman V.; Otten, Renee; Phung, Lien A.; Schilder, Jesika T.; Kern, Dorothee; Ubbink, Marcellus

    2015-01-01

    NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements

  13. Role of water in the enzymatic catalysis: study of ATP + AMP → 2ADP conversion by adenylate kinase.

    Science.gov (United States)

    Adkar, Bharat V; Jana, Biman; Bagchi, Biman

    2011-04-28

    The catalytic conversion ATP + AMP → 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a phosphate transfer and then the release of two ADP molecules. We have computed a novel two-dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, while considering explicit water interactions. Our computed 2DCFES clearly reveals the existence of a stable half-open half-closed (HOHC) intermediate state of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for the reaction by about 20 kJ/mol. We find that the stability of the HOHC state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle.

  14. Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

    Energy Technology Data Exchange (ETDEWEB)

    Shyy, Y.J.; Tian, G.; Tsai, M.D.

    1987-10-06

    Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

  15. Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules.

    Directory of Open Access Journals (Sweden)

    Anna Zdyb

    Full Text Available Jasmonic acid (JA, its derivatives and its precursor cis-12-oxo phytodienoic acid (OPDA form a group of phytohormones, the jasmonates, representing signal molecules involved in plant stress responses, in the defense against pathogens as well as in development. Elevated levels of JA have been shown to play a role in arbuscular mycorrhiza and in the induction of nitrogen-fixing root nodules. In this study, the gene families of two committed enzymes of the JA biosynthetic pathway, allene oxide synthase (AOS and allene oxide cyclase (AOC, were characterized in the determinate nodule-forming model legume Lotus japonicus JA levels were to be analysed in the course of nodulation. Since in all L. japonicus organs examined, JA levels increased upon mechanical disturbance and wounding, an aeroponic culture system was established to allow for a quick harvest, followed by the analysis of JA levels in whole root and shoot systems. Nodulated plants were compared with non-nodulated plants grown on nitrate or ammonium as N source, respectively, over a five week-period. JA levels turned out to be more or less stable independently of the growth conditions. However, L. japonicus nodules formed on aeroponically grown plants often showed patches of cells with reduced bacteroid density, presumably a stress symptom. Immunolocalization using a heterologous antibody showed that the vascular systems of these nodules also seemed to contain less AOC protein than those of nodules of plants grown in perlite/vermiculite. Hence, aeroponically grown L. japonicus plants are likely to be habituated to stress which could have affected JA levels.

  16. Stimulation of soluble guanylate cyclase prevents cigarette smoke-induced pulmonary hypertension and emphysema.

    Science.gov (United States)

    Weissmann, Norbert; Lobo, Borja; Pichl, Alexandra; Parajuli, Nirmal; Seimetz, Michael; Puig-Pey, Raquel; Ferrer, Elisabet; Peinado, Víctor I; Domínguez-Fandos, David; Fysikopoulos, Athanasios; Stasch, Johannes-Peter; Ghofrani, Hossein A; Coll-Bonfill, Núria; Frey, Reiner; Schermuly, Ralph T; García-Lucio, Jéssica; Blanco, Isabel; Bednorz, Mariola; Tura-Ceide, Olga; Tadele, Elsa; Brandes, Ralf P; Grimminger, Jan; Klepetko, Walter; Jaksch, Peter; Rodriguez-Roisin, Robert; Seeger, Werner; Grimminger, Friedrich; Barberà, Joan A

    2014-06-01

    Chronic obstructive pulmonary disease (COPD) is a major cause of death worldwide. No therapy stopping progress of the disease is available. To investigate the role of the soluble guanylate cyclase (sGC)-cGMP axis in development of lung emphysema and pulmonary hypertension (PH) and to test whether the sGC-cGMP axis is a treatment target for these conditions. Investigations were performed in human lung tissue from patients with COPD, healthy donors, mice, and guinea pigs. Mice were exposed to cigarette smoke (CS) for 6 hours per day, 5 days per week for up to 6 months and treated with BAY 63-2521. Guinea pigs were exposed to CS from six cigarettes per day for 3 months, 5 days per week and treated with BAY 41-2272. Both BAY compounds are sGC stimulators. Gene and protein expression analysis were performed by quantitative real-time polymerase chain reaction and Western blotting. Lung compliance, hemodynamics, right ventricular heart mass alterations, and alveolar and vascular morphometry were performed, as well as inflammatory cell infiltrate assessment. In vitro assays of cell adhesion, proliferation, and apoptosis have been done. The functionally essential sGC β1-subunit was down-regulated in patients with COPD and in CS-exposed mice. sGC stimulators prevented the development of PH and emphysema in the two different CS-exposed animal models. sGC stimulation prevented peroxynitrite-induced apoptosis of alveolar and endothelial cells, reduced CS-induced inflammatory cell infiltrate in lung parenchyma, and inhibited adhesion of CS-stimulated neutrophils. The sGC-cGMP axis is perturbed by chronic exposure to CS. Treatment of COPD animal models with sGC stimulators can prevent CS-induced PH and emphysema.

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

    Science.gov (United States)

    Waldman, Scott A

    2010-01-01

    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. PMID:20592492

  18. 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.

  19. [Attenuation of inhibitory influence of hormones on adenylyl cyclase systems in the myocardium and brain of rats with obesity and type 2 diabetes mellitus and effect of intranasal insulin on it].

    Science.gov (United States)

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

    2014-01-01

    The functional state of the adenylyl cyclase signaling system (ACSS) and its regulation by hormones, the inhibitors of adenylyl cyclase (AC)--somatostatin (SST) in the brain and myocardium and 5-nonyloxytryptamine (5-NOT) in the brain of rats of different ages (5- and 7-month-old) with experimental obesity and a combination of obesity and type 2 diabetes mellitus (DM2), and the effect of long-term treatment of animals with intranasally administered insulin (II) on ACSS were studied. It was shown that the basal AC activity in rats with obesity and DM2 was increased in the myocardium, and to the lesser extent in the brain, the treatment with II reducing this parameter. The AC stimulating effects of forskolin are decreased in the myocardium, but not in the brain, of rats with obesity and DM2. The treatment with II restored the AC action of forskolin in the 7-month-old animals, but has little effect on it in the 5-month-old rats. In obesity the basal AC activity and its stimulation by forskolin varied insignificantly and weakly changed in treatment of animals with II. The AC inhibitory effects of SST and 5-NOT in the investigated pathology are essentially attenuated, the effect of SST to the greatest extent, which we believe to be associated with a reduction in the functional activity of Gi-proteins. The II treatment of animals with obesity and with a combination of obesity and DM2 restored completely or partially the AC inhibiting effects of hormones, to the greatest extent in the brain. Since impaired functioning of ACSS is one of the causes of the metabolic syndrome and DM2, their elimination by treatments with II can be an effective approach to treat these diseases and their CNS and cardiovascular system complications.

  20. Expression of heterologous lycopene β-cyclase gene in flax can cause silencing of its endogenous counterpart by changes in gene-body methylation and in ABA homeostasis mechanism.

    Science.gov (United States)

    Boba, Aleksandra; Kostyn, Kamil; Preisner, Marta; Wojtasik, Wioleta; Szopa, Jan; Kulma, Anna

    2018-03-21

    Previously we described flax plants with expression of Arabidopsis lycopene β-cyclase (lcb) gene in which decreased expression of the endogenous lcb and increased resistance to fungal pathogen was observed. We suggested that co-suppression was responsible for the change. In this study we investigated the molecular basis of the observed effect in detail. We found that methylation changes in the Lulcb gene body might be responsible for repression of the gene. Treatment with azacitidine (DNA methylation inhibitor) confirmed the results. Moreover, we studied how the manipulation of carotenoid biosynthesis pathway increased ABA level in these plants. We suggest that elevated ABA levels may be responsible for the increased resistance of the flax plants to pathogen infection through activation of chitinase (PR gene). Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  1. Differential control of Yersinia pestis biofilm formation in vitro and in the flea vector by two c-di-GMP diguanylate cyclases.

    Directory of Open Access Journals (Sweden)

    Yi-Cheng Sun

    2011-04-01

    Full Text Available Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE, encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP and two DGCs (HmsT and Y3730 control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis.

  2. Differential Intraocular Pressure Measurements by Tonometry and Direct Cannulation After Treatment with Soluble Adenylyl Cyclase Inhibitors.

    Science.gov (United States)

    Gandhi, Jarel K; Roy Chowdhury, Uttio; Manzar, Zahid; Buck, Jochen; Levin, Lonny R; Fautsch, Michael P; Marmorstein, Alan D

    2017-10-01

    To validate the increase in intraocular pressure (IOP) caused by soluble adenylyl cyclase (sAC) inhibitors and determine reasons behind variation in IOP measurements performed by tonometry. C57BL/6J mice were administered DMSO solubilized sAC inhibitors (KH7 or LRE-1) by intraperitoneal injection. Two hours post-treatment, mice were anesthetized with avertin or ketamine/xylazine/acepromazine (KXA). IOP was measured by a rebound tonometer or direct cannulation of the anterior chamber. Spectral-domain optical coherence tomography was used to measure anterior chamber depth and corneal thickness in live mice. Outflow facility was measured in perfused, enucleated mouse eyes. Compared with DMSO controls, KH7 treatment caused an increased IOP in avertin- and KXA-anesthetized mice when measured by direct cannulation [avertin: 14.4 ± 2.1 mmHg vs. 11.1 ± 1.0 mmHg (P = 0.003); KXA: 14.4 ± 1.0 mmHg vs. 11.3 ± 0.8 mmHg (P < 0.001)] and tonometry [avertin: 10.8 ± 1.4 mmHg vs. 7.4 ± 0.6 mmHg (P < 0.001); KXA: 11.9 ± 0.9 mmHg vs. 10.3 ± 1.7 mmHg (P = 0.283)]. However, treatment with KH7 in nonanesthetized mice showed a significant decrease in IOP measured by tonometry and compared with DMSO-treated animals [13.1 ± 2.6 mmHg vs. 15.6 ± 0.5 mmHg (P = 0.003)]. Both KH7- and DMSO-treated groups anesthetized with avertin showed increased corneal thickness, whereas KH7-treated mice anesthetized with KXA exhibited a shallower anterior chamber compared with untreated mice. KH7 decreased outflow facility by 85.1% in nonanesthetized, enucleated eyes (P < 0.003). Systemically administered DMSO and anesthesia have significant effects on anterior chamber characteristics, resulting in altered IOP readings measured by tonometry. In the presence of DMSO and anesthesia, tonometry IOP readings should be confirmed with direct cannulation.

  3. 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

  4. Effects of Alkaline Phosphatase Activity on Nucleotide Measurements in Aquatic Microbial Communities †

    OpenAIRE

    Karl, D. M.; Craven, D. B.

    1980-01-01

    Alkaline phosphatase (APase) activity was detected in aquatic microbial assemblages from the subtropics to Antarctica. The occurrence of APase in environmental nucleotide extracts was shown to significantly affect the measured concentrations of cellular nucleotides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, guanosine triphosphate, uridine triphosphate, and cytidine triphosphate), adenylate energy charge, and guanosine triphosphate/adenosine triphosphate ratios, w...

  5. Somatic 'soluble' adenylyl cyclase isoforms are unaffected in Sacy tm1Lex/Sacy tm1Lex 'knockout' mice.

    Directory of Open Access Journals (Sweden)

    Jeanne Farrell

    Full Text Available BACKGROUND: Mammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacy(tm1Lex/Sacy(tm1Lex knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference. PRINCIPAL FINDINGS: We now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which 'escapes' the design of the Sacy(tm1Lex knockout allele. CONCLUSIONS/SIGNIFICANCE: These studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.

  6. Adenylyl cyclase alpha and cAMP signaling mediate Plasmodium sporozoite apical regulated exocytosis and hepatocyte infection.

    Directory of Open Access Journals (Sweden)

    Takeshi Ono

    2008-02-01

    Full Text Available Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei and human (P. falciparum Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase alpha (ACalpha, a gene containing regions with high homology to adenylyl cyclases. PbACalpha-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACalpha, as re-introduction of ACalpha in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACalpha and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.

  7. Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice

    Directory of Open Access Journals (Sweden)

    Margarita O. Shleeva

    2017-08-01

    Full Text Available Earlier we demonstrated that the adenylyl cyclase (AC encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis (Mtb, the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro, the AC-overexpressing pMindRv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo, AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMindRv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo.

  8. Cyclic AMP system in muscle tissue during prolonged hypokinesia

    Science.gov (United States)

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

    1980-01-01

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

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

    International Nuclear Information System (INIS)

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

    1993-01-01

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

  10. 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, ...

  11. The GIP receptor displays higher basal activity than the GLP-1 receptor but does not recruit GRK2 or arrestin3 effectively.

    Directory of Open Access Journals (Sweden)

    Suleiman Al-Sabah

    Full Text Available Glucagon-like peptide-1 (GLP-1 and glucose-dependent insulinotropic polypeptide (GIP are important regulators of insulin secretion, and their functional loss is an early characteristic of type 2 diabetes mellitus (T2DM. Pharmacological levels of GLP-1, but not GIP, can overcome this loss. GLP-1 and GIP exert their insulinotropic effects through their respective receptors expressed on pancreatic β-cells. Both the GLP-1 receptor (GLP-1R and the GIP receptor (GIPR are members of the secretin family of G protein-coupled receptors (GPCRs and couple positively to adenylate cyclase. We compared the signalling properties of these two receptors to gain further insight into why GLP-1, but not GIP, remains insulinotropic in T2DM patients.GLP-1R and GIPR were transiently expressed in HEK-293 cells, and basal and ligand-induced cAMP production were investigated using a cAMP-responsive luciferase reporter gene assay. Arrestin3 (Arr3 recruitment to the two receptors was investigated using enzyme fragment complementation, confocal microscopy and fluorescence resonance energy transfer (FRET.GIPR displayed significantly higher (P<0.05 ligand-independent activity than GLP-1R. Arr3 displayed a robust translocation to agonist-stimulated GLP-1R but not to GIPR. These observations were confirmed in FRET experiments, in which GLP-1 stimulated the recruitment of both GPCR kinase 2 (GRK2 and Arr3 to GLP-1R. These interactions were not reversed upon agonist washout. In contrast, GIP did not stimulate recruitment of either GRK2 or Arr3 to its receptor. Interestingly, arrestin remained at the plasma membrane even after prolonged (30 min stimulation with GLP-1. Although the GLP-1R/arrestin interaction could not be reversed by agonist washout, GLP-1R and arrestin did not co-internalise, suggesting that GLP-1R is a class A receptor with regard to arrestin binding.GIPR displays higher basal activity than GLP-1R but does not effectively recruit GRK2 or Arr3.

  12. [Peptide 612-627 of thyrotropin receptor and its modified derivatives as the regulators of adenylyl cyclase in the rat thyroid gland].

    Science.gov (United States)

    Shpakov, A O; Shpakova, E A; Tarasenko, I I; Derkach, K V

    2014-01-01

    The regulation of the specific activity of the thyroid gland is carried by thyroid-stimulating hormone (TSH) through TSH receptor (TSHR). This receptor is coupled to different types of G-proteins, including the G(s)-proteins, through which TSH stimulates the enzyme adenylyl cyclase (AC). As the application of TSH in medicine is limited, the development of selective regulators of TSHR with agonistic and antagonistic activity is carried out. One of the approaches to their creation is to develop the peptides corresponding to functionally important regions of TSHR which are located in its intracellular loops (ICL) and are involved in the binding and activation of G-proteins. We have synthesized peptide corresponding to the C-terminal region 612-627 of the third ICL of TSHR and its derivatives modified by palmitic acid residue (at the N- or the C-terminus) or by polylysine dendrimer (at the N-terminus), and studied their effect on the basal and TSH-stimulated AC activity in the membrane fraction isolated from the rat thyroid. The most active was peptide 612-627-K(Pal)A modified by palmitate at the C-terminus, where in TSHR the hydrophobic transmembrane region is located. At the micromolar concentrations the peptide increased AC activity and reduced the AC stimulating effect of TSH. The action of the 612-627-K(Pal)A has been directed onto TSHR homologous to it, as indicated by the following facts: 1) the inhibition of G(s)-protein, the downstream component of AC system, by treating the membranes with cholera toxin led to the blocking of peptide AC effect, 2) this effect was not detected in the tissues where no TSHR, 3) the peptide did not significantly affect the AC stimulating effects of hormones acting via other receptors. The unmodified peptide and the peptide with N-terminal dendrimer are far behind the 612-627-K(Pal)A in their ability to activate AC in the thyroid, while the peptide modified by palmitate at the N-terminus was inactive. At the same time, the peptide

  13. Structure of the D-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Bera, A.K.; Robinson, H.; Atanasova, V.; Gamage, S.; Parsons, J. F.

    2010-06-01

    The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound D-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

  14. Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis

    International Nuclear Information System (INIS)

    Kladova, A. V.; Gavel, O. Yu.; Mukhopaadhyay, A.; Boer, D. R.; Teixeira, S.; Shnyrov, V. L.; Moura, I.; Moura, J. J. G.; Romão, M. J.; Trincão, J.; Bursakov, S. A.

    2009-01-01

    Adenylate kinase (AK) from D. gigas was purified and crystallized in three different metal-bound forms: Zn 2+ –AK, Co 2+ –AK and Fe 2+ –AK. Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 Å resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 Å resolution, respectively. Zn 2+ –AK and Fe 2+ –AK crystallized in space group I222 with similar unit-cell parameters, whereas Co 2+ –AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn 2+ –AK and Fe 2+ –AK forms and a dimer was present for the Co 2+ –AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes

  15. Expression of the diguanylate cyclase GcbA is regulated by FleQ in response to cyclic di-GMP in Pseudomonas putida KT2440.

    Science.gov (United States)

    Xiao, Yujie; Nie, Hailing; Liu, Huizhong; Chen, Wenli; Huang, Qiaoyun

    2016-12-01

    Cyclic di-GMP (c-di-GMP), a ubiquitous bacterial second messenger that regulates diverse cellular processes, is synthesized by diguanylate cyclase (DGC) and degraded by phosphodiesterase (PDE). GcbA is a well conserved DGC among Pseudomonas species, and has been reported to influence biofilm formation and flagellar motility in Pseudomonas fluorescens and Pseudomonas aeruginosa. Here we confirm the function of GcbA in Pseudomonas putida and reveal that expression of GcbA is regulated by FleQ in response to c-di-GMP. GcbA deletion impaired initial biofilm formation and enhanced swimming motility, but showed no influence on biofilm maturation in Pseudomonas putida. Deletion of the c-di-GMP effector FleQ led to a significant decrease in transcription of gcbA. Moreover, reducing c-di-GMP levels promoted gcbA transcription in a FleQ dependent way, while enhancing c-di-GMP levels abolished the promotion. In in vitro experiments we found that FleQ bound to gcbA promoter DNA and the binding was inhibited by c-di-GMP. Besides, FleN, an anti-activator of FleQ, and the sigma factor RpoN also participated in transcription of gcbA. Our finding expands the complexity of FleQ-dependent regulation and reveals a self-regulation function of c-di-GMP by regulating GcbA expression via FleQ. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. Role of the metabolism of parathyroid hormone. [Rats

    Energy Technology Data Exchange (ETDEWEB)

    Teitelbaum, Anne P. [Univ. of Rochester, NY (United States)

    1978-01-01

    The heterogeneity of parathyroid hormone (PTH) in plasma has prompted investigations of the metabolism of PTH and its relationship to hormone action. The time course of tissue distribution and metabolism of electrolytically iodinated PTH (E-PTH) previously shown to retain biological activity was compared with that of inactive PTH iodinated with Chloramine-T (CT-PTH). Labeled PTH (0.4 μg) was injected in the saphenous veins of anesthetized rats which were sacrificed at 1, 3, 5, 10, and 20 min. Tissue extracts from kidney, liver, and serum were chromatographed to separate intact PTH from its metabolites. In the kidney, the initial rate of degradation of E-PTH was greater than that of CT-PTH. The difference in initial rates of metabolism may be due, in part, to receptor-specific hydrolysis on peritubular cell membranes which selectively act on biologically active PTH molecules. PTH-responsive adenyl cyclase activity in isolated kidney cortex plasma membranes was measured and PTH metabolism was monitored simultaneously. When degradation was completely blocked by histone f3 (1 mg/ml), adenyl cyclase activity was significantly increased over control. In addition, when adenyl cyclase activity was negligible, the rate of PTH degradation by the membranes was not significantly diminished. Consistent with the in vivo data was the observation that E-PTH is metabolized by these membranes at a greater rate than CT-PTH. The data demonstrate the existence of a receptor-specific metabolism at sites which are independent of PTH receptor mediated adenyl cyclase activity.

  17. Role of the metabolism of parathyroid hormone

    International Nuclear Information System (INIS)

    Teitelbaum, A.P.

    1978-01-01

    The heterogeneity of parathyroid hormone (PTH) in plasma has prompted investigations of the metabolism of PTH and its relationship to hormone action. The time course of tissue distribution and metabolism of electrolytically iodinated PTH (E-PTH) previously shown to retain biological activity was compared with that of inactive PTH iodinated with Chloramine-T (CT-PTH). Labeled PTH (0.4 μg) was injected in the saphenous veins of anesthetized rats which were sacrificed at 1, 3, 5, 10, and 20 min. Tissue extracts from kidney, liver, and serum were chromatographed to separate intact PTH from its metabolites. In the kidney, the initial rate of degradation of E-PTH was greater than that of CT-PTH. The difference in initial rates of metabolism may be due, in part, to receptor-specific hydrolysis on peritubular cell membranes which selectively act on biologically active PTH molecules. PTH-responsive adenyl cyclase activity in isolated kidney cortex plasma membranes was measured and PTH metabolism was monitored simultaneously. When degradation was completely blocked by histone f 3 (1 mg/ml), adenyl cyclase activity was significantly increased over control. In addition, when adenyl cyclase activity was negligible, the rate of PTH degradation by the membranes was not significantly diminished. Consistent with the in vivo data was the observation that E-PTH is metabolized by these membranes at a greater rate than CT-PTH. The data demonstrate the existence of a receptor-specific metabolism at sites which are independent of PTH receptor mediated adenyl cyclase activity

  18. Pharmacological analysis of feeding in a caterpillar: different transduction pathways for umami and saccharin?

    Science.gov (United States)

    Pszczolkowski, Maciej A.; Durden, Kevin; Marquis, Juleah; Ramaswamy, Sonny B.; Brown, John J.

    2009-05-01

    Neonate larvae of codling moth, Cydia pomonella (L.), modify their behavior in the presence of saccharin, monosodium glutamate (MSG), or L(+)-2-amino-4-phosphonobutyric acid (L-AP4) by commencing their feeding earlier. Previously published pharmacological analysis demonstrated that phagostimulatory effects of MSG and L-AP4 (which elicit umami taste sensation in humans) are reversed by adenylate cyclase activator and phosphodiesterase inhibitor. In this study, by measuring the time needed to start ingestion of foliage treated with mixtures of phagostimulants and signal transduction modulators, we show that phagostimulatory effects of l-aspartate (the third hallmark umami substance) are also abolished by both adenylate cyclase activator and phosphodiesterase inhibitor, but not by phospholipase C inhibitor. However, stimulatory effects of hemicalcium saccharin were affected only by phospholipase C inhibitor. The results suggest that codling moth neonates use different transduction pathways for perception of hemicalcium saccharin and umami.

  19. Inhibition of cAMP-Dependent PKA Activates β2-Adrenergic Receptor Stimulation of Cytosolic Phospholipase A2 via Raf-1/MEK/ERK and IP3-Dependent Ca2+ Signaling in Atrial Myocytes.

    Science.gov (United States)

    Pabbidi, M R; Ji, X; Maxwell, J T; Mignery, G A; Samarel, A M; Lipsius, S L

    2016-01-01

    -AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.

  20. Cenderitide: structural requirements for the creation of a novel dual particulate guanylyl cyclase receptor agonist with renal-enhancing in vivo and ex vivo actions.

    Science.gov (United States)

    Lee, Candace Y W; Huntley, Brenda K; McCormick, Daniel J; Ichiki, Tomoko; Sangaralingham, S Jeson; Lisy, Ondrej; Burnett, John C

    2016-04-01

    Cenderitide is a novel dual natriuretic peptide (NP) receptor chimeric peptide activator, which targets the particulate guanylyl cyclase B (pGC-B) receptor and pGC-A unlike native NPs. Cenderitide was engineered to retain the anti-fibrotic properties of C-type natriuretic peptide (CNP)/pGC-B with renal-enhancing actions facilitated by fusion to the carboxyl terminus of Dendroaspis NP (DNP), a pGC-A agonist, to CNP. Here, we address significance of the DNP carboxyl terminus in dual pGC receptor activation and actions of cenderitide compared with CNP on renal function and cyclic guanosine monophosphate (cGMP) in vivo and ex vivo in normal canines. In vitro, only cenderitide and not CNP or three CNP-based variants was a potent dual pGC-A/pGC-B activator of cGMP production (from 5 to 237 pmol/mL) in human embryonic kidney (HEK) 293 cells overexpressing human pGC-A while in pGC-B overexpressing cells cenderitide increased cGMP production (from 4 to 321 pmol/mL) while the three CNP-based variants were weak agonists. Based upon our finding that the DNP carboxyl terminus is a key structural requirement for dual pGC-A/pGC-B activation, we defined in vivo the renal-enhancing actions of cenderitide compared with CNP. Cenderitide increased urinary cGMP excretion (from 989 to 5977 pmol/mL), net generation of renal cGMP (821-4124 pmol/min), natriuresis (12-242 μEq/min), and glomerular filtration rate (GFR) (37-51 mL/min) while CNP did not. We then demonstrated the transformation of CNP ex vivo into a renal cGMP-activating peptide which increased cGMP in freshly isolated glomeruli eight-fold greater than CNP. The current study establishes that dual pGC-A and pGC-B activation with CNP requires the specific carboxyl terminus of DNP. In normal canines in vivo and in glomeruli ex vivo, the carboxyl terminus of DNP transforms CNP into a natriuretic and GFR-enhancing peptide. Future studies of cenderitide are warranted in cardiorenal disease states to explore its efficacy in overall

  1. C-type natriuretic peptide and the guanylyl cyclase receptors in the rat ovary are modulated by the estrous cycle.

    Science.gov (United States)

    Jankowski, M; Reis, A M; Mukaddam-Daher, S; Dam, T V; Farookhi, R; Gutkowska, J

    1997-01-01

    We have previously shown that rat ovaries synthesize atrial natriuretic peptide (ANP) and express the cognate guanylyl cyclase (GC-A and GC-B) receptors for ANP. Since another natriuretic peptide, termed the C-type natriuretic peptide (CNP), can also interact with these receptors, we have investigated whether rat ovaries express CNP and if so, whether the concentration of this natriuretic peptide and the guanylyl-cyclase receptors are influenced by the estrous cycle. CNP mRNA was detected in rat ovaries using a reverse transcription (RT) polymerase chain reaction (PCR) strategy. RIA of ovarian extracts, obtained at the individual days of the estrous cycle, revealed the presence of immunoreactive CNP. The highest levels of CNP were detected at proestrus and were approximately 4-fold higher than the levels seen at any other stage of the cycle. GC-A and GC-B receptors were detected using quantitative autoradiography after application of either [125I]ANP or [125I]-tyr0CNP to sections of frozen ovaries. The highest specific binding of each radiolabeled ligand was seen in ovaries from proestrous animals. The GC-B receptors were localized to the membrana granulosa of developing ovarian follicles. Using quantitative PCR, we determined that levels of GC-A and GC-B mRNAs were highest in the ovaries of proestrous animals and were approximately 2- to 3-fold higher than the levels seen at diestrus. These findings demonstrate that a natriuretic peptide system, consisting of ligands and receptors, is present in the rat ovary. Since CNP and the GC receptors show coordinate estrous cycle-dependent variation with maximal expression at proestrus, we speculate that the natriuretic peptides may play an important role in either the development of ovulatory follicles or in the ovulatory process.

  2. Molecular Mechanisms Elicited by d-Aspartate in Leydig Cells and Spermatogonia

    OpenAIRE

    Maria Maddalena Di Fiore; Alessandra Santillo; Sara Falvo; Salvatore Longobardi; Gabriella Chieffi Baccari

    2016-01-01

    A bulk of evidence suggests that d-aspartate (d-Asp) regulates steroidogenesis and spermatogenesis in vertebrate testes. This review article focuses on intracellular signaling mechanisms elicited by d-Asp possibly via binding to the N-methyl-d-aspartate receptor (NMDAR) in both Leydig cells, and spermatogonia. In Leydig cells, the amino acid upregulates androgen production by eliciting the adenylate cyclase-cAMP and/or mitogen-activated protein kinase (MAPK) pathways. d-Asp treatment enhances...

  3. Response of substances co-expressed in hypothalamic magnocellular neurons to osmotic challenges in normal and Brattleboro rats

    DEFF Research Database (Denmark)

    Bundzikova, J.; Pirnik, Z.; Zelena, D.

    2008-01-01

    , including cholecystokinin, chromogranin/secretogranin, dynorphin, endothelin-1, enkephalin, ferritin protein, interleukin 6, kininogen, neurokinin B, neuropeptide Y, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, TAFA5 protein, thyrotropin releasing hormone, tyrosine...... hydroxylase, and urocortin. In this brief review, also the responses of these substances to different hyperosmotic and hypoosmotic challenges are pointed out. Based on the literature data published recently, the functional implication of the majority of co-localized substances is still better understood...

  4. Neuropeptides in Lower Urinary Tract (LUT) Function

    OpenAIRE

    Arms, Lauren; Vizzard, Margaret A.

    2011-01-01

    Numerous neuropeptide/receptor systems including vasoactive intestinal polypeptide, pituitary adenylate cyclase-activating polypeptide, calcitonin gene-related peptide, substance P, neurokinin A, bradykinin, and endothelin-1 are expressed in the lower urinary tract (LUT) in both neural and non-neural (e.g., urothelium) components. LUT neuropeptide immunoreactivity is present in afferent and autonomic efferent neurons innervating the bladder and urethra and in the urothelium of the urinary bla...

  5. The RNA Chaperone Hfq Is Required for Virulence of Bordetella pertussis

    Czech Academy of Sciences Publication Activity Database

    Bíbová, Ilona; Škopová, Karolína; Mašín, Jiří; Černý, Ondřej; Hot, D.; Šebo, Peter; Večerek, Branislav

    2013-01-01

    Roč. 81, č. 11 (2013), s. 4081-4090 ISSN 0019-9567 R&D Projects: GA ČR(CZ) GAP302/11/1940; GA ČR GAP302/12/0460 Institutional support: RVO:61388971 Keywords : ADENYLATE-CYCLASE TOXIN * ISLET-ACTIVATING PROTEIN * ESCHERICHIA-COLI HFQ Subject RIV: EC - Immunology Impact factor: 4.156, year: 2013

  6. Mr 40,000 and Mr 39,000 pertussis toxin substrates are increased in surgically denervated dog ventricular myocardium

    Energy Technology Data Exchange (ETDEWEB)

    Hershberger, R.E.; Feldman, A.M.; Anderson, F.L.; Kimball, J.A.; Wynn, J.R.; Bristow, M.R. (Univ. of Utah School of Medicine, Salt Lake City (USA))

    1991-04-01

    To test the general hypothesis that cardiac innervation may participate in myocardial G protein regulation, we examined the effects of complete intrapericardial surgical denervation or sham operation in dogs. In particulate fractions of dog left ventricular (LV) myocardium harvested 28-33 days after denervation or sham operation, Mr 40,000 and Mr 39,000 pertussis toxin-sensitive substrates (G proteins) were increased by 31% (1.31 +/- 0.084 vs 1.00 +/- 0.058 OD, arbitrary units, p less than 0.01) and 40% (1.40 +/- 0.117 vs. 1.000 +/- 0.084 OD, arbitrary units, p less than 0.02), respectively, as compared with sham-operated controls. The Mr 40,000 pertussis toxin-sensitive band comigrated with a pertussis toxin-sensitive substrate in human erythrocyte membranes known to contain an alpha Gi species. In these same preparations basal, GTP and GppNHp stimulated adenylate cyclase activities were decreased in denervated heart by 20, 26, and 19%, respectively, consistent with increased activity of an inhibitory G protein. In contrast, Gs function was not altered, because cyc(-) membranes reconstituted with membrane extracts and fluoride and beta-receptor-stimulated adenylate cyclase activity were not different between groups. Furthermore, adenylate cyclase catalytic subunit function as assessed with forskolin and manganese stimulation was not different between preparations of control and denervated heart. We conclude that in preparations of surgically denervated dog myocardium Mr 40,000 and Mr 39,000 pertussis toxin-sensitive G proteins are increased by 31 and 40%, respectively, and that functional alterations in adenylate cyclase activity exist, consistent with increased inhibitory G-protein function.

  7. Mr 40,000 and Mr 39,000 pertussis toxin substrates are increased in surgically denervated dog ventricular myocardium

    International Nuclear Information System (INIS)

    Hershberger, R.E.; Feldman, A.M.; Anderson, F.L.; Kimball, J.A.; Wynn, J.R.; Bristow, M.R.

    1991-01-01

    To test the general hypothesis that cardiac innervation may participate in myocardial G protein regulation, we examined the effects of complete intrapericardial surgical denervation or sham operation in dogs. In particulate fractions of dog left ventricular (LV) myocardium harvested 28-33 days after denervation or sham operation, Mr 40,000 and Mr 39,000 pertussis toxin-sensitive substrates (G proteins) were increased by 31% (1.31 +/- 0.084 vs 1.00 +/- 0.058 OD, arbitrary units, p less than 0.01) and 40% (1.40 +/- 0.117 vs. 1.000 +/- 0.084 OD, arbitrary units, p less than 0.02), respectively, as compared with sham-operated controls. The Mr 40,000 pertussis toxin-sensitive band comigrated with a pertussis toxin-sensitive substrate in human erythrocyte membranes known to contain an alpha Gi species. In these same preparations basal, GTP and GppNHp stimulated adenylate cyclase activities were decreased in denervated heart by 20, 26, and 19%, respectively, consistent with increased activity of an inhibitory G protein. In contrast, Gs function was not altered, because cyc(-) membranes reconstituted with membrane extracts and fluoride and beta-receptor-stimulated adenylate cyclase activity were not different between groups. Furthermore, adenylate cyclase catalytic subunit function as assessed with forskolin and manganese stimulation was not different between preparations of control and denervated heart. We conclude that in preparations of surgically denervated dog myocardium Mr 40,000 and Mr 39,000 pertussis toxin-sensitive G proteins are increased by 31 and 40%, respectively, and that functional alterations in adenylate cyclase activity exist, consistent with increased inhibitory G-protein function

  8. Altered Regulation of the Diguanylate Cyclase YaiC Reduces Production of Type 1 Fimbriae in a Pst Mutant of Uropathogenic Escherichia coli CFT073.

    Science.gov (United States)

    Crépin, Sébastien; Porcheron, Gaëlle; Houle, Sébastien; Harel, Josée; Dozois, Charles M

    2017-12-15

    The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC ( adrA in Salmonella ) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA , affecting at the same time the inversion of the fim promoter switch ( fimS ). In the pst mutant, inactivation of yaiC restored fim -dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC , which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the

  9. Choleragen-stimulated release of guanyl nucleotides from turkey erythrocyte membranes.

    Science.gov (United States)

    Burns, D L; Moss, J; Vaughan, M

    1982-01-10

    Choleragen stimulates adenylate cyclase by ADP ribosylating a guanyl nucleotide-binding regulatory protein (G/F). beta-Adrenergic hormones also activate the adenylate cyclase of turkey erythrocytes, and it is currently believed that they do so in part by decreasing the affinity of G/F factor for GDP, an effect which is manifested by a hormone-stimulated release of guanyl nucleotides from the membranes. Since choleragen might also activate adenylate cyclase by a similar mechanism, the effect of toxin treatment on the release of guanyl nucleotides from turkey erythrocyte membranes was examined. In the presence of NAD, choleragen was found to stimulate release of guanyl nucleotides from membranes which had been preloaded with radiolabeled GTP. No stimulation of release was observed with cAMP or when NAD was replaced by NADP, which does not serve as a substrate for choleragen-catalyzed ADP ribosylation. While either isoproterenol or choleragen can stimulate release of guanyl nucleotides from the membranes, the amount of guanyl nucleotide released in the presence of both isoproterenol and choleragen was no greater than that released by isoproterenol alone. Furthermore, when membranes were first treated with choleragen and NAD, the subsequent release of guanyl nucleotides induced by isoproterenol was reduced to approximately 15% of that observed with membranes not treated with the toxin. Therefore, choleragen may enhance release of guanine nucleotides from sites on the membranes that are also affected by beta-adrenergic agonists, sites which are thought to correspond to G/F. These data are consistent with the hypothesis that choleragen may stimulate adenylate cyclase, in part, by enhancing release of guanyl nucleotides, a mechanism similar to that of beta-adrenergic agonists.

  10. Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis.

    Science.gov (United States)

    Ayabe, T; Park, S K; Takenaka, H; Sumida, M; Uesugi, S; Takenaka, O; Hamada, M

    1996-11-01

    To elucidate lysine residues in the N-terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site-directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady-state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2-. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2-. In human AK, the epsilon-amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2- and AMP2- during transition state.

  11. Structure of the d-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    International Nuclear Information System (INIS)

    Bera, Asim K.; Atanasova, Vesna; Gamage, Swarna; Robinson, Howard; Parsons, James F.

    2010-01-01

    The structure of EhpF from P. agglomerans has been solved alone and in complex with phenazine-1,6-dicarboxylate. Apo EhpF was solved and refined in two different space groups at 1.95 and 2.3 Å resolution and the EhpF–phenazine-1,6-dicarboxylate complex structure was determined at 2.8 Å resolution. The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound d-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate