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

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

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

Adenylate cyclase regulation in intact cultured myocardial cells

International Nuclear Information System (INIS)

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

1987-01-01

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

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

International Nuclear Information System (INIS)

Anderson, P.H.; Nielson, M.

1987-01-01

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

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

Science.gov (United States)

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

2015-02-01

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

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

Directory of Open Access Journals (Sweden)

Ma’ayan Israeli

2016-08-01

Full Text Available Edema Factor (EF, the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP, and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules.

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

Energy Technology Data Exchange (ETDEWEB)

Chatterjee, A; Bhattacharya, A K

1988-06-01

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

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

International Nuclear Information System (INIS)

Chatterjee, A.; Bhattacharya, A.K.

1988-01-01

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

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

Science.gov (United States)

Chatterjee, A; Bhattacharya, A K

1988-06-01

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

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

KAUST Repository

Hussein, Rana

2012-01-01

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

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.

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

DEFF Research Database (Denmark)

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

2005-01-01

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

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...

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

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

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

International Nuclear Information System (INIS)

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

1986-01-01

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

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.

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.

Adenylate cyclase activity in fish gills in relation to salt adaptation

International Nuclear Information System (INIS)

Guibbolini, M.E.; Lahlou, B.

1987-01-01

The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of [α- 32 P] - ATP into [α- 32 P] - cAMP) was investigated in gill plasma membranes of rainbow trout (Salmo gairdneri) adapted to various salinities (deionized water, DW; fresh water, FW; 3/4 sea water, 3/4 SW; sea water, SW) and in sea water adapted- mullet (Mugil sp.). Basal activity declined by a factor of 2 in trout with increasing external salinity (pmoles cAMP/mg protein/10 min: 530 in DW, 440 in FW, 340 in 3/4 SW; 250 in SW) and was very low in SW adapted-mullet: 35. The Km for ATP was similar (0.5 mM) in both FW adapted- and SW adapted- trout in either the absence (basal activity) or in the presence of stimulating agents (isoproterenol; NaF) while the Vm varied. Analysis of stimulation ratios with respect to basal levels of the enzyme showed that hormones and pharmacological substances (isoproterenol, NaF) display a greater potency in high salt than in low salt adapted- fish gills. In contrast, salt adaptation did not have any effect on the regulation of adenylate cyclase by PGE 1 . These results are interpreted in relation to the general process of osmoregulation. 27 references, 6 figures

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.

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

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

International Nuclear Information System (INIS)

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

1980-01-01

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

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

DEFF Research Database (Denmark)

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

2014-01-01

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

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

International Nuclear Information System (INIS)

Beebe, L.E.

1989-01-01

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

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

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

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

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

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

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

Czech Academy of Sciences Publication Activity Database

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

2018-01-01

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

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

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

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

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

International Nuclear Information System (INIS)

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

1986-01-01

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

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

Science.gov (United States)

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

1996-05-31

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

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

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

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

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

International Nuclear Information System (INIS)

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

1987-01-01

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

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.

Adenyl cyclases and cAMP in plant signaling - Past and present

KAUST Repository

Gehring, Christoph A

2010-01-01

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

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

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)

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

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

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

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

International Nuclear Information System (INIS)

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

1986-01-01

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

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

Science.gov (United States)

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

2015-01-01

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

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

Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture

International Nuclear Information System (INIS)

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

1986-01-01

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

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

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

Science.gov (United States)

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

2006-12-01

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

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

DEFF Research Database (Denmark)

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

1995-01-01

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

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

DEFF Research Database (Denmark)

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

1994-01-01

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

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

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...

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

International Nuclear Information System (INIS)

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

1990-01-01

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

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

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.

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

Science.gov (United States)

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

1982-03-10

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

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.

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

Energy Technology Data Exchange (ETDEWEB)

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

1982-11-01

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

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

Science.gov (United States)

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

2012-01-01

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

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

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

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

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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.

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.

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.

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

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.

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

International Nuclear Information System (INIS)

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

1980-01-01

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

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.

Adenylate cyclase regulates elongation of mammalian primary cilia

Energy Technology Data Exchange (ETDEWEB)

Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J. [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada); Rattner, Jerome B. [Department of Cell Biology and Anatomy, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada); Hoorn, Frans A. van der, E-mail: fvdhoorn@ucalgary.ca [Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 (Canada)

2009-10-01

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

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

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

Science.gov (United States)

Salmon, Didier

2018-04-25

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

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

Directory of Open Access Journals (Sweden)

Didier Salmon

2018-04-01

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

Pituitary adenylate cyclase activating peptide (PACAP participates in adipogenesis by activating ERK signaling pathway.

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Tatjana Arsenijevic

Full Text Available Pituitary adenylate cyclase activating peptide (PACAP belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP family. Its action can be mediated by three different receptor subtypes: PAC1, which has exclusive affinity for PACAP, and VPAC1 and VPAC2 which have equal affinity for PACAP and VIP. We showed that all three receptors are expressed in 3T3-L1 cells throughout their differentiation into adipocytes. We established the activity of these receptors by cAMP accumulation upon induction by PACAP. Together with insulin and dexamethasone, PACAP induced adipogenesis in 3T3-L1 cell line. PACAP increased cAMP production within 15 min upon stimulation and targeted the expression and phosphorylation of MAPK (ERK1/2, strengthened by the ERK1/2 phosphorylation being partially or completely abolished by different combinations of PACAP receptors antagonists. We therefore speculate that ERK1/2 activation is crucial for the activation of CCAAT/enhancer- binding protein β (C/EBPβ.

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)

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.

A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate

OpenAIRE

Pirger, Zsolt; László, Zita; Kemenes, Ildikó; Tóth, Gábor; Reglődi, Dóra; Kemenes, György

2010-01-01

Similar to other invertebrate and vertebrate animals, cAMP dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unkno...

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

Pituitary adenylate cyclase activating polypeptide in stress-related disorders: data convergence from animal and human studies.

Science.gov (United States)

Hammack, Sayamwong E; May, Victor

2015-08-01

The maladaptive expression and function of several stress-associated hormones have been implicated in pathological stress and anxiety-related disorders. Among these, recent evidence has suggested that pituitary adenylate cyclase activating polypeptide (PACAP) has critical roles in central neurocircuits mediating stress-related emotional behaviors. We describe the PACAPergic systems, the data implicating PACAP in stress biology, and how altered PACAP expression and signaling may result in psychopathologies. We include our work implicating PACAP signaling within the bed nucleus of the stria terminalis in mediating the consequences of stressor exposure and relatedly, describe more recent studies suggesting that PACAP in the central nucleus of the amygdala may impact the emotional aspects of chronic pain states. In aggregate, these results are consistent with data suggesting that PACAP dysregulation is associated with posttraumatic stress disorder in humans. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Investigation and characterization of receptors for pituitary adenylate cyclase-activating polypeptide in human brain by radioligand binding and chemical cross-linking

International Nuclear Information System (INIS)

Suda, K.; Smith, D.M.; Ghatei, M.A.; Murphy, J.K.; Bloom, S.R.

1991-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel peptide of hypothalamic origin which increases adenylate cyclase activity in rat anterior pituitary cell cultures. The 38-amino acid peptide shows a close sequence homology to vasoactive intestinal peptide (VIP). Binding sites for PACAP in membranes from postmortem human brain tissue were studied using [ 125 I]PACAP27 as the radioligand. High specific binding sites (amount of specific binding measured at 0.25 nM [ 125 I]PACAP27 in femtomoles per mg protein +/- SEM; n = 4) were present in hypothalamus (344.5 +/- 13.0), brain stem (343.0 +/- 29.3), cerebellum (292.0 +/- 21.1), cortex (259.6 +/- 19.8), and basal ganglia (259.2 +/- 50.3). Specific binding sites in pituitary, although present, were less abundant (35.0 +/- 8.9). Binding of [ 125 I]PACAP27 was reversible and time, pH, and temperature dependent. Despite the homology with VIP, VIP was a poor inhibitor of [ 125 I]PACAP27 binding (IC50, greater than 1 microM) compared with PACAP27 (IC50, 0.5-1.3 nM) and PACAP38 (IC50, 0.2-1.3 nM). Scatchard plots of [ 125 I]PACAP27 binding showed the presence of both high and lower affinity sites. Chemical cross-linking of PACAP-binding sites revealed that [ 125 I]PACAP27 was bound to polypeptide chains of 67,000 and 48,000 mol wt. Thus, we have demonstrated the presence of PACAP-specific receptors in human brain which are not VIP receptors. This opens the possibility of PACAP functioning as a novel neurotransmitter/neuromodulator in human brain

Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.

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Merriam, Laura A; Baran, Caitlin N; Girard, Beatrice M; Hardwick, Jean C; May, Victor; Parsons, Rodney L

2013-03-06

After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

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

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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.

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

Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

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Mounira Tlili

2015-01-01

Full Text Available The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP, we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC and cytokines (IL-1α and TNF-α in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders.

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

Phorbol esters alter adenylate cyclase responses to vasoactive intestinal peptide and forskolin in the GH cell line

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Summers, S.; Florio, T.; Cronin, M.

1986-05-01

Activation of protein kinase C with phorbol ester modifies cyclic AMP production in several anterior pituitary cell systems. In the GH cell line from a rat pituitary tumor, exposure to phorbol 12-myristate 13-acetate (PMA: 100 nM) for 30 minutes significantly reduces vasoactive intestinal peptide (VIP: 100 nM) stimulated adenylate cyclase (AC) activity in subsequent membrane preparations to 62 + 4% of control (n = 6 independent studies). In contrast, these same membrane preparations respond to forskolin (1 ..mu..M) with significantly more activity, 130 +/- 6% of controls (n = 6 independent studies). Finally, phorbol ester does not block an inhibitory hormone input into the AC system; somatostatin (100 nM) reduction of VIP-stimulated AC activity is not significantly different in membrane preparations from PMA treated and control cells (n = 3 independent studies). These other findings lead the authors to propose that protein kinase C can modify several sites in the AC complex in anterior pituitary cells.

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.

Science.gov (United States)

Mori, Takaaki; Kamiya, Koki; Tomita, Masahiro; Yoshimura, Tetsuro; Tsumoto, Kanta

2014-06-01

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.

Glomerular Podocytes Express Type 1 Adenylate Cyclase: Inactivation Results in Susceptibility to Proteinuria

Science.gov (United States)

Xiao, Zhijie; He, Liqun; Takemoto, Minoru; Jalanko, Hannu; Chan, Guy C.; Storm, Daniel R.; Betsholtz, Christer; Tryggvason, Karl; Patrakka, Jaakko

2011-01-01

Background/Aims The organization of actin cytoskeleton in podocyte foot processes plays a critical role in the maintenance of the glomerular filtration barrier. The cAMP pathway is an important regulator of the actin network assembly in cells. However, the role of the cAMP pathway in podocytes is not well understood. Type 1 adenylate cyclase (Adcy1), previously thought to be specific for neuronal tissue, is a member of the family of enzymes that catalyses the formation of cAMP. In this study, we characterized the expression and role of Adcy1 in the kidney. Methods Expression of Adcy1 was studied by RT-PCR, Northern blotting and in situ hybridization. The role of Adcy1 in podocytes was investigated by analyzing Adcy1 knockout mice (Adcy1–/–). Results and Conclusion: Adcy1 is expressed in the kidney specifically by podocytes. In the kidney, Adcy1 does not have a critical role in normal physiological functioning as kidney histology and function are normal in Adcy1–/– mice. However, albumin overload resulted in severe albuminuria in Adcy1–/– mice, whereas wild-type control mice showed only mild albumin leakage to urine. In conclusion, we have identified Adcy1 as a novel podocyte signaling protein that seems to have a role in compensatory physiological processes in the glomerulus. PMID:21196775

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

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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.

Presence and Effects of Pituitary Adenylate Cyclase Activating Polypeptide Under Physiological and Pathological Conditions in the Stomach

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Dora Reglodi

2018-03-01

Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a multifunctional neuropeptide with widespread occurrence throughout the body including the gastrointestinal system. In the small and large intestine, effects of PACAP on cell proliferation, secretion, motility, gut immunology and blood flow, as well as its importance in bowel inflammatory reactions and cancer development have been shown and reviewed earlier. However, no current review is available on the actions of PACAP in the stomach in spite of numerous data published on the gastric presence and actions of the peptide. Therefore, the aim of the present review is to summarize currently available data on the distribution and effects of PACAP in the stomach. We review data on the localization of PACAP and its receptors in the stomach wall of various mammalian and non-mammalian species, we then give an overview on PACAP’s effects on secretion of gastric acid and various hormones. Effects on cell proliferation, differentiation, blood flow and gastric motility are also reviewed. Finally, we outline PACAP’s involvement and changes in various human pathological conditions.

A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate.

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Pirger, Zsolt; László, Zita; Kemenes, Ildikó; Tóth, Gábor; Reglodi, Dóra; Kemenes, György

2010-10-13

Similar to other invertebrate and vertebrate animals, cAMP-dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) and for the AC-activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this "strong" food-reward conditioning paradigm the activation of AC by PACAP was necessary for LTM to form. We found that in a "weak" multitrial food-reward conditioning paradigm, lip touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multitrial tactile conditioning accelerated the formation of transcription-dependent memory. Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning.

Snf1 Phosphorylates Adenylate Cyclase and Negatively Regulates Protein Kinase A-dependent Transcription in Saccharomyces cerevisiae.

Science.gov (United States)

Nicastro, Raffaele; Tripodi, Farida; Gaggini, Marco; Castoldi, Andrea; Reghellin, Veronica; Nonnis, Simona; Tedeschi, Gabriella; Coccetti, Paola

2015-10-09

In eukaryotes, nutrient availability and metabolism are coordinated by sensing mechanisms and signaling pathways, which influence a broad set of cellular functions such as transcription and metabolic pathways to match environmental conditions. In yeast, PKA is activated in the presence of high glucose concentrations, favoring fast nutrient utilization, shutting down stress responses, and boosting growth. On the contrary, Snf1/AMPK is activated in the presence of low glucose or alternative carbon sources, thus promoting an energy saving program through transcriptional activation and phosphorylation of metabolic enzymes. The PKA and Snf1/AMPK pathways share common downstream targets. Moreover, PKA has been reported to negatively influence the activation of Snf1/AMPK. We report a new cross-talk mechanism with a Snf1-dependent regulation of the PKA pathway. We show that Snf1 and adenylate cyclase (Cyr1) interact in a nutrient-independent manner. Moreover, we identify Cyr1 as a Snf1 substrate and show that Snf1 activation state influences Cyr1 phosphorylation pattern, cAMP intracellular levels, and PKA-dependent transcription. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

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.

Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm.

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Boros, Akos; Somogyi, Ildikó; Engelmann, Péter; Lubics, Andrea; Reglodi, Dóra; Pollák, Edit; Molnár, László

2010-03-01

Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.

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

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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.

Stress-related disorders, pituitary adenylate cyclase-activating peptide (PACAP)ergic system, and sex differences.

Science.gov (United States)

Ramikie, Teniel S; Ressler, Kerry J

2016-12-01

Trauma-related disorders, such as posttraumatic stress disorder (PTSD) are remarkably common and debilitating, and are often characterized by dysregulated threat responses. Across numerous epidemiological studies, females have been found to have an approximately twofold increased risk for PTSD and other stress-related disorders. Understanding the biological mechanisms of this differential risk is of critical importance. Recent data suggest that the pituitary adenylate cyclase-activating polypeptide (PACAP) pathway is a critical regulator of the stress response across species. Moreover, increasing evidence suggests that this pathway is regulated by both stress and estrogen modulation and may provide an important window into understanding mechanisms of sex differences in the stress response. We have recently shown that PACAP and its receptor (PAC1R) are critical mediators of abnormal processes after psychological trauma. Notably, in heavily traumatized human subjects, there appears to be a robust sex-specific association of PACAP blood levels and PAC1R gene variants with fear physiology, PTSD diagnosis, and symptoms, specifically in females. The sex-specific association occurs within a single-nucleotide polymorphism (rs2267735) that resides in a putative estrogen response element involved in PAC1R gene regulation. Complementing these human data, the PAC1R messenger RNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP-PAC1R pathway are regulated by estrogen and are involved in abnormal fear responses underlying PTSD.

In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases

KAUST Repository

Raji, Misjudeen; Gehring, Christoph A

2017-01-01

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

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.

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

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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.

Effects of pituitary adenylate cyclase activating polypeptide in the urinary system, with special emphasis on its protective effects in the kidney.

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Reglodi, Dora; Kiss, Peter; Horvath, Gabriella; Lubics, Andrea; Laszlo, Eszter; Tamas, Andrea; Racz, Boglarka; Szakaly, Peter

2012-04-01

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widespread neuropeptide with diverse effects in the nervous system and peripheral organs. One of the most well-studied effects of PACAP is its cytoprotective action, against different harmful stimuli in a wide variety of cells and tissues. PACAP occurs in the urinary system, from the kidney to the lower urinary tract. The present review focuses on the nephroprotective effects of PACAP and summarizes data obtained regarding the protective effects of PACAP in different models of kidney pathologies. In vitro data show that PACAP protects tubular cells against oxidative stress, myeloma light chain, cisplatin, cyclosporine-A and hypoxia. In vivo data provide evidence for its protective effects in ischemia/reperfusion, cisplatin, cyclosporine-A, myeloma kidney injury, diabetic nephropathy and gentamicin-induced kidney damage. Results accumulated on the renoprotective effects of PACAP suggest that PACAP is an emerging candidate for treatment of human kidney pathologies. Copyright © 2011 Elsevier Ltd. All rights reserved.

Homologous desensitization of adenylate cyclase: the role of β-adrenergic receptor phosphorylation and dephosphorylation

International Nuclear Information System (INIS)

Sibley, D.R.; Strasser, R.H.; Daniel, K.; Lefkowitz, R.J.

1986-01-01

The authors utilized the frog erythrocyte (FE) as a β-adreneric receptor (βAR) model system in which to study homologous desensitization. Preincubation with isoproterenol (ISO) leads to a 50% decline in ISO-stimulated adenylate cyclase (AC) activity without significant changes in basal, PGE 1 -, NaF-, GppNHp-, forskolin-, or MnCl 2 -stimulated AC activities. ISO treatment also induces the sequestration of βAR from the cell surface as evidenced by a 35% decline in [ 3 H]CGP-12177 binding sites on the surface of intact FE. Treatment of intact FE with ISO also promotes βAR phosphorylation to 2 mol PO 4 /mol of βAR. At 25 0 C, the time courses of ISO-induced AC desensitization, βAR sequestration and βAR phosphorylation are identical occurring without a lag and exhibiting a t 1/2 of 30 min and a maximal response at 2.5 hrs. The sequestered βAR can be partially recovered upon cell lysis in a light membrane fraction (LMF), separable from the plasma membranes using sucrose gradients or differential centrifugation. βAR phosphorylation is reversed in the sequestered LMF exhibiting a PO 4 /βAR stoichiometry of 0.7 mol/mol - similar to that observed under basal conditions. These data suggest that phosphorylation of βAR in the plasma membrane promotes their translocation away from the cell surface into a sequestered membrane domain where the phosphorylation is reversed, thus, enabling the return of βAR back to the cell surface and recoupling with AC

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

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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

Luteinizing hormone-stimulated pituitary adenylate cyclase-activating polypeptide system and its role in progesterone production in human luteinized granulosa cells.

Science.gov (United States)

Park, Hyun-Jeong; Choi, Bum-Chae; Song, Sang-Jin; Lee, Dong-Sik; Roh, Jaesook; Chun, Sang-Young

2010-01-01

The present study examined the gonadotropin regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP type I receptor (PAC(1)-R) expression, and its role in progesterone production in the human luteinized granulosa cells. The stimulation of both PACAP and PAC(1)-R mRNA levels by LH was detected using a competitive reverse transcription-polymerase chain reaction (RT-PCR). PACAP transcript was stimulated by LH reaching maximum levels at 12 hours in a dose dependent manner. LH treatment also stimulated PAC(1)-R mRNA levels within 24 hours. Addition of PACAP-38 (10(-7) M) as well as LH significantly stimulated progesterone production during 48 hours culture. Furthermore, co-treatment with PACAP antagonist partially inhibited LH-stimulated progesterone production. Treatment with vasoactive intestinal peptide, however, did not affect progesterone production. Taken together, the present study demonstrates that LH causes a transient stimulation of PACAP and PAC(1)-R expression and that PACAP stimulates progesterone production in the human luteinized granulosa cells, suggesting a possible role of PACAP as a local ovarian regulator in luteinization.

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

Adrenalectomy mediated alterations in adrenergic activation of adenylate cyclase in rat liver

International Nuclear Information System (INIS)

El-Refai, M.; Chan, T.

1986-01-01

Adrenalectomy caused a large increase in the number of β-adrenergic binding sites on liver plasma membranes as measured by 125 I-iodocyanopindolol (22 and 102 fmol/mg protein for control and adrenalectomized (ADX) rats). Concomitantly an increase in the number of binding sites for 3 H-yohimbine was also observed (104 and 175 fmol/mg protein for control and adx membranes). Epinephrine-stimulated increase in cyclic AMP accumulation in isolated hepatocytes were greater in cells from ADX rats. This increase in β-adrenergic mediated action was much less than what may be expected as a result of the increase in the β-adrenergic binding in ADX membranes. In addition phenoxybenzamine (10 μM) further augmented this action of epinephrine in both control and ADX cells. To test the hypothesis that the increase in the number of the inhibitory α 2 -adrenergic receptors in adrenalectomy is responsible for the muted β-adrenergic response, the authors injected rats with pertussis toxin (PT). This treatment may cause the in vivo ribosylation of the inhibitory binding protein (Ni). Adenylate cyclase (AC) activity in liver plasma membranes prepared from treated and untreated animals was measured. In contrast with control rats, treatment of ADX rats with PT resulted in a significant increase in the basal activity of AC (5.5 and 7.7 pmol/mg protein/min for untreated and treated rats respectively). Isoproterenol (10 μM), caused AC activity to increase to 6.5 and 8.4 pmol/mg protein/min for membranes obtained from ADX untreated and ADX treated rats respectively. The α-adrenergic antagonists had no significant effect on the β-adrenergic-mediated activation of AC in liver plasma membranes from PT treated control and ADX rats. The authors conclude that the β-adrenergic activation of AC is attenuated by Ni protein both directly and as a result of activation of α-adrenergic receptors

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

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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

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

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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.

{beta}-adrenergic receptor density and adenylate cyclase activity in lead-exposed rat brain after cessation of lead exposure

Energy Technology Data Exchange (ETDEWEB)

Chang, Huoy-Rou [I-Shou University, Department of Biomedical Engineering, Dashu Shiang, Kaohsiung County (Taiwan); Tsao, Der-An [Fooyin University of Technology, Department of Medical Technology (Taiwan); Yu, Hsin-Su [Taiwan University, Department of Dermatology, College of Medicine (Taiwan); Ho, Chi-Kung [Kaohsiung Medical University, Occupational Medicine (Taiwan); Kaohsiung Medical University, Graduate Institute of Medicine, Research Center for Occupational Disease (Taiwan)

2005-01-01

To understanding the reversible or irreversible harm to the {beta}-adrenergic system in the brain of lead-exposed rats, this study sets up an animal model to estimate the change in the sympathetic nervous system of brain after lead exposure was withdrawn. We address the following topics in this study: (a) the relationship between withdrawal time of lead exposure and brain {beta}-adrenergic receptor, blood lead level, and brain lead level in lead-exposed rats after lead exposure was stopped; and (b) the relationship between lead level and {beta}-adrenergic receptor and cyclic AMP (c-AMP) in brain. Wistar rats were chronically fed with 2% lead acetate and water for 2 months. Radioligand binding was assayed by a method that fulfilled strict criteria of {beta}-adrenergic receptor using the ligand [{sup 125}I]iodocyanopindolol. The levels of lead were determined by electrothermal atomic absorption spectrometry. The c-AMP level was determined by radioimmunoassay. The results showed a close relationship between decreasing lead levels and increasing numbers of brain {beta}-adrenergic receptors and brain adenylate cyclase activity after lead exposure was withdrawn. The effect of lead exposure on the {beta}-adrenergic system of the brain is a partly reversible condition. (orig.)

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

Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin.

Science.gov (United States)

Gonyar, Laura A; Gray, Mary C; Christianson, Gregory J; Mehrad, Borna; Hewlett, Erik L

2017-06-01

Pertussis (whooping cough), caused by Bordetella pertussis , is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species. Copyright © 2017 American Society for Microbiology.

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

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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

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

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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.

Pituitary Adenylate Cyclase-Activating Polypeptide Disrupts Motivation, Social Interaction, and Attention in Male Sprague Dawley Rats.

Science.gov (United States)

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

2016-12-15

Severe or prolonged stress can trigger psychiatric illnesses including mood and anxiety disorders. Recent work indicates that pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in regulating stress effects. In rodents, exogenous PACAP administration can produce persistent elevations in the acoustic startle response, which may reflect anxiety-like signs including hypervigilance. We investigated whether PACAP causes acute or persistent alterations in behaviors that reflect other core features of mood and anxiety disorders (motivation, social interaction, and attention). Using male Sprague Dawley rats, we examined if PACAP (.25-1.0 µg, intracerebroventricular infusion) affects motivation as measured in the intracranial self-stimulation test. We also examined if PACAP alters interactions with a conspecific in the social interaction test. Finally, we examined if PACAP affects performance in the 5-choice serial reaction time task, which quantifies attention and error processing. Dose-dependent disruptions in motivation, social interaction, and attention were produced by PACAP, as reflected by increases in reward thresholds, decreases in social behaviors, and decreases in correct responses and alterations in posterror accuracy. Behavior normalized quickly in the intracranial self-stimulation and 5-choice serial reaction time task tests but remained dysregulated in the social interaction test. Effects on attention were attenuated by the corticotropin-releasing factor receptor-1 antagonist antalarmin but not the κ opioid receptor antagonist JDTic. Our findings suggest that PACAP affects numerous domains often dysregulated in mood and anxiety disorders, but that individual signs depend on brain substrates that are at least partially independent. This work may help to devise therapeutics that mitigate specific signs of these disorders. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

Science.gov (United States)

Li, Shengjie; Han, Linqiang; Bai, Junjie; Ma, Dongmei; Quan, Yingchun; Fan, Jiajia; Jiang, Peng; Yu, Lingyun

2015-03-01

Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass ( Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

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

Regulation of the beta-adrenergic receptor-adenylate cyclase complex of 3T3-L1 fibroblasts by sodium butyrate

International Nuclear Information System (INIS)

Stadel, J.M.; Poksay, K.S.; Nakada, M.T.; Crooke, S.T.

1986-01-01

Mouse 3T3-L1 fibroblasts contain beta-adrenergic receptors (BAR), predominantly of the B 1 subtype. Incubation of these cells with 2-10 mM sodium butyrate (SB) for 24-48 hr results in a switch in the BAR subtype from B 1 to B 2 and promotes a 1.5 to 2.5 fold increase in total BAR number. Other short chain acids were not as effective as SB in promoting changes in BAR. BAR were assayed in membranes prepared from the 3T3-L1 cells using the radiolabeled antagonist [ 125 I]-cyanopindolol and the B 2 selective antagonist ICI 118.551. BAR subtype switch was confirmed functionally by measuring cellular cAMP accumulation in response to agonists. The structure and amount of the alpha subunits of the guanine nucleotide regulatory proteins N/sub s/ and N/sub i/ were determined by ADP-ribosylation using 32 P-NAD and either cholera toxin or pertussis toxin for labeling of the respective subunits. Preincubation of cells with 5 mM SB for 48 hr resulted in a 2-3 fold increase in the labeling of the alpha subunits of both N/sub s/ and N/sub i/. A protein of M/sub r/ = 44,000 showed enhanced labeling by cholera toxin following SB treatment of the cells. These data indicate SB concomitantly regulates expression of BAR subtype and components of the adenylate cyclase in 3T3-L1 cells

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

Science.gov (United States)

Juhász, Tamás; Szentléleky, Eszter; Szűcs Somogyi, Csilla; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

2015-01-01

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. PMID:26230691

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.

VIP/PACAP receptors in cerebral arteries of rat

DEFF Research Database (Denmark)

Erdling, André; Sheykhzade, Majid; Maddahi, Aida

2013-01-01

BACKGROUND: Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP)-containing nerves surround cerebral blood vessels. The peptides have potent vasodilator properties via smooth muscle cell receptors and activation of adenylate cyclase. The purpose of this s......BACKGROUND: Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP)-containing nerves surround cerebral blood vessels. The peptides have potent vasodilator properties via smooth muscle cell receptors and activation of adenylate cyclase. The purpose...

Pituitary adenylate cyclase-activating polypeptide precursor is processed solely by prohormone convertase 4 in the gonads.

Science.gov (United States)

Li, M; Mbikay, M; Arimura, A

2000-10-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) is abundant not only in the brain, but also in the testis. Immunohistochemical studies have shown that PACAP-LI in rat testis is expressed stage specifically in spermatids. This suggests that testicular PACAP participates in the regulatory mechanism of spermatogenesis. Additionally, the ovary contains a relatively small amount of PACAP, conceivably involved in the regulation of folliculogenesis. PACAP is synthesized as a preprohormone and is processed by prohormone convertases, such as PC1, PC2, and PC4. PC4 is expressed only in the testis and ovary, where neither PC1 nor PC2 is expressed. However, whether PC4 is the sole endoprotease for the PACAP precursor in the gonads remains unknown. Recent studies using PC4-transgenic mice revealed that male PC4-null mice exhibited severely impaired fertility, although spermatogenesis appeared to be normal. The female PC4-null mice exhibited delayed folliculogenesis in the ovaries. To examine whether PC4 is the sole processing enzyme for the PACAP precursor in the gonads, we analyzed testicular and ovarian extracts from the PC4-null and wild-type mice for PACAP (PACAP38 and PACAP27) and its messenger RNA using reverse phase HPLC combined with specific RIAs and ribonuclease protection assay, respectively. For RIAs, three different polyclonal antisera with different recognition sites were used to identify PACAP38, PACAP27, and its precursor. Neither the testis nor the ovary from the PC4-null mice expressed PACAP38 or PACAP27, but the levels of PACAP transcripts in the testis and ovary of homozygous PC4-deficient mice were considerably elevated compared with those of the wild-type and heterozygous animals. The findings indicate that PC4 is the sole processing enzyme for the precursor of PACAP in the testis and ovary of mice. The possibility that the absence of bioactive PACAP in the testis and ovary of PC4-null mice caused severely impaired fertility in the males and

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

KAUST Repository

Al-Younis, Inas

2018-01-01

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

Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

KAUST Repository

Gehring, Christoph A; Turek, Ilona S.

2017-01-01

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

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.

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

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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.

Pituitary adenylate cyclase activating polypeptide (PACAP signalling exerts chondrogenesis promoting and protecting effects: implication of calcineurin as a downstream target.

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Tamás Juhász

Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is an important neurotrophic factor influencing differentiation of neuronal elements and exerting protecting role during traumatic injuries or inflammatory processes of the central nervous system. Although increasing evidence is available on its presence and protecting function in various peripheral tissues, little is known about the role of PACAP in formation of skeletal components. To this end, we aimed to map elements of PACAP signalling in developing cartilage under physiological conditions and during oxidative stress. mRNAs of PACAP and its receptors (PAC1,VPAC1, VPAC2 were detectable during differentiation of chicken limb bud-derived chondrogenic cells in micromass cell cultures. Expression of PAC1 protein showed a peak on days of final commitment of chondrogenic cells. Administration of either the PAC1 receptor agonist PACAP 1-38, or PACAP 6-38 that is generally used as a PAC1 antagonist, augmented cartilage formation, stimulated cell proliferation and enhanced PAC1 and Sox9 protein expression. Both variants of PACAP elevated the protein expression and activity of the Ca-calmodulin dependent Ser/Thr protein phosphatase calcineurin. Application of PACAPs failed to rescue cartilage formation when the activity of calcineurin was pharmacologically inhibited with cyclosporine A. Moreover, exogenous PACAPs prevented diminishing of cartilage formation and decrease of calcineurin activity during oxidative stress. As an unexpected phenomenon, PACAP 6-38 elicited similar effects to those of PACAP 1-38, although to a different extent. On the basis of the above results, we propose calcineurin as a downstream target of PACAP signalling in differentiating chondrocytes either in normal or pathophysiological conditions. Our observations imply the therapeutical perspective that PACAP can be applied as a natural agent that may have protecting effect during joint inflammation and/or may promote

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

KAUST Repository

Al-Younis, Inas M.

2018-05-01

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

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

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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

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

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.

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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.

Comparative effects of sub-stimulating concentrations of non-human versus human Luteinizing Hormones (LH) or chorionic gonadotropins (CG) on adenylate cyclase activation by forskolin in MLTC cells.

Science.gov (United States)

Nguyen, Thi-Mong Diep; Filliatreau, Laura; Klett, Danièle; Combarnous, Yves

2018-05-15

We have compared various Luteinizing Hormone (LH) and Chorionic Gonadotropin (CG) preparations from non-human and human species in their ability to synergize with 10 µM forskolin (FSK) for cyclic AMP intracellular accumulation, in MLTC cells. LH from rat pituitary as well as various isoforms of pituitary ovine, bovine, porcine, equine and human LHs and equine and human CG were studied. In addition, recombinant human LH and CG were also compared with the natural human and non-human hormones. Sub-stimulating concentrations of all LHs and CGs (2-100 pM) were found to stimulate cyclic AMP accumulation in MLTC cells in the presence of an also non-stimulating FSK concentration (10 µM). Like rat LH, the most homologous available hormone for mouse MLTC cells, all non-human LHs and CG exhibit a strong potentiating effect on FSK response. The human, natural and recombinant hLH and hCG also do so but in addition, they were found to elicit a permissive effect on FSK stimulation. Indeed, when incubated alone with MLTC cells at non-stimulating concentrations (2-70 pM) hLH and hCG permit, after being removed, a dose-dependent cyclic AMP accumulation with 10 µM FSK. Our data show a clearcut difference between human LH and CG compared to their non-human counterparts on MLTC cells adenylate cyclase activity control. This points out the risk of using hCG as a reference ligand for LHR in studies using non-human cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Retention of differentiated characteristics by cultures of defined rabbit kidney epithelia.

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Wilson, P D; Anderson, R J; Breckon, R D; Nathrath, W; Schrier, R W

1987-02-01

Rabbit nephron segments of proximal convoluted tubules (PCT); proximal straight tubules (PST); cortical and medullary thick ascending limbs of Henle's loop (CAL, MAL); and cortical, outer medullary, and inner medullary collecting tubules (CCT, OMCT, IMCT) were individually microdissected and grown in monolayer culture in hormone supplemented, defined media. Factors favoring a rapid onset of proliferation included young donor age, distal tubule origin, and the addition of 3% fetal calf serum to the medium. All primary cultures had polarized morphology with apical microvilli facing the medium and basement membrane-like material adjacent to the dish. Differentiated properties characteristic of the tubular epithelium of origin retained in cultures included ultrastructural characteristics and cytochemically demonstrable marker enzyme proportions. PCT and PST were rich in alkaline phosphatase; CAL stained strongly for NaK-ATPase; CCT contained two cell populations with regard to cytochrome oxidase reaction. A CCT-specific anti-keratin antibody (aLEA) was immunolocalized in CCT cultures, and a PST cytokeratin antibody stained PST cultures. The biochemical response of adenylate cyclase to putative stimulating agents was the same in primary cultures as in freshly isolated tubules. In PCT and PST adenylate cyclase activity was stimulated by parathyroid hormone (PTH) but not by arginine vasopressin (AVP); CAL and MAL adenylate cyclase was stimulated by neither PTH nor AVP; CCT, OMCT, and IMCT adenylate cyclase was stimulated by AVP but not by PTH. NaF stimulated adenylate cyclase activity in every cultured segment. It is concluded that primary cultures of individually microdissected rabbit PCT, PST, CAL, MAL, CCT, OMCT, and IMCT retain differentiated characteristics with regard to ultrastructure, marker enzymes, cytoskeletal proteins, and hormone response of adenylate cyclase and provide a new system for studying normal and abnormal functions of the heterogeneous tubular

Comparison of renal and osseous binding of parathyroid hormone and hormonal fragments

International Nuclear Information System (INIS)

Demay, M.; Mitchell, J.; Goltzman, D.

1985-01-01

The authors compared receptor binding and adenylate cyclase stimulation of intact bovine parathyroid hormone (bPTH)-(1-84) and the synthetic amino-terminal fragments, bPTH-(1-34) and rat PTH (rPTH)-(1-34). In both canine renal membranes and cloned rat osteosarcoma cells the amino-terminal fragments bound to a single order of sites; the affinity of rPTH-(1-34) exceeded that of bPTH-(1-34), correlating with its higher potency in stimulating adenylate cyclase. In studies with oxidized bPTH-(1--84), the middle and carboxyl regions of intact PTH were found to bind to both tissues but with higher affinity to osteosarcoma cells than to renal membranes. Our results demonstrate that rPTH-(1--34) is the most favorable probe of amino-terminal PTH binding and the most potent of the PTH peptides in stimulating renal and osseous adenylate cyclase. The results also show that midregion and carboxyl determinants within intact PTH contribute to hormone binding, which does not correlate with adenylate cyclase activation and appears more significant for skeletal than for renal binding

6-hydroxydopamine-induced degeneration of nigral dopamine neurons: differential effect on nigral and striatal D-1 dopamine receptors

International Nuclear Information System (INIS)

Porceddu, M.L.; Giorgi, O.; De Montis, G.; Mele, S.; Cocco, L.; Ongini, E.; Biggio, G.

1987-01-01

Dopamine-sensitive adenylate cyclase and 3 H-SCH 23390 binding parameters were measured in the rat substantia nigra and striatum 15 days after the injection of 6-hydroxydopamine into the medial forebrain bundle. The activity of nigral dopamine-sensitive adenylate cyclase and the binding of 3 H-SCH 23390 to rat nigral D-1 dopamine receptors were markedly decreased after the lesion. On the contrary, 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway enhanced both adenylate cyclase activity and the density of 3 H-SCH 23390 binding sites in striatal membrane preparations. The changes in 3 H-SCH 23390 binding found in both nigral and striatal membrane preparations were associated with changes in the total number of binding sites with no modifications in their apparent affinity. The results indicate that: a) within the substantia nigra a fraction (30%) of D-1 dopamine receptors coupled to the adenylate cyclase is located on cell bodies and and/or dendrites of dopaminergic neurons; b) striatal D-1 dopamine receptors are tonically innervated by nigrostriatal afferent fibers. 24 references, 1 figure, 1 table

Cytosolic adenylate changes during exercise in prawn muscle

International Nuclear Information System (INIS)

Thebault, M.T.; Raffin, J.P.; Pichon, R.

1994-01-01

31 P NMR and biochemical analysis were used to assess the effect of heavy exercise on cytosolic adenylate levels in Palaemon serratus abdominal muscle. At rest, the MgATP level corresponded to 85.5% of the total ATP content. The cytosolic adenylate concentrations of the prawn muscle are considerably different from that of vertebrates. The percentage of ADP bound to myofilaments was lower in the prawn muscle. Consequently, the level of free cytosolic AMP was greatly higher (thirty fold higher) than in vertebrate muscle. During vigorous work, the concentration of MgATP dropped and the cytosolic AMP accumulated, while the cytosolic adenine nucleotide pool decreased significantly. The phosphorylation potential value and the ATP/ADP ratio, calculated from the cytosolic adenylate, dropped acutely during the whole period of muscular contractions. On the contrary, the adenylate energy charge calculated from the cytosolic adenylate decreased slightly. Therefore, even in muscle displaying no AMP deamination, the adenylate charge is stabilized during exercise by the dynamic changes between cytosolic and bound adenylate species. (author). 21 refs., 2 tabs

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

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

International Nuclear Information System (INIS)

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

1986-01-01

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

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

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.

Pharmacological characterization of VIP and PACAP receptors in the human meningeal and coronary artery

DEFF Research Database (Denmark)

Chan, Kayi Y; Baun, Michael; de Vries, René

2011-01-01

We pharmacologically characterized pituitary adenylate cyclase-activating polypeptides (PACAPs), vasoactive intestinal peptide (VIP) and the VPAC(1), VPAC(2) and PAC(1) receptors in human meningeal (for their role in migraine) and coronary (for potential side effects) arteries.......We pharmacologically characterized pituitary adenylate cyclase-activating polypeptides (PACAPs), vasoactive intestinal peptide (VIP) and the VPAC(1), VPAC(2) and PAC(1) receptors in human meningeal (for their role in migraine) and coronary (for potential side effects) arteries....

Event timing in associative learning: from biochemical reaction dynamics to behavioural observations.

Directory of Open Access Journals (Sweden)

Ayse Yarali

Full Text Available Associative learning relies on event timing. Fruit flies for example, once trained with an odour that precedes electric shock, subsequently avoid this odour (punishment learning; if, on the other hand the odour follows the shock during training, it is approached later on (relief learning. During training, an odour-induced Ca(++ signal and a shock-induced dopaminergic signal converge in the Kenyon cells, synergistically activating a Ca(++-calmodulin-sensitive adenylate cyclase, which likely leads to the synaptic plasticity underlying the conditioned avoidance of the odour. In Aplysia, the effect of serotonin on the corresponding adenylate cyclase is bi-directionally modulated by Ca(++, depending on the relative timing of the two inputs. Using a computational approach, we quantitatively explore this biochemical property of the adenylate cyclase and show that it can generate the effect of event timing on associative learning. We overcome the shortage of behavioural data in Aplysia and biochemical data in Drosophila by combining findings from both systems.

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.

Calmodulin-regulated adenylyl cyclases and neuromodulation.

Science.gov (United States)

Xia, Z; Storm, D R

1997-06-01

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

Characterization of phosphorylated beta-adrenergic receptors from desensitized turkey erythrocytes

International Nuclear Information System (INIS)

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

1986-01-01

Catecholamine-induced desensitization of turkey erythrocyte (TE) adenylate cyclase results in a 40-50 percent decrease in agonist stimulated cyclase activity. Desensitization is accompanied by decreased mobility on SDS-PAGE of beta-adrenergic receptor (BAR) proteins photoaffinity labeled with [ 125 I]-p-azidobenzylcarazolol compared to control. Using a low crosslinked gel, the M/sub r/ = 42,000 band of BAR from desensitized TE was further resolved into a doublet compared to a single M/sub r/ = 38,000 band for control. The formation of the doublet appears to correlate with the amount of adenylate cyclase desensitization. Preincubating TE for 20 hr at 37 0 C with 32 P-/sub i/ labels BAR. 32 P-BAR was partially purified by affinity chromatography over alprenolol-Sepharose. Limited digest peptide maps of 32 P-BAR using papain identified a unique peptide (M/sub r/ = 2800) from BAR of desensitized TE which was absent in control. This unique 32 P-peptide was found only in the upper band of the doublet of BAR from desensitized TE. These data indicate that BAR is not uniformly phosphorylated following agonist-induced desensitization of TE and identify a peptide of BAR which is a site of phosphorylation correlating with desensitization of TE adenylate cyclase

Characterization of D1 dopamine receptors in the central nervous system

International Nuclear Information System (INIS)

Hess, E.J.

1987-01-01

Several lines of evidence suggest an association of central nervous system dopaminergic systems in the etiology of the schizophrenia. Interest in the role of D 1 dopamine receptors has revived with the advent of selective drugs for this dopamine receptor, particularly the D 1 dopamine receptor antagonists, SCH23390. [ 3 H]SCH23390 represents a superior radioligand for labeling the two-state striatal D 1 dopamine receptor in that its high percent specific binding makes it especially suitable for detailed mechanistic studies of this receptor. Striatal D 1 dopamine receptors have been shown to mediate the stimulation of adenylate cyclase activity via a guanine nucleotide regulatory subunit. Forskolin acts in a synergistic manner with dopamine agonists, guanine nucleotides or sodium fluoride to potentiate the stimulation of rat striatal adenylate cyclase activity mediated by these reagents. By using the aforementioned reagents and the irreversible receptor modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2,-dihydroquinoline, we demonstrated that the D 1 dopamine receptor population in rat striatum is not a stoichiometrically-limiting factor in agonist stimulation of adenylate cyclase activity

Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study

Energy Technology Data Exchange (ETDEWEB)

Emson, P C; Westmore, K; Augood, S J [MRC Molecular Neuroscience Group, The Department of Neurobiology, The Babraham Institute, Babraham, Cambridge (United Kingdom)

1996-12-11

The chemical phenotype of proneurotensin messenger RNA-expressing cells was determined in the acute haloperidol-treated rat striatum using a combination of [{sup 35}S]-labelled and alkaline phosphatase-labelled oligonucleotides. Cellular sites of proneurotensin messenger RNA expression were visualized simultaneously on tissue sections processed to reveal cellular sites of preproenkephalin A messenger RNA or the dopamine and adenylate cyclase phosphoprotein-32, messenger RNA. The cellular co-expression of preproenkepahlin A and preprotachykinin messenger RNA was also examined within forebrain structures. Cellular sites of preproenkephalin A and dopamine and adenylate cyclase phosphoprotein-32 messenger RNAs were visualized using alkaline phosphatase-labelled oligonucleotides whilst sites of preprotachykinin and proneurotensin messenger RNA expression were detected using [{sup 35}S]-labelled oligos. Cellular sites of enkephalin and dopamine and adenylate cyclase phosphoprotein-32 gene expression were identified microscopically by the concentration of purple alkaline phosphatase reaction product within the cell cytoplasm, whereas sites of substance P and proneurotensin gene expression were identified by the dense clustering of silver grains overlying cells.An intense hybridization signal was detected for all three neuropeptide messenger RNAs in the striatum, the nucleus accumbens and septum. Dopamine and adenylate cyclase phosphoprotein-32 messenger RNA was detected within the neostriatum but not within the septum. In all forebrain regions examined, with the exception of the islands of Cajella, the cellular expression of enkephalin messenger RNA and substance P messenger RNA was discordant; the two neuropeptide messenger RNAs were detected essentially in different cells, although in the striatum and nucleus accumbens occasional isolated cells were detected which contained both hybridization signals; dense clusters of silver grains overlay alkaline phosphatase

Phenotypic characterization of neurotensin messenger RNA-expressing cells in the neuroleptic-treated rat striatum: a detailed cellular co-expression study

International Nuclear Information System (INIS)

Emson, P.C.; Westmore, K.; Augood, S.J.

1996-01-01

The chemical phenotype of proneurotensin messenger RNA-expressing cells was determined in the acute haloperidol-treated rat striatum using a combination of [ 35 S]-labelled and alkaline phosphatase-labelled oligonucleotides. Cellular sites of proneurotensin messenger RNA expression were visualized simultaneously on tissue sections processed to reveal cellular sites of preproenkephalin A messenger RNA or the dopamine and adenylate cyclase phosphoprotein-32, messenger RNA. The cellular co-expression of preproenkepahlin A and preprotachykinin messenger RNA was also examined within forebrain structures. Cellular sites of preproenkephalin A and dopamine and adenylate cyclase phosphoprotein-32 messenger RNAs were visualized using alkaline phosphatase-labelled oligonucleotides whilst sites of preprotachykinin and proneurotensin messenger RNA expression were detected using [ 35 S]-labelled oligos. Cellular sites of enkephalin and dopamine and adenylate cyclase phosphoprotein-32 gene expression were identified microscopically by the concentration of purple alkaline phosphatase reaction product within the cell cytoplasm, whereas sites of substance P and proneurotensin gene expression were identified by the dense clustering of silver grains overlying cells.An intense hybridization signal was detected for all three neuropeptide messenger RNAs in the striatum, the nucleus accumbens and septum. Dopamine and adenylate cyclase phosphoprotein-32 messenger RNA was detected within the neostriatum but not within the septum. In all forebrain regions examined, with the exception of the islands of Cajella, the cellular expression of enkephalin messenger RNA and substance P messenger RNA was discordant; the two neuropeptide messenger RNAs were detected essentially in different cells, although in the striatum and nucleus accumbens occasional isolated cells were detected which contained both hybridization signals; dense clusters of silver grains overlay alkaline phosphatase-positive cells

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

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.

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

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.

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

Computational identification of candidate nucleotide cyclases in higher plants

KAUST Repository

Wong, Aloysius Tze; Gehring, Christoph A

2013-01-01

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

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.

Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

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Laakso Kati

2007-10-01

Full Text Available Abstract Background Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1 and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis. Results Here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyB1 and mcyC genes in a diverse selection of microcystin producing cyanobacteria. Our results provide support for complex evolutionary processes taking place at the mcyB1 and mcyC adenylation domains which recognize and activate the amino acids found at X and Z positions. We find evidence for recent recombination between mcyB1 and mcyC in strains of the genera Anabaena, Microcystis, and Hapalosiphon. We also find clear evidence for independent adenylation domain conversion of mcyB1 by unrelated peptide synthetase modules in strains of the genera Nostoc and Microcystis. The recombination events replace only the adenylation domain in each case and the condensation domains of mcyB1 and mcyC are not transferred together with the adenylation domain. Our findings demonstrate that the mcyB1 and mcyC adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. Conclusion Recombination is thought to be one of the main mechanisms driving the diversification of NRPSs. However, there is very little information on how recombination takes place in nature. This study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.

Science.gov (United States)

Duckworth, Benjamin P; Wilson, Daniel J; Aldrich, Courtney C

2016-01-01

Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.

Role of coronary endothelium in cyclic AMP formation by the heart

International Nuclear Information System (INIS)

Kroll, K.; Schrader, J.

1986-01-01

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

Production of non-stimulatory immunoglobulins that inhibit TSH binding in Graves' disease after radioiodine administration

International Nuclear Information System (INIS)

Bech, K.; Bliddal, H.; Siersbaek-Nielsen, K.; Friis, T.

1982-01-01

The effect of single dose of 131 I upon thyroid stimulating immunoglobulins has been studied in twenty-two patients with Graves' disease. The thyroid stimulating immunoglobulins were assessed by parallel measurements of thyrotrophin receptor binding inhibitory immunoglobulins (TBII) and of thyroid adenylate cyclase stimulating immunoglobulins (TACSI) in serum by radioreceptor assay and stimulation of adenylate cyclase respectively. The present study thus confirms that radioiodine therapy is followed by an increase of TBII and TACSI in most patients with Graves' disease. The level of TBII can probably provide a marker for development of hypothyroidism following 131 I therapy and might be involved in its pathogenesis. (author)

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.

Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme.

Science.gov (United States)

Wang, Nan; Rudolf, Jeffrey D; Dong, Liao-Bin; Osipiuk, Jerzy; Hatzos-Skintges, Catherine; Endres, Michael; Chang, Chin-Yuan; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2018-06-04

Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.

Pituitary adenylate cyclase-activating polypeptide stimulates glucose production via the hepatic sympathetic innervation in rats.

Science.gov (United States)

Yi, Chun-Xia; Sun, Ning; Ackermans, Mariette T; Alkemade, Anneke; Foppen, Ewout; Shi, Jing; Serlie, Mireille J; Buijs, Ruud M; Fliers, Eric; Kalsbeek, Andries

2010-07-01

The unraveling of the elaborate brain networks that control glucose metabolism presents one of the current challenges in diabetes research. Within the central nervous system, the hypothalamus is regarded as the key brain area to regulate energy homeostasis. The aim of the present study was to investigate the hypothalamic mechanism involved in the hyperglycemic effects of the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP). Endogenous glucose production (EGP) was determined during intracerebroventricular infusions of PACAP-38, vasoactive intestinal peptide (VIP), or their receptor agonists. The specificity of their receptors was examined by coinfusions of receptor antagonists. The possible neuronal pathway involved was investigated by 1) local injections in hypothalamic nuclei, 2) retrograde neuronal tracing from the thoracic spinal cord to hypothalamic preautonomic neurons together with Fos immunoreactivity, and 3) specific hepatic sympathetic or parasympathetic denervation to block the autonomic neuronal input to liver. Intracerebroventricular infusion of PACAP-38 increased EGP to a similar extent as a VIP/PACAP-2 (VPAC2) receptor agonist, and intracerebroventricular administration of VIP had significantly less influence on EGP. The PACAP-38 induced increase of EGP was significantly suppressed by preinfusion of a VPAC2 but not a PAC1 receptor antagonist, as well as by hepatic sympathetic but not parasympathetic denervation. In the hypothalamus, Fos immunoreactivity induced by PACAP-38 was colocalized within autonomic neurons in paraventricular nuclei projecting to preganglionic sympathetic neurons in the spinal cord. Local infusion of PACAP-38 directly into the PVN induced a significant increase of EGP. This study demonstrates that PACAP-38 signaling via sympathetic preautonomic neurons located in the paraventricular nucleus is an important component in the hypothalamic control of hepatic glucose production.

Central actions of a novel and selective dopamine antagonist

International Nuclear Information System (INIS)

Schulz, D.W.

1985-01-01

Receptors for the neurotransmitter dopamine traditionally have been divided into two subgroups: the D 1 class, which is linked to the stimulation of adenylate cyclase-activity, and the D 2 class which is not. There is much evidence suggesting that it is the D 2 class which is not. There is much evidence suggesting that it is the D 2 dopamine receptor that mediates the physiological and behavioral actions of dopamine in the intact animal. However, the benzazepine SCH23390 is a dopamine antagonist which has potent behavioral actions while displaying apparent neurochemical selectivity for the D 1 class of dopamine receptors. The purpose of this dissertation was to (1) confirm and characterize this selectivity, and (2) test certain hypothesis related to possible modes of action of SCH233390. The inhibition of adenylate cyclase by SCH23390 occurred via an action at the dopamine receptor only. A radiolabeled analog of SCH23390 displayed the receptor binding properties of a specific high-affinity ligand, and regional receptor densities were highly correlated with dopamine levels. The subcellular distribution of [ 3 H]-SCH23390 binding did not correspond completely with that of dopamine-stimulated adenylate cyclase. The neurochemical potency of SCH23390 as a D 1 receptor antagonist was preserved following parental administration. A variety of dopamine agonists and antagonists displayed a high correlation between their abilities to compete for [ 3 H]-SCH23390 binding in vitro and to act at an adenylate cyclase-linked receptor. Finally, the relative affinities of dopamine and SCH23390 for both D 1 receptors and [ 3 H]-SCH23390 binding sites were comparable. It is concluded that the behavioral effects of SCH23390 are mediated by actions at D 1 dopamine receptors only, and that the physiological importance of this class of receptors should be reevaluated

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

Science.gov (United States)

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

2007-07-10

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

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.

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

The guanylyl cyclase family at Y2K.

Science.gov (United States)

Wedel, B; Garbers, D

2001-01-01

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

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

Erratum

African Journals Online (AJOL)

EB

5-hydroxytryptamine (serotonin) receptor 7 (adenylate cyclase-coupled). 3363. Hs.73739 ... 3034. Hs.190783. 5. response to chemical substance. 0.027 .... involving alteration of. RNA secondary structure such as translation initiation,.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

Science.gov (United States)

Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

2015-05-29

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Molecular characterization of a novel intracellular ADP-ribosyl cyclase.

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Dev Churamani

2007-08-01

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

Parasympathetic denervation increases responses to VIP in isolated rat parotid acini

International Nuclear Information System (INIS)

McMillian, M.K.; Talamo, B.R.

1989-01-01

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in the salivary glands of many species, including the rat parotid gland. Parasympathetic denervation has been reported to deplete VIP in the rat parotid gland and to lead to supersensitivity to this peptide in vivo. We have compared the effects of VIP on acini isolated from parasympathetically denervated and unoperated parotid glands to examine possible supersensitivity to the peptide in vitro. VIP normally produced responses similar to those obtained with a low concentration of the beta adrenergic agonist isoproterenol (ISO), but strikingly different from the effects obtained with the muscarinic agonist carbachol (CARB). In parotid membrane preparations, VIP stimulated adenylate cyclase activity. Dissociated acini treated with VIP showed increases in cAMP accumulation and amylase release which were potentiated by forskolin and also by inhibition of phosphodiesterase. After parasympathetic denervation, maximal effects of VIP on adenylate cyclase, cAMP accumulation and amylase release in intact cells were increased two- to five-fold over contralateral control (or unoperated) parotid responses. The increase in adenylate cyclase-mediated responses after denervation was specific to VIP; there was no increased response nor increased sensitivity of any of these responses to ISO. Specific [125I]VIP binding to parotid acini increased two-fold per gland and three-fold per mg of protein after denervation; this probably explains the observed increases in the response to VIP

Molecular approaches to contraceptive development

Indian Academy of Sciences (India)

Unknown

drugs; PACAP, pituitary adenyl cyclase activating peptide; PK, progesterone receptor; RA, retinoic acid. ... Institute for Research in Reproduction, JM Street, Parel, Mumbai 400 012, India .... antagonistic properties devoid of side effects such as.

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

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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.

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.

Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors

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Thomas Pleli

2018-03-01

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

Overexpression of functional human oxidosqualene cyclase in Escherichia coli

DEFF Research Database (Denmark)

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

2015-01-01

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

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

NARCIS (Netherlands)

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

2002-01-01

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

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.

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

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

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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.

Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

Energy Technology Data Exchange (ETDEWEB)

Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

2011-02-18

Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

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)....

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

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.

Mechanistic Insights from the Crystal Structure of Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase Complexed with the Adenylate Intermediate.

Science.gov (United States)

Chen, Yaozong; Jiang, Yiping; Guo, Zhihong

2016-12-06

o-Succinylbenzoyl-CoA (OSB-CoA) synthetase, or MenE, catalyzes an essential step in vitamin K biosynthesis and is a valuable drug target. Like many other adenylating enzymes, it changes its structure to accommodate substrate binding, catalysis, and product release along the path of a domain alternation catalytic mechanism. We have determined the crystal structure of its complex with the adenylation product, o-succinylbenzoyl-adenosine monophosphate (OSB-AMP), and captured a new postadenylation state. This structure presents unique features such as a strained conformation for the bound adenylate intermediate to indicate that it represents the enzyme state after completion of the adenylation reaction but before release of the C domain in its transition to the thioesterification conformation. By comparison to the ATP-bound preadenylation conformation, structural changes are identified in both the reactants and the active site to allow inference about how these changes accommodate and facilitate the adenylation reaction and to directly support an in-line backside attack nucleophilic substitution mechanism for the first half-reaction. Mutational analysis suggests that the conserved His196 plays an important role in desolvation of the active site rather than stabilizing the transition state of the adenylation reaction. In addition, comparison of the new structure with a previously determined OSB-AMP-bound structure of the same enzyme allows us to propose a release mechanism of the C domain in its alteration to form the thioesterification conformation. These findings allow us to better understand the domain alternation catalytic mechanism of MenE as well as many other adenylating enzymes.

Structural basis of the interaction of MbtH-like proteins, putative regulators of nonribosomal peptide biosynthesis, with adenylating enzymes.

Science.gov (United States)

Herbst, Dominik A; Boll, Björn; Zocher, Georg; Stehle, Thilo; Heide, Lutz

2013-01-18

The biosynthesis of nonribosomally formed peptides (NRPs), which include important antibiotics such as vancomycin, requires the activation of amino acids through adenylate formation. The biosynthetic gene clusters of NRPs frequently contain genes for small, so-called MbtH-like proteins. Recently, it was discovered that these MbtH-like proteins are required for some of the adenylation reactions in NRP biosynthesis, but the mechanism of their interaction with the adenylating enzymes has remained unknown. In this study, we determined the structure of SlgN1, a 3-methylaspartate-adenylating enzyme involved in the biosynthesis of the hybrid polyketide/NRP antibiotic streptolydigin. SlgN1 contains an MbtH-like domain at its N terminus, and our analysis defines the parameters required for an interaction between MbtH-like domains and an adenylating enzyme. Highly conserved tryptophan residues of the MbtH-like domain critically contribute to this interaction. Trp-25 and Trp-35 form a cleft on the surface of the MbtH-like domain, which accommodates the alanine side chain of Ala-433 of the adenylating domain. Mutation of Ala-433 to glutamate abolished the activity of SlgN1. Mutation of Ser-23 of the MbtH-like domain to tyrosine resulted in strongly reduced activity. However, the activity of this S23Y mutant could be completely restored by addition of the intact MbtH-like protein CloY from another organism. This suggests that the interface found in the structure of SlgN1 is the genuine interface between MbtH-like proteins and adenylating enzymes.

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

International Nuclear Information System (INIS)

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

1991-01-01

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

Adenylate Nucleotides and 2,3-Biphosphoglycerate Concentration in Erythrocytes of Growing Wielkopolska Stallions

OpenAIRE

M. Suska; E. Skotnicka; W. Dudzińska; W. Orowicz; M. Brzezinska

2006-01-01

The aim of this study was to examine the relationships between the concentrations of adenylate nucleotides (ATP, ADP, AMP), total nucleotide pool (TAN), adenylate energy charge (AEC) and 2,3-biphosphoglycerate (2,3-BPG) in the erythrocytes of young horses in the period of their rapid growth and development. The studies were conducted on 10 young Wielkopolska breed stallions for two years; Group A: 1-month-old, Group B: 3-month-old, Group C: 6-month-old, Group D: 1-year-old, and Group E: 2-yea...

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.

MELATONIN-INDUCED SUPPRESSION OF PC12 CELL GROWTH IS MEDIATED BY ITS GI COUPLED TRANSMEMBRANE RECEPTORS. (R826248)

Science.gov (United States)

The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the mela...

Bacillus anthracis Edema Toxin Inhibits Staphylococcus aureus Enterotoxin B Effects in Vitro: A Potential Protein Therapeutic?

Science.gov (United States)

2005-10-01

5). Inherent characteristics of edema toxin and other procaryotic adenylate cyclases from Bordetella pertussis, Pseudomonas aeruginosa, and Yersinia...by mouse peritoneal macrophages: the role of cellular cyclic AMP. Immunology 64:719–724. 12. Krakauer, T. 1999. Induction of CC chemokines in human

Effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane of S49 lymphoma cells

International Nuclear Information System (INIS)

Bode, D.C.; Molinoff, P.B.

1988-01-01

The effects of chronic exposure to ethanol on the physical and functional properties of the plasma membrane were examined with cultured S49 lymphoma cells. The β-adrenergic receptor-coupled adenylate cyclase system was used as a probe of the functional properties of the plasma membrane. Steady-state fluorescence anisotropy of diphenylhexatriene and the lipid composition of the plasma membrane were used as probes of the physical properties of the membrane. Cells were grown under conditions such that the concentration of ethanol in the growth medium remained stable and oxidation of ethanol to acetaldehyde was not detected. Chronic exposure of S49 cells to 50 mM ethanol or growth of cells at elevated temperature resulted in a decrease in adenylate cyclase activity. There were no changes in the density of receptors or in the affinity of β-adrenergic receptors for agonists or antagonists following chronic exposure to ethanol. The fluorescence anisotropy of diphenylhexatriene was lower in plasma membranes prepared from cells that had been treated with 50 mM ethanol than in membranes prepared from control cells. However, this change was not associated with changes in the fatty acid composition or the cholesterol to phospholipid ratio of the plasma membrane. There was a small but statistically significant decrease in the amount of phosphatidylserine and an increase in the amount of phosphatidylethanolamine. These changes cannot account for the decrease in anisotropy. In contrast to the effect of ethanol, a decrease in adenylate cyclase activity following growth of S49 cells at 40 0 C was not associated with a change in anisotropy

Pharmacological differences between the D-2 autoreceptor and the D-1 dopamine receptor in rabbit retina

International Nuclear Information System (INIS)

Dubocovich, M.L.; Weiner, N.

1985-01-01

The effect of dopamine receptor agonists and antagonists was studied on the calcium-dependent release of [ 3 H]dopamine elicited by field stimulation at 3 Hz for a duration of 1 min (20 mA, 2 msec) from the rabbit retina in vitro and on adenylate cyclase activity in homogenates of rabbit retina. The relative order of potency of dopamine receptor agonists to inhibit the stimulation-evoked [ 3 H]dopamine release was pergolide greater than bromocriptine greater than apomorphine greater than LY 141865 greater than N,N-di-n-propyldopamine greater than or equal to dopamine. The relative order of potencies of dopamine receptor antagonists to increase [ 3 H]dopamine release was: S-sulpiride greater than or equal to domperidone greater than or equal to spiroperidol greater than metoclopramide greater than fluphenazine greater than or equal to R-sulpiride. alpha-Flupenthixol (0.01-1 microM) and (+)-butaclamol (0.01-1 microM) did not increase [ 3 H]dopamine overflow when added alone, but they antagonized the concentration-dependent inhibitory effect of apomorphine (0.1-10 microM). These results suggest that the dopamine inhibitory autoreceptor involved in the modulation of dopamine release from the rabbit retina possesses the pharmacological characteristics of a D-2 dopamine receptor. Maximal stimulation by 30 microM dopamine resulted in a 3-fold increase in adenylate cyclase activity with half-maximal stimulation occurring at a concentration of 2.46 microM. Apomorphine and pergolide elicited a partial stimulation of adenylate cyclase activity. However, at low concentrations both compounds were more potent than dopamine

Loss-of-function variants in ADCY3 increase risk of obesity and type 2 diabetes

DEFF Research Database (Denmark)

Grarup, Niels; Moltke, Ida; Andersen, Mette K

2018-01-01

We have identified a variant in ADCY3 (encoding adenylate cyclase 3) associated with markedly increased risk of obesity and type 2 diabetes in the Greenlandic population. The variant disrupts a splice acceptor site, and carriers have decreased ADCY3 RNA expression. Additionally, we observe...

Localisation of the neuropeptide PACAP and its receptors in the rat parathyroid and thyroid glands

DEFF Research Database (Denmark)

Fahrenkrug, Jan; Hannibal, Jens

2011-01-01

PACAP (pituitary adenylate cyclase activating polypeptide) is widely distributed neuropeptide acting via three subtypes of receptors, PAC(1), VPAC(1) and VPAC(2). Here we examined the localisation and nature of PACAP-immunoreactive nerves in the rat thyroid and parathyroid glands and defined the ...

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

Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins.

Science.gov (United States)

Zhang, Wenjun; Heemstra, John R; Walsh, Christopher T; Imker, Heidi J

2010-11-23

Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (∼70-residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways.

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...

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

Science.gov (United States)

The importance of cAMP signaling in fungal development and pathogenesis has been well documented in many fungal species including several phytopathogenic Fusarium spp. Two key components of the cAMP-PKA pathway, adenylate cyclase (AC) and catalytic subunit of PKA (CPKA), have been functionally chara...

An Increase in Antimycobacterial Th1-Cell Responses by Prime-Boost Protocols of Immunization Does Not Enhance Protection against Tuberculosis

Czech Academy of Sciences Publication Activity Database

Majlessi, L.; Šimšová, Marcela; Jarvis, Zdeňka; Brodin, P.; Rojas, M. J.; Bauche, C.; Nouzé, C.; Ladant, D.; Cole, S. T.; Šebo, Peter; Leclerc, C.

2006-01-01

Roč. 74, č. 4 (2006), s. 2128-2137 ISSN 0019-9567 R&D Projects: GA AV ČR IBS5020311 Institutional research plan: CEZ:AV0Z50200510 Keywords : adenylate cyclase * mycobacterium tuberculosis * immunity Subject RIV: EE - Microbiology, Virology Impact factor: 4.004, year: 2006

Postulated Role of Vasoactive Neuropeptide-Related Immunopathology of the Blood Brain Barrier and Virchow-Robin Spaces in the Aetiology of Neurological-Related Conditions

Directory of Open Access Journals (Sweden)

D. R. Staines

2008-01-01

Full Text Available Vasoactive neuropeptides (VNs such as pituitary adenylate cyclase-activating polypeptide (PACAP and vasoactive intestinal peptide (VIP have critical roles as neurotransmitters, vasodilators including perfusion and hypoxia regulators, as well as immune and nociception modulators. They have key roles in blood vessels in the central nervous system (CNS including maintaining functional integrity of the blood brain barrier (BBB and blood spinal barrier (BSB. VNs are potent activators of adenylate cyclase and thus also have a key role in cyclic AMP production affecting regulatory T cell and other immune functions. Virchow-Robin spaces (VRSs are perivascular compartments surrounding small vessels within the CNS and contain VNs. Autoimmunity of VNs or VN receptors may affect BBB and VRS function and, therefore, may contribute to the aetiology of neurological-related conditions including multiple sclerosis, Parkinson's disease, and amyotrophic lateral sclerosis. VN autoimmunity will likely affect CNS and immunological homeostasis. Various pharmacological and immunological treatments including phosphodiesterase inhibitors and plasmapheresis may be indicated.

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...

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

Science.gov (United States)

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

Transmission of Mycobacterium tuberculosis Undetected by Tuberculin Skin Testing

Czech Academy of Sciences Publication Activity Database

Anderson, S. T.; Williams, A. J.; Brown, J. R.; Newton, S. M.; Šimšová, Marcela; Nicol, M. P.; Šebo, Peter; Levin, M.; Wilkinson, R. J.; Wilkinson, K. A.

2006-01-01

Roč. 173, - (2006), s. 1038-1042 ISSN 1073-449X R&D Projects: GA AV ČR IAA5020406 Institutional research plan: CEZ:AV0Z50200510 Keywords : adenylate cyclase * diagnostic tests and procedures * mycobacterium tuberculosis Subject RIV: EE - Microbiology, Virology Impact factor: 9.091, year: 2006

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

Energy Technology Data Exchange (ETDEWEB)

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

2011-09-20

The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

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

Science.gov (United States)

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

2016-03-01

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

Adenylate kinase amplification of ATP bioluminescence for hygiene monitoring in the food and beverage industry.

Science.gov (United States)

Corbitt, A J; Bennion, N; Forsythe, S J

2000-06-01

Fourteen food residues, Escherichia coli O157:H7 and Staphylococcus aureus on stainless steel surfaces were detected using a combined assay with adenylate kinase as a cellular marker and ATP bioluminescence. The limit of sensitivity ranged from 0.02 to 708 microg for minced meat and broccoli, respectively. Both methods gave the same detection limit (105 cfu) for E. coli and Staph. aureus on stainless steel surfaces. The combined adenylate kinase-ATP assay is applicable to monitor the hygiene of work surfaces, especially those prone to contamination by meat and vegetable residues.

Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

OpenAIRE

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

2007-01-01

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

Human adenylate kinases – classification, structure, physiological and pathological importance

Directory of Open Access Journals (Sweden)

Magdalena Wujak

2015-01-01

Full Text Available Adenylate kinase (AK, EC 2.7.4.3 is a ubiquitous phosphotransferase which catalyzes the reversible transfer of high-energy β – and γ-phosphate groups between nucleotides. All classified AKs show a similar structure: they contain a large central CORE region, nucleoside monophosphate and triphosphate binding domains (NMPbd and NTPbd and the LID domain. Analysis of amino acid sequence similarity revealed the presence of as many as nine human AK isoenzymes, which demonstrate different organ-tissue and intercellular localization. Among these kinases, only two, AK1 and AK2, fulfill the structural and functional criterion by the highest affinity for adenine nucleotides and the utilization of only AMP or dAMP as phosphate acceptors. Human AK isoenzymes are involved in nucleotide homeostasis and monitor disturbances of cell energy charge. Participating in large regulatory protein complexes, AK supplies high energy substrates for controlling the functions of channels and transporters as well as ligands for extracellular P2 nucleotide receptors. In pathological conditions AK can take over the function of other kinases, such as creatine kinase in oxygen-depleted myocardium. Directed mutagenesis and genetic studies of diseases (such as aleukocytosis, hemolytic anemia, primary ciliary dyskinesia (PCD link the presence and activity of AK with etiology of these disturbances. Moreover, AK participates in regulation of differentiation and maturation of cells as well as in apoptosis and oncogenesis. Involvement of AK in a wide range of processes and the correlation between AK and etiology of diseases support the medical potential for the use of adenylate kinases in the diagnosis and treatment of certain diseases. This paper summarizes the current knowledge on the structure, properties and functions of human adenylate kinase.

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.

Radioreceptor assay for TBII in the spectrum of thyroid stimulating immunoglobulins

Energy Technology Data Exchange (ETDEWEB)

Bliddal, H.

1986-01-01

Thyroid stimulating immunoglobulins have been measured by several different methods among which the radioreceptor assay for TBII has been mostly employed. This technique is review in detail and it is concluded that the present method has the advantage of a larger stability aquired by the use of a purified plasma membrane fraction produced from autopsy material. TBII has been found in 60 to 80 percent in untreated Graves' disease while the percentage of positive patients decreases after any kind of treatment. Thus, TBII descreases during long term antithyroid treatment and by the end of such treatment TBII has a certain prognostic value. In the present study TBII was correlated to several other methods of measurement, primarily with the thyroid adenylate cyclase stimulating antibodies. In Graves's disease a general correlation was found with significant differences in TBII and TSAb activity in some cases. In contrast, a dissociation between two tests was often found in Hashimoto's thyroiditis and several other diseases with a low occurence of these antibodies, e.g. IDDM. Based on the present results it is concluded that thyroid stimulating immunoglobulins are heterogenous and probably polyclonal antibodies directed against several different epitopes on the TSH receptor with different affinity for the binding side of TSH. An activation in vitro by adenylate cyclase is apparently possible by-passing this binding side, and in some sera antibodies have been described which block the adenylate cyclase in vitro without affecting the binding of TSH. It is concluded that these antibodies are a major factor in the pathogenesis of thyrotoxicosis in Graves' disease, while differences in expression and affinity of antibodies against the TSH receptor may explain the lack of clinical correlation in other diseases. 193 refs.

The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

Czech Academy of Sciences Publication Activity Database

Romero, Rodrigo, Villarino; Bíbová, Ilona; Černý, Ondřej; Večerek, Branislav; Wald, Tomáš; Benada, Oldřich; Zavadilová, J.; Osička, Radim; Šebo, Peter

2013-01-01

Roč. 81, č. 8 (2013), s. 2761-2767 ISSN 0019-9567 R&D Projects: GA ČR(CZ) GAP302/11/0580; GA ČR(CZ) GAP302/11/1940 Institutional support: RVO:61388971 Keywords : ADENYLATE CYCLASE-HEMOLYSIN * T-CELL EPITOPES * IMMUNE-RESPONSES Subject RIV: EC - Immunology Impact factor: 4.156, year: 2013

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

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.

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.

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

Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation.

Science.gov (United States)

Segall-Shapiro, Thomas H; Nguyen, Peter Q; Dos Santos, Edgardo D; Subedi, Saurav; Judd, Justin; Suh, Junghae; Silberg, Jonathan J

2011-02-11

The extent to which thermostability influences the location of protein fragmentation sites that allow retention of function is not known. To evaluate this, we used a novel transposase-based approach to create libraries of vectors that express structurally-related fragments of Bacillus subtilis adenylate kinase (BsAK) and Thermotoga neapolitana adenylate kinase (TnAK) with identical modifications at their termini, and we selected for variants in each library that complement the growth of Escherichia coli with a temperature-sensitive adenylate kinase (AK). Mutants created using the hyperthermophilic TnAK were found to support growth with a higher frequency (44%) than those generated from the mesophilic BsAK (6%), and selected TnAK mutants complemented E. coli growth more strongly than homologous BsAK variants. Sequencing of functional clones from each library also identified a greater dispersion of fragmentation sites within TnAK. Nondisruptive fission sites were observed within the AMP binding and core domains of both AK homologs. However, only TnAK contained sites within the lid domain, which undergoes dynamic fluctuations that are critical for catalysis. These findings implicate the flexible lid domain as having an increased sensitivity to fission events at physiological temperatures. In addition, they provide evidence that comparisons of nondisruptive fission sites in homologous proteins could be useful for finding dynamic regions whose conformational fluctuations are important for function, and they show that the discovery of protein fragments that cooperatively function in mesophiles can be aided by the use of thermophilic enzymes as starting points for protein design. Copyright © 2010 Elsevier Ltd. All rights reserved.

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.

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

Sensitive method for the assay of guanylate cyclase activity

Energy Technology Data Exchange (ETDEWEB)

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

1978-07-01

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

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

Science.gov (United States)

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

2016-09-01

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

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 hy...... hydrogenation chemistry with the use of a heterogeneous palladium catalyst and carrier-free tritium gas on a tritium manifold system....

Effect of small doses of ionizing radiation on motility, rosette formation, and antioxidant state of leukocytes under modification of G-proteins by cholera and pertussis toxins

International Nuclear Information System (INIS)

Zhirnov, V.V.; Luik, A.I.; Metelitsa, L.A.; Mogilevich, S.E.; Charochkina, L.L.

2000-01-01

The responses of motility and rosette formation of leukocytes to small radioactive doses (from 6 centre dot 10 -10 to 6 centre dot 10 -4 Gy) are studied. The influence of these doses on cell functions and oxidative homeostasis are investigated under the modification of transducing components of membrane signal pathways (adenylate cyclase and polyphosphoinositide cascades) with pertussis and cholera toxins

Modifying influence of incorporated 137Cs upon the mechanisms of adrenergic control over contractile myucard function

International Nuclear Information System (INIS)

Lobanok, L.M.; Bulanova, K.Ya.; Gerasimovich, N.V.; Sineleva, M.V.; Milyutin, A.A.

1994-01-01

Incorporated 137 Cs (absorbed dose of 0.26 Gy) causes decrease of myocard's contractile function and intropic response to β-adrenagonists effect, isoproterenol-stimulated adenylate cyclase activity and β-adrenoreceptors affinity. Adrenergic effects, mediated by α-adrenergic structures on heart contractile function, on the contrary, become stronger, that is due to the increase of the receptors' dencity on sarcolemma surface

Structural studies of Schistosoma mansoni adenylate kinases

International Nuclear Information System (INIS)

Marques, I.A.; Pereira, H.M.; Garrat, R.C.

2012-01-01

Full text: Parasitic diseases are a major cause of death in developing countries, however receive little or no attention from pharmaceutical companies for the development of novel therapies. In this respect, the Center for Structural Molecular Biology (CBME) of the Institute of Physics of Sao Carlos (IFSC / USP) has developed expertise in all stages of the development of active compounds against target enzymes from parasitic diseases. The present work focuses on the adenylate kinase enzymes (ADK's) from Schistosoma mansoni. These enzymes are widely distributed and catalyze the reaction of phosphoryl exchange between nucleotides in the reaction 2ADP to ATP + AMP, which is critical for the cells life cycle. Due to the particular property of the reaction catalyzed, the ADK's are recognized as reporters of the cells energetic state, translating small changes in the balance between ATP and ADP into a large change in concentration of AMP. The genome of S. mansoni was recently sequenced by the Sanger Center in England. On performing searches for genes encoding adenylate kinases we found two such genes. The corresponding gene products were named ADK1 (197 residues) and ADK2 (239 residues), and the two sequences share only 28 percent identity. Both have been cloned into the pET-28a(+)vector, expressed in E. coli and purified. Preliminary tests of activity have been performed only for ADK1 showing it to be catalytically active. Crystallization trials were performed for both proteins and thus far, crystals of ADK1 have been obtained which diffract to 2.05 at the LNLS beamline MX2 and the structure solved by molecular replacement. Understanding, at the atomic level, the function of these enzymes may help in the development of specific inhibitors and may provide tools for developing diagnostic tests for schistosomiasis. (author)

Multilevel control of glucose homeostasis by adenylyl cyclase 8

NARCIS (Netherlands)

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

2015-01-01

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

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

Adenylate control contributes to thermal acclimation of sugar maple fine-root respiration in experimentally warmed soil.

Science.gov (United States)

Jarvi, Mickey P; Burton, Andrew J

2018-03-01

We investigated the occurrence of and mechanisms responsible for acclimation of fine-root respiration of mature sugar maple (Acer saccharum) after 3+ years of experimental soil warming (+4 to 5 °C) in a factorial combination with soil moisture addition. Potential mechanisms for thermal respiratory acclimation included changes in enzymatic capacity, as indicated by root N concentration; substrate limitation, assessed by examining nonstructural carbohydrates and effects of exogenous sugar additions; and adenylate control, examined as responses of root respiration to a respiratory uncoupling agent. Partial acclimation of fine-root respiration occurred in response to soil warming, causing specific root respiration to increase to a much lesser degree (14% to 26%) than would be expected for a 4 to 5 °C temperature increase (approximately 55%). Acclimation was greatest when ambient soil temperature was warmer or soil moisture availability was low. We found no evidence that enzyme or substrate limitation caused acclimation but did find evidence supporting adenylate control. The uncoupling agent caused a 1.4 times greater stimulation of respiration in roots from warmed soil. Sugar maple fine-root respiration in warmed soil was at least partially constrained by adenylate use, helping constrain respiration to that needed to support work being performed by the roots. © 2017 John Wiley & Sons Ltd.

Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors.

Directory of Open Access Journals (Sweden)

Shiva Kumar

Full Text Available 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 of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.

The low-dose combination preparation Vertigoheel activates cyclic nucleotide pathways and stimulates vasorelaxation.

Science.gov (United States)

Heinle, H; Tober, C; Zhang, D; Jäggi, R; Kuebler, W M

2010-01-01

Vertigo of various and often unknown aetiologies has been associated with and attributed to impaired microvascular perfusion in the inner ear or the vertebrobasilar system. Vertigoheel is a low-dose combination preparation of proven value in the symptomatic treatment of vertigo. In the present study we tested the hypothesis that Vertigoheel's anti-vertiginous properties may in part be due to a vasodilatory effect exerted via stimulation of the adenylate and/or guanylate cyclase pathways. Thus, the influence of Vertigoheel or its single constituents on synthesis and degradation of cyclic nucleotides was measured. Furthermore, vessel myography was used to observe the effect of Vertigoheel on the vasoreactivity of rat carotid arteries. Vertigoheel and one of its constituents, Anamirta cocculus, stimulated adenylate cyclase activity, while another constituent, Conium maculatum, inhibited phosphodiesterase 5, suggesting that the individual constituents of Vertigoheel contribute differentially to a synergistic stimulation of cyclic nucleotide signalling pathways. In rat carotid artery rings, Vertigoheel counteracted phenylephrine-induced tonic vasoconstriction. The present data demonstrate a vasorelaxant effect of Vertigoheel that goes along with a synergistic stimulation of cyclic nucleotide pathways and may provide a mechanistic basis for the documented anti-vertiginous effects of this combination preparation.

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

International Nuclear Information System (INIS)

Heuschneider, G.; Schwartz, R.D.

1989-01-01

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

Effects of isomers of apomorphines on dopamine receptors in striatal and limbic tissue of rat brain

Energy Technology Data Exchange (ETDEWEB)

Kula, N.S.; Baldessarini, R.J.; Bromley, S.; Neumeyer, J.L.

1985-09-16

The optical isomers of apomorphine (APO) and N-propylnorapomorphine (NPA) were interacted with three biochemical indices of dopamine (Da) receptors in extrapyramidal and limbic preparations of rat brain tissues. There were consistent isomeric preferences for the R(-) configuration of both DA analogs in stimulation adenylate cyclase (D-1 sites) and in competing for high affinity binding of /sup 3/H-spiroperidol (D-2 sites) and of /sup 3/H-ADTN (DA agonist binding sites) in striatal tissue, with lesser isomeric differences in the limbic tissue. The S(+) apomorphines did not inhibit stimulation of adenylate cyclase by DA. The tendency for greater activity of higher apparent affinity of R(-) apomorphines in striatum may reflect the evidently greater abundance of receptor sites in that region. There were only small regional differences in interactions of the apomorphine isomers with all three receptor sites, except for a strong preference of (-)NPA for striatal D-2 sites. These results do not parallel our recent observations indicating potent and selective antidopaminergic actions of S(+) apomorphines in the rat limbic system. They suggest caution in assuming close parallels between current biochemical functional, especially behavioral, methods of evaluating dopamine receptors of mammalian brain.

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

OpenAIRE

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

2012-01-01

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

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

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

Science.gov (United States)

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

2012-07-31

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

PACAP and VIP inhibit the invasiveness of glioblastoma cells exposed to hypoxia through the regulation of HIFs and EGFR expression

OpenAIRE

Grazia eMaugeri; Agata Grazia eD'Amico; Agata Grazia eD'Amico; Rita eReitano; Gaetano eMagro; Sebastiano eCavallaro; Salvatore eSalomone; Velia eD'Agata

2016-01-01

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) through the binding of vasoactive intestinal peptide receptors (VIPRs), perform a wide variety of effects in human cancers, including glioblastoma multiforme (GBM). This tumor is characterized by extensive areas of hypoxia, which triggers the expression of hypoxia-inducible factors (HIFs). HIFs not only mediate angiogenesis but also tumor cell migration and invasion. Furthermore, HIFs activation...

Hydrodynamic properties of the gonadotropin receptor from a murine Leydig tumor cell line are altered by desensitization

International Nuclear Information System (INIS)

Rebois, R.V.; Bradley, R.M.; Titlow, C.C.

1987-01-01

The murine Leydig tumor cell line 1 (MLTC-1) contains gonadotropin receptors (GR) that are coupled to adenylate cyclase through the stimulatory guanine nucleotide binding protein (G/sub s/). The binding of human choriogonadotropin (hGC) causes MLTC-1 cells to accumulate cAMP. With time, the ability of MLTC-1 cells to respond to hCG is attenuated by a process called desensitization. The hydrodynamic properties of GR from control and desensitized MLTC-1 cells were studied. Sucrose density gradient sedimentation in H 2 O and D 2 O and gel filtration chromatography were used to estimate the Stokes radius (a), partial specific volume (v/sub c/), sedimentation coefficient (s/sub 20,w/), and molecular weight (M/sub r/) of the detergent-solubilized hormone-receptor complex (hCG-GR). [ 125 I]hCG was bound to MLTC-1 cells under conditions that allow (37 0 C) or prevent (0 0 C) desensitization, and hCG-GR was solubilized in Triton X-100. In the absence of desensitization, control hCG-GR had a M/sub r/ of 213,000, whereas desensitized hCG-GR had a M/sub r/ of 158,000. Deglycosylated hCG (DG-HCG) is an antagonist that binds to GR with high affinity but fails to stimulate adenylate cyclase or cause desensitization. [ 125 I]DG-hCG was bound to MLTC-1 cells and DG-hCG-GR solubilized in Triton X-100. The hydrodynamic properties of DG-hCG-GR were the same as that for control hCG-GR. There was no evidence for the association of adenylate cyclase or G/sub s/ with GR in Triton X-100 solubilized preparations. When hCG was cross-linked to GR and solubilized with sodium dodecyl sulfate (SDS), the M/sub r/ was found to be 116,000, which was similar to that determined by SDS-polyacrylamide gel electrophoresis and less than that of the Triton X-100 solubilized control hCG-GR

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.

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

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

Science.gov (United States)

Nestler, Eric J

2016-08-15

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

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

A human D1 dopamine receptor gene is located on chromosome 5 at q35.1 and identifies an EcoRI RFLP.

OpenAIRE

Grandy, D K; Zhou, Q Y; Allen, L; Litt, R; Magenis, R E; Civelli, O; Litt, M

1990-01-01

Dopaminergic neurons have been shown to affect voluntary movement, hormone secretion, and emotional tone. Mediating these activities are two receptor subtypes, D1 and D2, which are biochemically and pharmacologically distinct. The D1 subtype, the most abundant form of dopamine receptor in the central nervous system, stimulates adenylate cyclase, modulates D2 receptor activity, regulates neuron growth and differentiation, and mediates several behavioral responses. Recently we reported the clon...

m-Acetylanilido-GTP, a novel photoaffinity label for GTP-binding proteins: synthesis and application.

OpenAIRE

Zor, T; Halifa, I; Kleinhaus, S; Chorev, M; Selinger, Z

1995-01-01

A novel photoaffinity label, m-acetylanilido-GTP (m-AcAGTP), was synthesized and used to identify GTP-binding proteins (G-proteins). This GTP analogue is easily prepared and can be used for photoaffinity labelling of G-proteins without chromatographic purification. In the presence of the beta-adrenergic agonist isoprenaline, it activates turkey erythrocyte adenylate cyclase. This activation persists even when the beta-adrenergic receptor is subsequently blocked by antagonist, indicating that ...

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

Science.gov (United States)

Ravichandran, Akshaya; Gu, Ganyu; Escano, Jerome; Lu, Shi-En; Smith, Leif

2014-01-01

Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and β-hydroxyasparagine variants coordinate the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides. PMID:23394257

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.

Reversible switching of fluorophore property based on intrinsic conformational transition of adenylate kinase during its catalytic cycle.

Science.gov (United States)

Fujii, Akira; Hirota, Shun; Matsuo, Takashi

2013-07-17

Adenylate kinase shows a conformational transition (OPEN and CLOSED forms) during substrate binding and product release to mediate the phosphoryl transfer between ADP and ATP/AMP. The protein motional characteristics will be useful to construct switching systems of fluorophore properties caused by the catalytic cycle of the enzyme. This paper demonstrates in situ reversible switching of a fluorophore property driven by the conformational transition of the enzyme. The pyrene-conjugated mutant adenylate kinase is able to switch the monomer/excimer emission property of pyrene on addition of ADP or P(1)P(5)-di(adenosine-5')pentaphosphate (Ap5A, a transition state analog). The observation under the dilute condition (~0.1 μM) indicates that the emission spectral change was caused by the motion of a protein molecule and not led by protein-protein interactions through π-π stacking of pyrene rings. The switching can be reversibly conducted by using hexokinase-coupling reaction. The fashion of the changes in emission intensities at various ligand concentrations is different between ADP, Mg(2+)-bound ADP, and Mg(2+)-bound Ap5A. The emission property switching is repeatable by a sequential addition of a substrate in a one-pot process. It is proposed that the property of a synthetic molecule on the enzyme surface is switchable in response to the catalytic cycle of adenylate kinase.

Functional changes in the properties of the β-adrenoreceptors of 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.

1986-01-01

The β-adrenoreceptors were solubilized from the plasma membranes of pigeon erythrocytes, treated with N-ethylmaleimide, using deoxycholate. The removal of the deoxycholate leads to incorporation of receptors into phospholipid vesicles and a restoration of their biological activity. After fusion of vesicles containing reconstituted receptors with vesicles containing the N/sub s/-protein and the catalytic component, a restoration of the hormonal activity of the enzyme was observed. If vesicles containing β-adrenoreceptors were incubated before fusion with the catalytic subunit of cAMP-dependent protein kinase, the hormonal activity of the preparation obtained was lowered by 45-50%. The decrease in activity occurred on account of an increase in the lag phase of activation of the enzyme in the presence of isoproterenol and GPP(NH)p, as well as on account of a decrease in the activity in the stationary phase of activation. Phosphorylation of the β-adrenoreceptors leads to a decrease in the content of the ternary isoproterenol-receptor-N/sub s/-protein complex, participating in the activation of adenylate cyclase. Thus, phosphorylation of the receptors leads to disruptions of the mechanism of transmission of the hormonal signal, analogous to those observed in the desensitization of adenylate cyclase

Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

International Nuclear Information System (INIS)

Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

1988-01-01

The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated 32 P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. 32 P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice

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 requir...

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.

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

Desynchronization of cells on the developmental path triggers the formation of spiral waves of cAMP during Dictyostelium aggregation

OpenAIRE

Lauzeral, Jacques; Halloy, José; Goldbeter, Albert

1997-01-01

Whereas it is relatively easy to account for the formation of concentric (target) waves of cAMP in the course of Dictyostelium discoideum aggregation after starvation, the origin of spiral waves remains obscure. We investigate a physiologically plausible mechanism for the spontaneous formation of spiral waves of cAMP in D. discoideum. The scenario relies on the developmental path associated with the continuous changes in the activity of enzymes such as adenylate cyclase and phosphodiesterase ...

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

Science.gov (United States)

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

1980-03-01

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

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

Science.gov (United States)

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

2016-03-01

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

Application of adenylate energy charge to problems of environmental impact assessment in aquatic organisms

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Ivanovici, A. M.

1980-03-01

Various physiological and biochemical methods have been proposed for assessing the effects of environmental perturbation on aquatic organisms. The success of these methods as diagnostic tools has, however, been limited. This paper proposes that adenylate energy charge overcomes some of these limitations. The adenylate energy charge (AEC) is calculated from concentrations of adenine nucleotides ([ATP+½ADP]/[ATP+ADP+AMP]), and is a reflection of metabolic potential available to an organism. Several features of this method are: correlation of specific values with physiological condition or growth state, a defined range of values, fast response times and high precision. Several examples from laboratory and field experiments are given to demonstrate these features. The test organisms used (mollusc species) were exposed to a variety of environmental perturbations, including salinity reduction, hydrocarbons and low doses of heavy metal. The studies performed indicate that the energy charge may be a useful measure in the assessment of environmental impact. Its use is restricted, however, as several limitations exist which need to be fully evaluated. Further work relating values to population characteristics of multicellular organisms needs to be completed before the method can become a predictive tool for management.

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

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

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

Science.gov (United States)

Melmed, RN; Karanian, PJ; Berlin, RD

1981-01-01

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

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

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Teresa eDuda

2014-03-01

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

Structural Studies of Archaealthermophilic Adenylate Kinase; TOPICAL

International Nuclear Information System (INIS)

Konisky, J.

2002-01-01

Through this DOE-sponsored program Konisky has studied the evolution and molecular biology of microbes that live in extreme environments. The emphasis of this work has been the determination of the structural features of thermophilic enzymes that allow them to function optimally at near 100 C. The laboratory has focused on a comparative study of adenylate kinase (ADK), an enzyme that functions to interconvert adenine nucleotides. Because of the close phylogenetic relatedness of members of the Methanococci, differences in the structure of their ADKs will be dominated by structural features that reflect contributions to their optimal temperature for activity, rather than differences due to phylogenetic divergence. We have cloned, sequenced and modeled the secondary structure for several methanococcal ADKs. Using molecular modeling threading approaches that are based on the solved structure for the porcine ADK, we have also proposed a general low resolution three dimensional structure for each of the methanococcal enzymes. These analyses have allowed us to propose structural features that confer hyperthermoactivity to those enzymes functioning in the hyperthermophilic members of the Methanococci. Using protein engineering methodologies, we have tested our hypotheses by examining the effects of selective structural changes on thermoactivity. Despite possessing between 68-81% sequence identity, the methanococcal AKs had significantly different stability against thermal denaturation, with melting points ranging from 69-103 C. The construction of several chimerical AKs by linking regions of the MVO and MJA AKs demonstrated the importance of cooperative interactions between amino- and carboxyl-terminal regions in influencing thermostability. Addition of MJA terminal fragments to the MVO AK increased thermal stability approximately 20 C while maintaining 88% of the mesophilic sequence. Further analysis using structural models suggested that hydrophobic interactions are

Demonstration of β-adrenergic receptors and catecholamine-mediated effects on cell proliferation in embryonic palatal tissue

International Nuclear Information System (INIS)

Pisano, M.M.

1986-01-01

The ability of catecholamines to modulate cell proliferation, differentiation and morphogenesis in other systems, and modulate adenylate cyclase activity in the developing palate during the period of cellular differentiation, made it of interest to determine their involvement in palatal ontogenesis. Catecholamines exert their physiologic effects via interaction with distinct membrane-bound receptors, one class being the B-adrenergic receptors which are coupled to stimulation of adenylate cyclase and the generation of cAMP. A direct radioligand binding technique utilizing the B-adrenergic antagonist [ 3 H]-dihydroalprenolol ([ 3 H]-DHA) was employed in the identification of B-adrenergic receptors in the developing murine secondary palate. Specific binding of [ 3 H]-DHA in embryonic (day 13) palatal tissue homogenates was saturable and of high affinity. The functionality of B-adrenergic receptor binding sites was assessed from the ability of embryonic palate mesenchmyal cells in vitro to respond to catecholamines with elevations of cAMP. Embryonic palate mesenchymal cells responded to various B-adrenergic catecholamine agonists with significant, dose-dependent accumulations of intracellular cAMP. Embryonic (day 13) maxillary tissue homogenates were analyzed for the presence of catecholamines by high performance liquid chromatography and radioenzymatic assay. Since normal palatal and craniofacial morphogenesis depends on proper temporal and spatial patterns of growth, the effect of B-adrenergic catecholamines on embryonic palate mesenchymal cell proliferation was investigated

Changes in expression of a functional Gi protein in cultured rat heart cells

International Nuclear Information System (INIS)

Allen, I.S.; Gaa, S.T.; Rogers, T.B.

1988-01-01

The muscarinic cholinergic agonist, carbachol, and pertussis toxin were used to examine the functional status of the guanine nucleotide-binding protein that inhibits adenylate cyclase (G i ) in cultured neonatal rat heart myocytes. The isoproterenol stimulation of adenylate cyclase activity in myocyte membranes and adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in intact cells (4 days in culture) were insensitive to carbachol. However, in cells cultured for 11 days, carbachol inhibited isoproterenol-stimulated cAMP accumulation by 30%. Angiotensin II (ANG II) was also found to inhibit isoproterenol-stimulated cAMP accumulation in day 11 cells in a dose-dependent manner. Pertussis toxin treatment reversed the inhibitory effects of both ANG II and carbachol, suggesting a role for G i in the process. Carbachol binding to membranes from day 4 cells was relatively insensitive to guanine nucleotides when compared with binding to membranes from day 11 or adult cells. Furthermore, pertussis toxin-mediated 32 P incorporation into a 39- to 41-kDa substrate in day 11 membranes was increased 3.2-fold over that measured in day 4 membranes. These findings support the view that, although G i is expressed, it is nonfunctional in 4-day-old cultured neonatal rat heart myocytes and acquisition of functional G i is dependent on culture conditions. Furthermore, the ANG II receptor can couple to G i in heart

Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

Directory of Open Access Journals (Sweden)

Yuan Hao

2015-08-01

Full Text Available Background/Aims: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (ISC in a human bronchial epithelial cell line (16HBE14o-, and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. Methods: The ISC measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca2+]i and cAMP were also quantified. Results: Nobiletin stimulated a concentration-dependent increase in ISC, which was due to Cl- secretion. The increase in ISC was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTRinh-172, but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS, Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated ISC was also sensitive to a protein kinase A (PKA inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in ISC in a cystic fibrosis (CF cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca2+]i. Conclusion: Nobiletin stimulated transepithelial Cl- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl- channels.

Transmitter-induced glycogenolysis and gluconeogenesis in leech segmental ganglia.

Science.gov (United States)

Pennington, A J; Pentreath, V W

1987-01-01

1. The utilization and control of glycogen stores were studied in the isolated segmental ganglia of the horse leech, Haemopis sanguisuga. The glycogen in the ganglia was extracted and assayed fluorimetrically and its cellular localization and turnover studied by autoradiography in conjunction with [3H] glucose. 2. The glycogen levels were measured after incubation with different neurotransmitters for 60 min at 28 degrees C. The results for each experimental ganglion were compared to a paired control ganglion, and the results analysed by paired t-tests. 3. Several transmitter substances (5-HT, octopamine, dopamine, noradrenaline, histamine) produced reductions in glycogen (glycogenolysis); other transmitters (glutamate, GABA) produced increases in glycogen (gluconeogenesis); others (adenosine, glycine) produced reductions or increases, depending on concentration. Acetylcholine had no effect on the glycogen levels. 4. Most of the glycogen in the ganglia is localized in the packet glial cells, which surround the neuron perikarya. Autoradiographic analysis demonstrated that the effects of histamine and dopamine were principally on the glycogen in the glial cells. 5. Adenylate cyclase was demonstrated by electron microscope histochemistry to be localized on the plasma membranes of the glial cells, and to a lesser extent on the neuronal membranes. 6. It is concluded that the changes in glycogen in the glial cells may be party controlled by transmitters via adenylate cyclase. This may provide a sensitive mechanism for coupling neuronal activity with energy metabolism.

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

Science.gov (United States)

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

2015-06-01

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

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

Science.gov (United States)

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

2017-04-01

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

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.

The PACAP receptor: a novel target for migraine treatment

DEFF Research Database (Denmark)

Schytz, Henrik W; Olesen, Jes; Ashina, Messoud

2010-01-01

The origin of migraine pain has not yet been clarified, but accumulating data point to neuropeptides present in the perivascular space of cranial vessels as important mediators of nociceptive input during migraine attacks. Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in s......) receptor, which suggests a possible signaling pathway implicated in migraine pain. This review summarizes the current evidence supporting the involvement of PACAP in migraine pathophysiology and the PAC(1) receptor as a possible novel target for migraine treatment....

The effect of immobilization and 3 (beta-aminoethyl)-1, 2, 4 triazol on the calcium content in gastric tissues of guinea pigs during the formation of experimental ulcers

Science.gov (United States)

Grechishkin, L. L.; Ritling, K.

1980-01-01

A sharp fall in the concentration of calcium in gastric tissues upon immobilization and after administration of the histamine analog was recorded. Similar shifts were seen to occur in the blood plasma as well. This implies that under the effect of different action, tissue dystrophy develops by following a common mechanism involving not only the adenyl cyclase system, but that of calcium ion metabolism as well. The calcium ion content in the blood plasma and gastric tissues were measured by atomic absorption spectrophotometry.

In vivo PTH provokes apical NHE3 and NaPi2 redistribution and Na-K-ATPase inhibition

DEFF Research Database (Denmark)

Zhang, Y; Norian, J M; Magyar, C E

1999-01-01

and to determine whether the same cellular signals drive the changes in apical and basolateral transporters. PTH-(1-34) (20 U), which couples to adenylate cyclase (AC), phospholipase C (PLC), and phospholipase A2 (PLA2), or [Nle8,18,Tyr34]PTH-(3-34) (10 U), which couples to PLC and PLA2 but not AC, were given....../diuresis and NHE3 and NaPi2 internalization, and that Na-K-ATPase inhibition is not secondary to depressed apical Na+ transport....

Signaling pathways in PACAP regulation of VIP gene expression in human neuroblastoma cells

DEFF Research Database (Denmark)

Falktoft, B.; Georg, B.; Fahrenkrug, J.

2009-01-01

Ganglia expressing the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) innervate vasoactive intestinal peptide (VIP) containing neurons suggesting a role of PACAP in regulating VIP expression. Human NB-1 neuroblastoma cells were applied to study PACAP regulated VIP gene...... in PACAP regulation of the FOS and VIP gene expressions suggest for the first time a role of FOS in PACAP-induced VIP gene expression in human NB-1 neuroblastoma cells. (C) 2009 Elsevier Ltd. All rights reserved Udgivelsesdato: 2009/10...

Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

Science.gov (United States)

Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

2015-01-01

Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

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

DEFF Research Database (Denmark)

Chen, Yicai; Yuan, Mingjun; Mohanty, Anee

2015-01-01

The nucleotide signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays an essential role in regulating microbial virulence and biofilm formation. C-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. One...

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

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

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

Cocoa procyanidins modulate transcriptional pathways linked to inflammation and metabolism in human dendritic cells

DEFF Research Database (Denmark)

Midttun, Helene L E; Ramsay, Aina; Mueller-Harvey, Irene

2018-01-01

the mechanistic basis of this inhibition, here we conducted transcriptomic analysis on DCs cultured with either LPS or LPS combined with oligomeric cocoa PC. Procyanidins suppressed a number of genes encoding cytokines and chemokines such as CXCL1, but also genes involved in the cGMP pathway such as GUCY1A3...... (encoding guanylate cyclase soluble subunit alpha-3). Upregulated genes were involved in diverse metabolic pathways, but notably two of the four most upregulated genes (NMB, encoding neuromedin B and ADCY3, encoding adenyl cyclase type 3) were involved in the cAMP signalling pathway. Gene-set enrichment...... analysis demonstrated that upregulated gene pathways were primarily involved in nutrient transport, carbohydrate metabolism and lysosome function, whereas down-regulated gene pathways involved cell cycle, signal transduction and gene transcription, as well as immune function. qPCR analysis verified...

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

International Nuclear Information System (INIS)

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

1987-01-01

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

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

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

DEFF Research Database (Denmark)

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

2015-01-01

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

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

Science.gov (United States)

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

2017-11-25

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

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

KAUST Repository

Wong, Aloysius Tze; Gehring, Christoph A

2013-01-01

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

Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

International Nuclear Information System (INIS)

Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

1987-01-01

Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent [ 3 H]acetylcholine release from rabbit retina labeled in vitro with [ 3 H]choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of [ 3 H]acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of [ 3 H]acetylcholine with the following order of potency: apomorphine ≤ SKF(R)82526 3 H]acetylcholine: SCH 23390 (IC50 = 1 nM) 3 H]acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by [ 3 H]SCH 23390, or as determined by adenylate cyclase activity. [ 3 H]SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of [ 3 H]SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate [ 3 H]acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at [ 3 H]SCH 23390 binding sites (r = 0.755, P < .05, n = 8)

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

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

Science.gov (United States)

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

2015-12-01

Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

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.

The extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches

Czech Academy of Sciences Publication Activity Database

Seydlová, G.; Beranová, I.; Bíbová, Ilona; Dienstbier, Ana; Držmíšek, Jakub; Mašín, Jiří; Fišer, R.; Konopásek, I.; Večerek, Branislav

2017-01-01

Roč. 292, č. 19 (2017), s. 8048-8058 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GF16-34825L; GA ČR(CZ) GA16-05919S; GA MZd(CZ) NV16-30782A; GA MŠk(CZ) EE2.3.20.0055; GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : PERTUSSIS ADENYLATE-CYCLASE * ESCHERICHIA-COLI * FATTY-ACIDS Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 4.125, year: 2016

Therapeutic effects of the joint administration of magnesium aspartate and adenosine monophosphate in gamma-irradiated mice

International Nuclear Information System (INIS)

Pospisil, M.; Netikova, J.; Pipalova, I.; Kozubik, A.

1990-01-01

The joint administration of magnesium aspartate and adenosine monophosphate, injected on days 1 to 4 post radiation, has been found to exert stimulatory effects on the recovery of hemopoietic functions in sublethally gamma-irradiated mice. These therapeutical effects were enhanced in animals protected by peroral administration of cystamine. The treatment scheme used did not modify survival of lethally irradiated mice. The therapeutic effects of magnesium aspartate and adenosine monophosphate in sublethally irradiated mice are explained by the stimulatory action of these drugs on the cell adenylate cyclase system, which influences the erythropoietic functions. (author)

Side effects in preventive maintenance therapy with neuroleptics with special emphasis on tardive dyskinesia.

Science.gov (United States)

Logothetis, J; Paraschos, A; Frangos, E

1981-01-01

Neuroleptics induce hypersensitivity reactions, and toxic, systemic and extrapyramidal manifestations. The latter mainly include acute dystonic reactions, other early dyskinesias, akathisia, parkinsonism and TD. These drugs have been implicated for DA antagonism exerted by an adenylate cyclase inhibition. Prolonged blockade of DA receptors is considered as the motivation for a counterbalancing mechanism inducing the DA supersensitivity from which TD results. Recent reports suggest cholinergic and GABA ergic insufficiency as secondary participants. The increasing frequency of TD calls for prevention by modifying treatment practices and searching for effective measures to combat the symptoms.

Integrated genome based studies of Shewanella ecophysiology

Energy Technology Data Exchange (ETDEWEB)

Saffarini, Daad A

2013-03-07

Progress is reported in these areas: Regulation of anaerobic respiration by cAMP receptor protein and role of adenylate cyclases; Identification of an octaheme c cytochrome as the terminal sulfite reductase in S. oneidensis MR-1; Identification and analysis of components of the electron transport chains that lead to reduction of thiosulfate, tetrathionate, and elemental sulfur in MR-1; Involvement of pili and flagella in metal reduction by S. oneidensis MR-1; and work suggesting that HemN1 is the major enzyme that is involved in heme biosynthesis under anaerobic conditions.

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

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

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

OpenAIRE

Zhang, Keya; Nelson, Kathryn M.; Bhuripanyo, Karan; Grimes, Kimberly D.; Zhao, Bo; Aldrich, Courtney C.; Yin, Jun

2013-01-01

The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative sub...

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

Czech Academy of Sciences Publication Activity Database

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

2014-01-01

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

Inferring biological functions of guanylyl cyclases with computational methods

KAUST Repository

Alquraishi, May Majed; Meier, Stuart Kurt

2013-01-01

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

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.

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

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

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

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

OpenAIRE

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

2012-01-01

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

Amarogentin, a secoiridoid glycoside, abrogates platelet activation through PLC γ 2-PKC and MAPK pathways.

Science.gov (United States)

Yen, Ting-Lin; Lu, Wan-Jung; Lien, Li-Ming; Thomas, Philip Aloysius; Lee, Tzu-Yin; Chiu, Hou-Chang; Sheu, Joen-Rong; Lin, Kuan-Hung

2014-01-01

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60  μM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

Amarogentin, a Secoiridoid Glycoside, Abrogates Platelet Activation through PLCγ2-PKC and MAPK Pathways

Directory of Open Access Journals (Sweden)

Ting-Lin Yen

2014-01-01

Full Text Available Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 μM inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLCγ2, protein kinase C (PKC, and mitogen-activated protein kinases (MAPKs. It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLCγ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

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

Science.gov (United States)

Andre, Christelle M; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; Cooney, Janine M; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; Laing, William A

2016-09-01

Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Subcellular Location of PKA Controls Striatal Plasticity: Stochastic Simulations in Spiny Dendrites

Science.gov (United States)

Oliveira, Rodrigo F.; Kim, MyungSook; Blackwell, Kim T.

2012-01-01

Dopamine release in the striatum has been implicated in various forms of reward dependent learning. Dopamine leads to production of cAMP and activation of protein kinase A (PKA), which are involved in striatal synaptic plasticity and learning. PKA and its protein targets are not diffusely located throughout the neuron, but are confined to various subcellular compartments by anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs). Experiments have shown that blocking the interaction of PKA with AKAPs disrupts its subcellular location and prevents LTP in the hippocampus and striatum; however, these experiments have not revealed whether the critical function of anchoring is to locate PKA near the cAMP that activates it or near its targets, such as AMPA receptors located in the post-synaptic density. We have developed a large scale stochastic reaction-diffusion model of signaling pathways in a medium spiny projection neuron dendrite with spines, based on published biochemical measurements, to investigate this question and to evaluate whether dopamine signaling exhibits spatial specificity post-synaptically. The model was stimulated with dopamine pulses mimicking those recorded in response to reward. Simulations show that PKA colocalization with adenylate cyclase, either in the spine head or in the dendrite, leads to greater phosphorylation of DARPP-32 Thr34 and AMPA receptor GluA1 Ser845 than when PKA is anchored away from adenylate cyclase. Simulations further demonstrate that though cAMP exhibits a strong spatial gradient, diffusible DARPP-32 facilitates the spread of PKA activity, suggesting that additional inactivation mechanisms are required to produce spatial specificity of PKA activity. PMID:22346744

Dynamics and molecular determinants of cytoplasmic lipid droplet clustering and dispersion.

Directory of Open Access Journals (Sweden)

David J Orlicky

Full Text Available Perilipin-1 (Plin1, a prominent cytoplasmic lipid droplet (CLD binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps.

Dynamics and molecular determinants of cytoplasmic lipid droplet clustering and dispersion.

Science.gov (United States)

Orlicky, David J; Monks, Jenifer; Stefanski, Adrianne L; McManaman, James L

2013-01-01

Perilipin-1 (Plin1), a prominent cytoplasmic lipid droplet (CLD) binding phosphoprotein and key physiological regulator of triglyceride storage and lipolysis in adipocytes, is thought to regulate the fragmentation and dispersion of CLD that occurs in response to β-adrenergic activation of adenylate cyclase. Here we investigate the dynamics and molecular determinants of these processes using cell lines stably expressing recombinant forms of Plin1 and/or other members of the perilipin family. Plin1 and a C-terminal CLD-binding fragment of Plin1 (Plin1CT) induced formation of single dense CLD clusters near the microtubule organizing center, whereas neither an N-terminal CLD-binding fragment of Plin1, nor Plin2 or Plin3 induced clustering. Clustered CLD coated by Plin1, or Plin1CT, dispersed in response to isoproterenol, or other agents that activate adenylate cyclase, in a process inhibited by the protein kinase A inhibitor, H89, and blocked by microtubule disruption. Isoproterenol-stimulated phosphorylation of CLD-associated Plin1 on serine 492 preceded their dispersion, and live cell imaging showed that cluster dispersion involved initial fragmentation of tight clusters into multiple smaller clusters, which then fragmented into well-dispersed individual CLD. siRNA knockdown of the cortical actin binding protein, moesin, induced disaggregation of tight clusters into multiple smaller clusters, and inhibited the reaggregation of dispersed CLD into tight clusters. Together these data suggest that the clustering and dispersion processes involve a complex orchestration of phosphorylation-dependent, microtubule-dependent and independent, and microfilament dependent steps.

Skeletal muscle beta-receptors and isoproterenol-stimulated vasodilation in canine heart failure

International Nuclear Information System (INIS)

Frey, M.J.; Lanoce, V.; Molinoff, P.B.; Wilson, J.R.

1989-01-01

To investigate whether heart failure alters beta-adrenergic receptors on skeletal muscle and its associated vasculature, the density of beta-adrenergic receptors, isoproterenol-stimulated adenylate cyclase activity, and coupling of the guanine nucleotide-binding regulatory protein were compared in 18 control dogs and 16 dogs with heart failure induced by 5-8 wk of ventricular pacing at 260 beats/min. Hindlimb vascular responses to isoproterenol were compared in eight controls and eight of the dogs with heart failure. In dogs with heart failure, the density of beta-receptors on skeletal muscle was reduced in both gastrocnemius (control: 50 +/- 5; heart failure: 33 +/- 8 fmol/mg of protein) and semitendinosus muscle (control: 43 +/- 9; heart failure: 27 +/- 9 fmol/mg of protein, both P less than 0.05). Receptor coupling to the ternary complex, as determined by isoproterenol competition curves with and without guanosine 5'-triphosphate (GTP), was unchanged. Isoproterenol-stimulated adenylate cyclase activity was significantly decreased in semitendinosus muscle (control: 52.4 +/- 4.6; heart failure: 36.5 +/- 9.5 pmol.mg-1.min-1; P less than 0.05) and tended to be decreased in gastrocnemius muscle (control: 40.1 +/- 8.5; heart failure: 33.5 +/- 4.5 pmol.mg-1.min-1; P = NS). Isoproterenol-induced hindlimb vasodilation was not significantly different in controls and in dogs with heart failure. These findings suggest that heart failure causes downregulation of skeletal muscle beta-adrenergic receptors, probably due to receptor exposure to elevated catecholamine levels, but does not reduce beta-receptor-mediated vasodilation in muscle

Interaction of sarcolysine with β-adrenergic receptors of tumor cells

International Nuclear Information System (INIS)

Belousova, A.K.; Solntseva, T.I.; Khabarov, S.V.

1986-01-01

The sites of specific binding of [L- 3 H]dihydroalprenolol ([ 3 H]DHA), possessing the properties of β-adrenergic receptors, coupled with adenylate cyclase, were detected by methods of competitive displacement and binding of β-adrenoblockers: [ 3 H]-DHA and L-propranolol on the surface of ascites sarcoma 37 cells. Specific binding of the ligand occurs rapidly and with saturation. The total number of binding sites in the case of total saturation is (30-40) x 10 3 per cell. An analysis of the results by the Scatchard method permitted the detection of two types of β-adrenoreceptors with high (K/sub d/ = 0.9-1.0 mM) and low (K/sub d/ = 15-20 nM) affinity for [ 3 H]DHA. The number of receptors of the first type is (5.0-7.5) x 10 3 , and of the second (20-30) x 10 3 per cell. Sarcolysine in 1-10 μM concentrations is capable of displacing [ 3 H]DHA bound to the β-adrenoreceptors, competing with it for common binding sites, and, like isoproterenol, inducing a brief increase in the content of cAMP in the tumor cells. Since sarcolysine noncompetitively inhibits cAMP phosphodiesterase of the plasma membranes of ascites sarcoma 37 cells in the same concentration range (2.5-25 μM), a possible functional association between the β-adrenoreceptors, adenylate cyclase, and the membrane cAMP phosphodiesterase and the participation of this complex in the antitumor effect of the cytostatic are suggested

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

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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.

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

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

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

2017-08-31

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

Hyperglycemia of Diabetic Rats Decreased by a Glucagon Receptor Antagonist

Science.gov (United States)

Johnson, David G.; Ulichny Goebel, Camy; Hruby, Victor J.; Bregman, Marvin D.; Trivedi, Dev

1982-02-01

The glucagon analog [l-Nα-trinitrophenylhistidine, 12-homoarginine]-glucagon (THG) was examined for its ability to lower blood glucose concentrations in rats made diabetic with streptozotocin. In vitro, THG is a potent antagonist of glucagon activation of the hepatic adenylate cyclase assay system. Intravenous bolus injections of THG caused rapid decreases (20 to 35 percent) of short duration in blood glucose. Continuous infusion of low concentrations of the inhibitor led to larger sustained decreases in blood glucose (30 to 65 percent). These studies demonstrate that a glucagon receptor antagonist can substantially reduce blood glucose levels in diabetic animals without addition of exogenous insulin.

Cholera toxin can catalyze ADP-ribosylation of cytoskeletal proteins

International Nuclear Information System (INIS)

Kaslow, H.R.; Groppi, V.E.; Abood, M.E.; Bourne, H.R.

1981-01-01

Cholera toxin catalyzes transfer of radiolabel from [ 32 P]NAD + to several peptides in particulate preparations of human foreskin fibroblasts. Resolution of these peptides by two-dimensional gel electrophoresis allowed identification of two peptides of M/sub r/ = 42,000 and 52,000 as peptide subunits of a regulatory component of adenylate cyclase. The radiolabeling of another group of peptides (M/sub r/ = 50,000 to 65,000) suggested that cholera toxin could catalyze ADP-ribosylation of cytoskeletal proteins. This suggestion was confirmed by showing that incubation with cholera toxin and [ 32 P]NAD + caused radiolabeling of purified microtubule and intermediate filament proteins

Serotonin receptors influencing cell proliferation in the jejunal crypt epithelium and in colonic adenocarcinomas.

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Tutton, P J; Barkla, D H

1986-01-01

Serotonin has previously been shown to stimulate cell proliferation in the jejunal crypt epithelium and in colonic tumours. The original classification of serotonin receptors into D and M groups was not conductive to the understanding of these observations. The more recent classification of serotonin receptors into 5HT1 and 5HT2 groups is considered in this report. On the balance of evidence it appears that similar receptors mediate the response to serotonin in the two tissues under consideration and that these receptors resemble those of the 5HT1 group. Such receptors are usually positively linked to adenylate cyclase.

Rapid increase of inositol 1,4,5-trisphosphate in the HeLa cells after hypergravity exposure

Science.gov (United States)

Kumei, Yasuhiro; Whitson, Peggy A.; Cintron, Nitza M.; Sato, Atsushige

1990-01-01

The IP3 level in HeLa cells has been elevated through the application in hypergravity in a time-dependent manner. The data obtained for the hydrolytic products of PIP2, IP3, and DG are noted to modulate c-myc gene expression. It is also established that the cAMP accumulation by the IBMX in hypergravity-exposed cells was suppressed relative to the control. In light of IP3 increase and cAMP decrease results, a single GTP-binding protein may play a role in the hypergravity signal transduction of HeLa cells by stimulating PLC while inhibiting adenylate cyclase.

Overproduction, Purification and Characterization of Adenylate Deaminase from Aspergillus oryzae.

Science.gov (United States)

Li, Shubo; Qian, Yi; Liang, Yunlong; Chen, Xinkuan; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

2016-12-01

Adenylate deaminase (AMPD, EC 3.5.4.6) is an aminohydrolase that widely used in the food and medicine industries. In this study, the gene encoding Aspergillus oryzae AMPD was cloned and expressed in Escherichia coli. Induction with 0.75 mM isopropyl β-D-l-thiogalactopyranoside resulted in an enzyme activity of 1773.9 U/mL. Recombinant AMPD was purified to electrophoretic homogeneity using nickel affinity chromatography, and its molecular weight was calculated as 78.6 kDa. Purified AMPD exhibited maximal activity at 35 °C, pH 6.0 and 30 mM K + , with apparent K m and V max values of 2.7 × 10 -4  M and 77.5 μmol/mg/min under these conditions. HPLC revealed that recombinant AMPD could effectively catalyse the synthesis of inosine-5'-monophosphate (IMP) with minimal by-products, indicating high specificity and suggesting that it could prove useful for IMP production.

Induction of chinook salmon growth hormone promoter activity by the adenosine 3',5'-monophosphate (cAMP)-dependent pathway involves two cAMP-response elements with the CGTCA motif and the pituitary-specific transcription factor Pit-1.

Science.gov (United States)

Wong, A O; Le Drean, Y; Liu, D; Hu, Z Z; Du, S J; Hew, C L

1996-05-01

In this study, the functional role of two cAMP-response elements (CRE) in the promoter of the chinook salmon GH gene and their interactions with the transcription factor Pit-1 in regulating GH gene expression were examined. A chimeric construct of the chloramphenicol acetyltransferase (CAT) reporter gene with the CRE-containing GH promoter (pGH.CAT) was transiently transfected into primary cultures of rainbow trout pituitary cells. The expression of CAT activity was stimulated by an adenylate cyclase activator forskolin as well as a membrane-permeant cAMP analog 8-bromo-cAMP. Furthermore, these stimulatory responses were inhibited by a protein kinase A inhibitor H89, suggesting that these CREs are functionally coupled to the adenylate cyclase-cAMP-protein kinase A cascade. This hypothesis is supported by parallel studies using GH4ZR7 cells, a rat pituitary cell line stably transfected with dopamine D2 receptors. In this cell line, D2 receptor activation is known to inhibit adenylate cyclase activity and cAMP synthesis. Stimulation with a nonselective dopamine agonist, apomorphine, or a D2-specific agonist, Ly171555, suppressed the expression of pGH.CAT in GH4ZR7 cells, and this inhibition was blocked by simultaneous treatment with forskolin. These results indicate that inhibition of the cAMP-dependent pathway reduces the basal promoter activity of the CRE-containing pGH.CAT. The functionality of these CREs was further confirmed by deletion analysis and site-specific mutagenesis. In trout pituitary cells, the cAMP inducibility of pGH.CAT was inhibited after deleting the CRE-containing sequence from the GH promoter. When the CRE-containing sequence was cloned into a CAT construct with a viral thymidine kinase promoter, a significant elevation of cAMP inducibility was observed. This stimulatory response, however, was abolished by mutating the core sequence, CGTCA, in these CREs, suggesting that these cis-acting elements confer cAMP inducibility to the salmon GH gene

Postulated vasoactive neuropeptide immunopathology affecting the blood–brain/blood–spinal barrier in certain neuropsychiatric fatigue-related conditions: A role for phosphodiesterase inhibitors in treatment?

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Sonya Marshall-Gradisnik

2008-10-01

Full Text Available Donald R Staines1,2, Ekua W Brenu2, Sonya Marshall-Gradisnik21Queensland Health, Gold Coast Population Health Unit, Southport, Gold Coast, Queensland, Australia; 2Faculty of Health Science and Medicine, Population Health and Neuroimmunology Unit, Bond University, Robina, Queensland, AustraliaAbstract: Neuropsychiatric symptoms occur in a number of neurological fatigue-related conditions including multiple sclerosis (MS, Parkinson’s disease (PD, amyotrophic lateral sclerosis (ALS, and chronic fatigue syndrome (CFS. These conditions have been attributed variably to neuroinflammatory and neurodegenerative processes. While autoimmune pathology, at least in part, has long been suspected in these conditions proof has been elusive. Autoimmune pathomechanisms affecting the blood–brain barrier (BBB or blood–spinal barrier (BSB may predispose the BBB/BSB to ‘leakiness’ and be a precursor to additional autoimmune events resulting in neuroinflammatory or neurodegenerative processes. The aim of the paper is to postulate immunopathology of the cerebrospinal perivascular compartment involving certain vasoactive neuropeptides, specifically pituitary adenylate cyclase-activating polypeptide (PACAP and vasoactive intestinal peptide (VIP, in the etiology of certain neuropsychiatric fatigue-related conditions such as MS, ALS, PD, and CFS. Vasoactive neuropeptides (VNs such as PACAP and VIP have critical roles as neurotransmitters, vasodilators including perfusion and hypoxia regulators, and immune and nociception modulators. PACAP and VIP are widely distributed in the central nervous system (CNS and have key roles in CNS blood vessels including maintaining functional integrity of the BBB and BSB. Autoimmunity affecting these VNs would likely have a detrimental effect on BBB and BSB functioning arguably predisposing to further pathological processes. Virchow–Robin spaces (VRS are perivascular compartments surrounding small vessels within the CNS which

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

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

2017-02-01

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

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

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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.

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

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Janet M Young

2007-09-01

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

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

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Roa, Jinae N; Tresguerres, Martin

2016-08-01

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

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

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Misquitta Stephanie A

2004-02-01

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

Effect of inhibition of microsomal Ca(2+)-ATPase on cytoplasmic calcium and enzyme secretion in pancreatic acini.

Science.gov (United States)

Metz, D C; Pradhan, T K; Mrozinski, J E; Jensen, R T; Turner, R J; Patto, R J; Gardner, J D

1994-01-13

We used thapsigargin (TG), 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ) and cyclopiazonic acid (CPA), each of which inhibits microsomal Ca(2+)-ATPase, to evaluate the effects of this inhibition on cytoplasmic free calcium ([Ca2+]i) and secretagogue-stimulated enzyme secretion in rat pancreatic acini. Using single-cell microspectrofluorimetry of fura-2-loaded acini we found that all three agents caused a sustained increase in [Ca2+]i by mobilizing calcium from inositol-(1,4,5)-trisphosphate-sensitive intracellular calcium stores and by promoting influx of extracellular calcium. Concentrations of all three agents that increased [Ca2+]i potentiated the stimulation of enzyme secretion caused by secretagogues that activate adenylate cyclase but inhibited the stimulation of enzyme secretion caused by secretagogues that activate phospholipase C. With BHQ, potentiation of adenylate cyclase-mediated enzyme secretion occurred immediately whereas inhibition of phospholipase C-mediated enzyme secretion occurred only after several min of incubation. In addition, the effects of BHQ and CPA on both [Ca2+]i and secretagogue-stimulated enzyme secretion were reversed completely by washing whereas the actions of TG could not be reversed by washing. Concentrations of BHQ in excess of those that caused maximal changes in [Ca2+]i inhibited all modes of stimulated enzyme secretion by a mechanism that was apparently unrelated to changes in [Ca2+]i. Finally, in contrast to the findings with TG and BHQ, CPA inhibited bombesin-stimulated enzyme secretion over a range of concentrations that was at least 10-fold lower than the range of concentrations over which CPA potentiated VIP-stimulated enzyme secretion.

Thyrotropin regulates IL-6 expression in CD34+ fibrocytes: clear delineation of its cAMP-independent actions.

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Nupur Raychaudhuri

Full Text Available IL-6 plays diverse roles in normal and disease-associated immunity such as that associated with Graves' disease (GD. In that syndrome, the orbit undergoes remodeling during a process known as thyroid-associated ophthalmopathy (TAO. Recently, CD34(+ fibrocytes were found to infiltrate the orbit in TAO where they transition into CD34(+ orbital fibroblasts. Surprisingly, fibrocytes display high levels of functional thyrotropin receptor (TSHR, the central antigen in GD. We report here that TSH and the pathogenic anti-TSHR antibodies that drive hyperthyroidism in GD induce IL-6 expression in fibrocytes and orbital fibroblasts. Unlike TSHR signaling in thyroid epithelium, that occurring in fibrocytes is completely independent of adenylate cyclase activation and cAMP generation. Instead TSH activates PDK1 and both AKT/PKB and PKC pathways. Expression and use of PKCβII switches to that of PKCµ as fibrocytes transition to TAO orbital fibroblasts. This shift is imposed by CD34(- orbital fibroblasts but reverts when CD34(+ fibroblasts are isolated. The up-regulation of IL-6 by TSH results from coordinately enhanced IL-6 gene promoter activity and increased IL-6 mRNA stability. TSH-dependent IL-6 expression requires activity at both CREB (-213 to -208 nt and NF-κB (-78 to -62 nt binding sites. These results provide novel insights into the molecular action of TSH and signaling downstream for TSHR in non-thyroid cells. Fibrocytes neither express adenylate cyclase nor generate cAMP and thus these findings are free from any influence of cAMP-related signaling. They identify potential therapeutic targets for TAO.

Effective stimulation of cardiac contractility and myocardial metabolism by impromidine and dimaprit--two new H2-agonistic compounds--in the surviving, catecholamine-insensitive myocardium after coronary occlusion

International Nuclear Information System (INIS)

Baumann, G.; Felix, S.B.; Riess, G.; Loher, U.; Ludwig, L.; Bloemer, H.

1982-01-01

Left ventricular infarctions were produced in guinea pigs, and the contractile response to beta-adrenergic and H2-histaminergic stimulation was tested in isolated perfused heart preparations. Adenylate cyclase activity and binding characteristics of sarcolemmal beta 1-, H2-, and muscarinic cholinergic receptors were determined in sarcolemmal membrane preparations of the uninvolved right ventricle of the same hearts. Three days after infarction, the positive inotropic effects of isoproterenol (2.8 X 10(-9) mol/L) and tyramine (5.5 X 10(-5) mol/L) were nearly abolished, while the inotropic effects of impromidine (4.6 X 10(-7) mol/L) and dimaprit (8.5 X 10(-6) mol/L) were not impaired. Stimulation rates of cardiac adenylate cyclase activity by isoproterenol were markedly reduced (-90%) whereas impromidine, dimaprit, and NaF revealed stimulation rates equivalent to the sham-operated control group. beta-Receptor binding studies with [ 3 H]dihydroalprenol revealed 90% loss and nearly 10 times lowered affinity (KD) of the remaining receptors, while specific binding of [ 3 H]tiotidine and [ 3 H]quinuclidinyl-benzylate was unchanged in the same preparations. All of the above alterations could be prevented to a similar extent by treatment with different beta-blocking agents, but differences between the drugs were seen with respect to survival rates and reduction of infarct size. In agreement with previous findings, we conclude that the observed alterations in the nonischemic surviving myocardium are the result of specific damage of sarcolemmal beta-receptors due to excessive exposure to increased catecholamines after infarction. The stimulation of the uninvolved H2-receptors may be of therapeutic value

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

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

International Nuclear Information System (INIS)

Deterre, Philippe

1983-01-01

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

Calcium-Driven Folding of RTX Domain beta-Rolls Ratchets Translocation of RTX Proteins through Type I Secretion Ducts

Czech Academy of Sciences Publication Activity Database

Bumba, Ladislav; Mašín, Jiří; Macek, Pavel; Wald, Tomáš; Motlová, Lucia; Bíbová, Ilona; Klímová, Nela; Bednárová, Lucie; Veverka, Václav; Kachala, M.; Svergun, D. I.; Bařinka, Cyril; Šebo, Peter

2016-01-01

Roč. 62, č. 1 (2016), s. 47-62 ISSN 1097-2765 R&D Projects: GA MŠk(CZ) LK11205; GA MŠk(CZ) LO1304; GA ČR(CZ) GA15-11851S; GA ČR GA13-14547S; GA ČR GAP302/12/0460; GA ČR(CZ) GAP207/11/0717; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 ; RVO:61388963 ; RVO:86652036 Keywords : ADENYLATE-CYCLASE TOXIN * GRAM-NEGATIVE BACTERIA * BORDETELLA-PERTUSSIS Subject RIV: CE - Biochemistry ; EB - Genetics ; Molecular Biology (BTO-N); CE - Biochemistry (UOCHB-X) Impact factor: 14.714, year: 2016

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

Science.gov (United States)

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

1991-01-01

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

Thermostability promotes the cooperative function of split adenylate kinases.

Science.gov (United States)

Nguyen, Peter Q; Liu, Shirley; Thompson, Jeremy C; Silberg, Jonathan J

2008-05-01

Proteins can often be cleaved to create inactive polypeptides that associate into functional complexes through non-covalent interactions, but little is known about what influences the cooperative function of the ensuing protein fragments. Here, we examine whether protein thermostability affects protein fragment complementation by characterizing the function of split adenylate kinases from the mesophile Bacillus subtilis (AKBs) and the hyperthermophile Thermotoga neapolitana (AKTn). Complementation studies revealed that the split AKTn supported the growth of Escherichia coli with a temperature-sensitive AK, but not the fragmented AKBs. However, weak complementation occurred when the AKBs fragments were fused to polypeptides that strongly associate, and this was enhanced by a Q16L mutation that thermostabilizes the full-length protein. To examine how the split AK homologs differ in structure and function, their catalytic activity, zinc content, and circular dichroism spectra were characterized. The reconstituted AKTn had higher levels of zinc, greater secondary structure, and >10(3)-fold more activity than the AKBs pair, albeit 17-fold less active than full-length AKTn. These findings provide evidence that the design of protein fragments that cooperatively function can be improved by choosing proteins with the greatest thermostability for bisection, and they suggest that this arises because hyperthermophilic protein fragments exhibit greater residual structure compared to their mesophilic counterparts.

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

OpenAIRE

Arango, Y; Heise, K P

1998-01-01

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

Bacillus anthracis-derived edema toxin (ET counter-regulates movement of neutrophils and macromolecules through the endothelial paracellular pathway

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Nguyen Chinh

2012-01-01

Full Text Available Abstract Background A common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear leukocytes (PMNs in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of anthrax is edema toxin (ET, which is formed by the combination of two proteins produced by the organism, edema factor (EF, which is an adenyl cyclase, and protective antigen (PA. Since cAMP, a product of adenyl cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of PMNs into tissues through closure of the paracellular pathway through which PMNs traverse. Results Pretreatment of human microvascular endothelial cell(ECs of the lung (HMVEC-L with ET decreased interleukin (IL-8-driven transendothelial migration (TEM of PMNs with a maximal reduction of nearly 60%. This effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial 14 C-albumin flux by ~ 50% compared to medium controls. Coadministration of ET with either tumor necrosis factor-α or bacterial lipopolysaccharide, each at 100 ng/mL, attenuated the increase of transendothelial 14 C-albumin flux caused by either agent alone. The inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA activity, but could not be blocked by inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP. Conclusions We conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of cAMP/PKA activity.

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

International Nuclear Information System (INIS)

Sonnenburg, W.K.

1987-01-01

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

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.

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.

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

Science.gov (United States)

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

2018-02-13

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

Thyroid Stimulating Immunoglobulin Bioassay Using Cultured Human Thyroid Cells; A Simplified Micromethod

International Nuclear Information System (INIS)

Lee, Myung Chul; Chung, June Key; Cho, Bo Youn; Koh, Chang Soon; Lee, Moon Ho; Ahn, Il Min; Ahn, Hee Kwon

1985-01-01

The activation of adenylate cyclase of human thymocytes in primary cell culture and the release of c-AMP into the medium are used to detect b-TSH and TSAb in sera of patients with autoimmune thyroid disease. Sera of patients are used directly as a part of cell culture without immunoglobulin precipitation. In the above TSI bioassay, TSAb pooled serum show c-AMP concentration between that of 1 mU/ml and 10 mU/ml b-TSH but normal control pooled serum doesn't show any detectable c-AMP response. Ninety five percent of untreated Graves' patients shows TSAb activity above normal range, 20% of Hashimoto's and 363/0 of euthyroid Graves' patients show detectable TSAb activity.

Emerging migraine treatments and drug targets

DEFF Research Database (Denmark)

Olesen, Jes; Ashina, Messoud

2011-01-01

Migraine has a 1-year prevalence of 10% and high socioeconomic costs. Despite recent drug developments, there is a huge unmet need for better pharmacotherapy. In this review we discuss promising anti-migraine strategies such as calcitonin gene-related peptide (CGRP) receptor antagonists and 5....... Tonabersat, a cortical spreading depression inhibitor, has shown efficacy in the prophylaxis of migraine with aura. Several new drug targets such as nitric oxide synthase, the 5-HT(1D) receptor, the prostanoid receptors EP(2) and EP(4), and the pituitary adenylate cyclase receptor PAC1 await development....... The greatest need is for new prophylactic drugs, and it seems likely that such compounds will be developed in the coming decade....

Watery diarrhea syndrome in an adult with ganglioneuroma-pheochromocytoma: identification of vasoactive intestinal peptide, calcitonin, and catecholamines and assessment of their biologic activity.

Science.gov (United States)

Trump, D L; Livingston, J N; Baylin, S B

1977-10-01

A case of adult ganglioneuroma-pheochromocytoma with an associated watery diarrhea syndrome is reported. High levels of vasoactive intestinal peptide (VIP) were found in preoperative serum and in tumor tissue. The serum VIP levels fell to normal, and the watery diarrhae syndrome completely ceased following removal of the tumor. In addition to containing VIP, the tumor was rich in catecholamines, and calcitonin. Peptide hormone-containing extracts and catecholamine extracts from the tumor both activated the adenyl cyclase system and increased lipolytic activity in a preparation of isolated rat fat cells. The findings in this patient further link VIP with neural crest tissues, and suggest the importance of determining catecholamine levels in patients with the watery diarrhea syndrome.

Physiological and biochemical characteristics of adrenergic receptors and pathways in brown adipocytes

Science.gov (United States)

Horwitz, B. A.

1975-01-01

Mechanisms involved in the thermogenic response of brown adipose tissue (BAT) to sympathetic nervous stimulation (e.g., by cold exposure) and to norepinephrine (NE) release are investigated. Three effects appear to play a role in the increased oxygen consumption (and heat production) of the adipocytes: increased membrane permeability, activation of the beta-adrenergic pathway, and enhancement of Na(+)/K(+) membrane pump activity. Increased passive influx of Na(+) and efflux of K(+) due to greater permeability raise the energy demands of the Na/K pump; the pump is also stimulated by increased cyclic AMP synthesis resulting from activation by NE of membrane-bound adenyl cyclase. Studies with inhibitors such as propanolol, phentolamine, and ouabain support this hypothesis.

Novel Neuroprotective Strategies in Ischemic Retinal Lesions

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Robert Gabriel

2010-02-01

Full Text Available Retinal ischemia can be effectively modeled by permanent bilateral common carotid artery occlusion, which leads to chronic hypoperfusion-induced degeneration in the entire rat retina. The complex pathways leading to retinal cell death offer a complex approach of neuroprotective strategies. In the present review we summarize recent findings with different neuroprotective candidate molecules. We describe the protective effects of intravitreal treatment with: (i urocortin 2; (ii a mitochondrial ATP-sensitive K+ channel opener, diazoxide; (iii a neurotrophic factor, pituitary adenylate cyclase activating polypeptide; and (iv a novel poly(ADP-ribose polymerase inhibitor (HO3089. The retinoprotective effects are demonstrated with morphological description and effects on apoptotic pathways using molecular biological techniques.

Gene expression profiling of gastric mucosa in mice lacking CCK and gastrin receptors

DEFF Research Database (Denmark)

Zhao, Chun-Mei; Kodama, Yosuke; Flatberg, Arnar

2014-01-01

normalized, which was associated with an up-regulated pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1). The basal part of the gastric mucosa expressed parathyroid hormone-like hormone (PTHLH) in a subpopulation of likely ECL cells (and possibly other cells) and vitamin D3 1α...... suggest a possible link between gastric PTHLH and vitamin D and bone metabolism.......The stomach produces acid, which may play an important role in the regulation of bone homeostasis. The aim of this study was to reveal signaling pathways in the gastric mucosa that involve the acid secretion and possibly the bone metabolism in CCK1 and/or CCK2 receptor knockout (KO) mice. Gastric...

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

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Victor L Jensen

2010-11-01

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

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

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Zhang, Ming; Wang, Hongbing

2013-01-01

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

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

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

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.

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

Science.gov (United States)

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

2014-01-17

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

Functional and biochemical responses of cultured heart cells to angiotensin II

International Nuclear Information System (INIS)

Allen, I.; Gaa, S.; Rogers, T.B.

1986-01-01

The authors have utilized a cultured neonatal rat heart myocyte system to study the molecular mechanisms involved in the stimulation of heart cells by angiotensin II (AII). The intact cultured cells, and membranes from these cells, have specific, high affinity receptors for 125 I-AII and for an AII antagonist, 125 I-Sar 1 ,Leu 8 -AII. Binding affinity was in the nanomolar range and was inhibited by guanine nucleotides. Functional studies on intact, beating cells revealed a maximal increase in contractile frequency of 50%, observed at 5 nM AII, with half maximal effects noted at around 1 nM. These responses were reversible and specific as the antagonist, Sar 1 , Ala 8 -AII, inhibited AII-induced chronotropic stimulation. AII (100 nM) had no effect on basal adenylate cyclase activity (20 pmoles cAMP/mg prot/min at 2.5mM Mg 2+ ) in cell membranes. Further, in membranes where cyclase activity was stimulated with isoproterenol (290 pmoles cAMP/mg prot/min at 2.5mM Mg 2+ ), addition of AII had no effect. The cyclase-inhibitory muscarinic agonist, carbachol, also failed to reduce isoproterenol-stimulated activity. In preliminary work with the intact cells, AII again did not alter basal cAMP levels (3-10 pmoles cAMP/mg prot). However, the hormone increased isoproterenol-stimulated cAMP levels by almost 50%. These cells are an excellent system for correlating AII receptor binding with functional and biochemical responses

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.

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

Science.gov (United States)

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

2006-10-12

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

Diminished but Not Abolished Effect of Two His351 Mutants of Anthrax Edema Factor in a Murine Model

Science.gov (United States)

Zhao, Taoran; Zhao, Xinghui; Liu, Ju; Meng, Yingying; Feng, Yingying; Fang, Ting; Zhang, Jinlong; Yang, Xiuxu; Li, Jianmin; Xu, Junjie; Chen, Wei

2016-01-01

Edema toxin (ET), which is composed of a potent adenylate cyclase (AC), edema factor (EF), and protective antigen (PA), is one of the major toxicity factors of Bacillus anthracis. In this study, we introduced mutations in full-length EF to generate alanine EF(H351A) and arginine EF(H351R) variants. In vitro activity analysis displayed that the adenylyl cyclase activity of both the mutants was significantly diminished compared with the wild-type EF. When the native and mutant toxins were administered subcutaneously in a mouse footpad edema model, severe acute swelling was evoked by wild-type ET, while the symptoms induced by mutant toxins were very minor. Systemic administration of these EF variants caused non-lethal hepatotoxicity. In addition, EF(H351R) exhibited slightly higher activity in causing more severe edema than EF(H351A). Our findings demonstrate that the toxicity of ET is not abolished by substitution of EF residue His351 by alanine or arginine. These results also indicate the potential of the mouse footpad edema model as a sensitive method for evaluating both ET toxicity and the efficacy of candidate therapeutic agents. PMID:26848687

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

Science.gov (United States)

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

2012-07-03

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

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, ...

Relaxation of soman-induced contracture of airway smooth muscle in vitro. (Reannouncement with new availability information)

Energy Technology Data Exchange (ETDEWEB)

Filbert, M.G.; Moore, D.H.; Adler, M.

1992-12-31

A possible role for beta-adrenergic agonists in the management of bronchoconstriction resulting from exposure to anticholinesterase compounds was investigated in vitro in canine tracheal smooth muscle. Norepinephrine, salbutamol and isoproterenol produced partial relaxation of soman-induced contractures. However, the relaxation induced was not sustained; muscle tensions returned to pretreatment levels within minutes despite the continued presence of beta-agonists. Increasing cAMP levels with the non beta-agonist bronchodilators such as thoophylline, a phosphodiesterase inhibitor, or forskolin, a specific stimulator of adenylate cyclase, resulted in more complete and longer lasting relaxation, suggesting that beta-adrenoceptor desensitization may contribute to the failure by beta-agonists to produce sustained relaxation. Nerve agents, Soman, Toxicity, Airway smooth muscle, In vitro, Physiology, Effects.

Transient alterations in neurotransmitter activity in the caudate nucleus of rat brain after a high dose of ionizing radiation

International Nuclear Information System (INIS)

Hunt, W.A.; Dalton, T.K.; Darden, J.H.

1979-01-01

A single 10,000-rad dose of high-energy electrons induced an increase in dopaminergic and cholinergic activity in the caudate nucleus of the rat brain as assessed by K + -stimulated dopamine release in vitro and high-affinity choline uptake. These alterations occur during early transient incapacitation (ETI) and dissipate as the animal recovers behaviorally, in about 30 min after irradiation. Although the responses observed resemble those that result from blockade of dopamine receptors, no radiation-induced changes were found in dopamine-sensitive adenylate cyclase activity and [ 3 H]haloperidol binding, two indices of dopaminergic receptor function. The data suggest that changes in dopaminergic and cholinergic activity are associated with the development of ETI and may play a role in the behavioral decrement observed under this condition

[Signal transudation pathways in parietal cells of the gastric mucosa in experimental stomach ulcer].

Science.gov (United States)

Ostapchenko, L I; Drobins'ka, O V; Chaĭka, V O; Bohun, L I; Bohdanova, O V; Kot, L I; Haĭda, L M

2009-01-01

The goal of the presented work was the research of signal transduction mechanism in the rat gastric parietal cells under stomach ulcer conditions. In these cells activation of adenylate cyclase (increase of cAMP level and proteinkinase A activity) and phosphoinositide (increases [Ca2+]i; cGMP and phoshatidylinocitole levels; proteinkinase C, proteinkinase G, and calmodulin-dependent-proteinkinase activity) of signals pathway was shown. An increase of plasma membrane phospholipids (PC, PS, PE, PI, LPC) level was shown. Under conditions of influence of the stress factor the membran enzymes activity (H+, K+ -ATPase, 5'-AMPase, Na+, K+ -ATPase, Ca2+, Mg2+ -ATPase and H+, K+ -ATPase) was considerably increased. The intensification of lipid peroxidation processes in rats was demonstrated.

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.

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

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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.

In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

International Nuclear Information System (INIS)

Kim, Hyo Joon; Nishikawa, Satoshi; Tokutomi, Yuiko; Uesugi, Seiichi; Takenaka, Hitoshi; Hamada, Minoru; Kuby, S.A.

1990-01-01

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP 2- and for AMP 2- in human cytosolic adenylate kinase, the authors have investigated the enzymic effects of replacement of the arginine residues, which had been assumed by Pai et al. to interact with the phosphoryl groups of AMP 2- and MgATP 2- . With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the K m,app values for AMP 2- of the mutant enzymes, the relatively small increases in the K m,app values for MgATP 2- , and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. have been reversed and that their ATP-binding site may be assigned as the AMP site

Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

KAUST Repository

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

2012-01-01

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

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.

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

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

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

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

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…

STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition.

Science.gov (United States)

Terra, Silvia R; Cardoso, João Carlos R; Félix, Rute C; Martins, Leo Anderson M; Souza, Diogo Onofre G; Guma, Fatima C R; Canário, Adelino Vicente M; Schein, Vanessa

2015-03-05

Stanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

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

Pituitary Adenylate Cyclase-Activating Polypeptide Stimulates Glucose Production via the Hepatic Sympathetic Innervation in Rats

NARCIS (Netherlands)

Yi, Chun-Xia; Sun, Ning; Ackermans, Mariette T.; Alkemade, Anneke; Foppen, Ewout; Shi, Jing; Serlie, Mireille J.; Buijs, Ruud M.; Fliers, Eric; Kalsbeek, Andries

2010-01-01

OBJECTIVE-The unraveling of the elaborate brain networks that control glucose metabolism presents one of the current challenges in diabetes research. Within the central nervous system, the hypothalamus is regarded as the key brain area to regulate energy homeostasis. The aim of the present study was

Adaptation of an L-proline adenylation domain to use 4-propyl-L-proline in the evolution of lincosamide biosynthesis.

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Stanislav Kadlčík

Full Text Available Clinically used lincosamide antibiotic lincomycin incorporates in its structure 4-propyl-L-proline (PPL, an unusual amino acid, while celesticetin, a less efficient related compound, makes use of proteinogenic L-proline. Biochemical characterization, as well as phylogenetic analysis and homology modelling combined with the molecular dynamics simulation were employed for complex comparative analysis of the orthologous protein pair LmbC and CcbC from the biosynthesis of lincomycin and celesticetin, respectively. The analysis proved the compared proteins to be the stand-alone adenylation domains strictly preferring their own natural substrate, PPL or L-proline. The LmbC substrate binding pocket is adapted to accommodate a rare PPL precursor. When compared with L-proline specific ones, several large amino acid residues were replaced by smaller ones opening a channel which allowed the alkyl side chain of PPL to be accommodated. One of the most important differences, that of the residue corresponding to V306 in CcbC changing to G308 in LmbC, was investigated in vitro and in silico. Moreover, the substrate binding pocket rearrangement also allowed LmbC to effectively adenylate 4-butyl-L-proline and 4-pentyl-L-proline, substrates with even longer alkyl side chains, producing more potent lincosamides. A shift of LmbC substrate specificity appears to be an integral part of biosynthetic pathway adaptation to the PPL acquisition. A set of genes presumably coding for the PPL biosynthesis is present in the lincomycin--but not in the celesticetin cluster; their homologs are found in biosynthetic clusters of some pyrrolobenzodiazepines (PBD and hormaomycin. Whereas in the PBD and hormaomycin pathways the arising precursors are condensed to another amino acid moiety, the LmbC protein is the first functionally proved part of a unique condensation enzyme connecting PPL to the specialized amino sugar building unit.

Effect of tributyltin on adenylate content and enzyme activities of teleost sperm: a biochemical approach to study the mechanisms of toxicant reduced spermatozoa motility.

Science.gov (United States)

Rurangwa, E; Biegniewska, A; Slominska, E; Skorkowski, E F; Ollevier, F

2002-03-01

The effects of tributyltin (TBT) on the energy metabolism and motility of fish spermatozoa were investigated in vitro in African catfish and common carp. A significant (PTBT for 24 h. Exposure of catfish spermatozoa to 2.7-27 microg/l TBT caused an instant decrease in ATP content. In the presence of 27 microg/l TBT approximately 55% of the initial ATP concentration in catfish semen was lost after 60 min incubation while AMP concentrations increased and the total adenine nucleotide (TAN) pool remained unchanged. The reduction in sperm ATP levels could not be attributed to cell death since viability decreased only slightly over the period of exposure. In carp by contrast, none of the adenylates concentrations studied (ATP, ADP and AMP) were affected by TBT exposure at any experimental condition. However, carp sperm motility was significantly reduced by exposure to 2.7 microg/l TBT. Among the enzymes investigated only lactate dehydrogenase (LDH) in catfish sperm was significantly (PTBT treatment with a reduction in activity of approximately 75%. Compared with carp sperm before TBT exposure, that of catfish had lower adenylate contents and overall lower enzymatic activities; this explains its slower sperm velocity and shorter duration of movement as measured by computer assisted sperm analysis (CASA). The present in vitro study shows that catfish spermatozoa are more sensitive to TBT exposure (and probably to other toxicants) than those of carp.

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

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

2017-11-16

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

Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.

Science.gov (United States)

Zhang, Keya; Nelson, Kathryn M; Bhuripanyo, Karan; Grimes, Kimberly D; Zhao, Bo; Aldrich, Courtney C; Yin, Jun

2013-01-24

The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains. Copyright © 2013 Elsevier Ltd. All rights reserved.

RNA-Seq analysis validates the use of culture-derived Trypanosoma brucei and provides new markers for mammalian and insect life-cycle stages.

Science.gov (United States)

Naguleswaran, Arunasalam; Doiron, Nicholas; Roditi, Isabel

2018-04-02

Trypanosoma brucei brucei, the parasite causing Nagana in domestic animals, is closely related to the parasites causing sleeping sickness, but does not infect humans. In addition to its importance as a pathogen, the relative ease of genetic manipulation and an innate capacity for RNAi extend its use as a model organism in cell and infection biology. During its development in its mammalian and insect (tsetse fly) hosts, T. b. brucei passes through several different life-cycle stages. There are currently four life-cycle stages that can be cultured: slender forms and stumpy forms, which are equivalent to forms found in the mammal, and early and late procyclic forms, which are equivalent to forms in the tsetse midgut. Early procyclic forms show coordinated group movement (social motility) on semi-solid surfaces, whereas late procyclic forms do not. RNA-Seq was performed on biological replicates of each life-cycle stage. These constitute the first datasets for culture-derived slender and stumpy bloodstream forms and early and late procyclic forms. Expression profiles confirmed that genes known to be stage-regulated in the animal and insect hosts were also regulated in culture. Sequence reads of 100-125 bases provided sufficient precision to uncover differential expression of closely related genes. More than 100 transcripts showed peak expression in stumpy forms, including adenylate cyclases and several components of inositol metabolism. Early and late procyclic forms showed differential expression of 73 transcripts, a number of which encoded proteins that were previously shown to be stage-regulated. Moreover, two adenylate cyclases previously shown to reduce social motility are up-regulated in late procyclic forms. This study validates the use of cultured bloodstream forms as alternatives to animal-derived parasites and yields new markers for all four stages. In addition to underpinning recent findings that early and late procyclic forms are distinct life-cycle stages

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

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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

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

Science.gov (United States)

Beste, Kerstin Y; Seifert, Roland

2013-02-01

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

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

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Chinnusamy Viswanathan

2010-04-01

Full Text Available Abstract Background Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B promoter from a green fruited S. habrochaites genotype EC520061. Results A 908 bp region upstream to the initiation codon of the Lycopene β-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908

Change in brain network connectivity during PACAP38-induced migraine attacks

DEFF Research Database (Denmark)

Amin, Faisal Mohammad; Hougaard, Anders; Magon, Stefano

2016-01-01

OBJECTIVE: To investigate resting-state functional connectivity in the salience network (SN), the sensorimotor network (SMN), and the default mode network (DMN) during migraine attacks induced by pituitary adenylate cyclase-activating polypeptide-38 (PACAP38). METHODS: In a double-blind, randomized...... connectivity with the bilateral opercular part of the inferior frontal gyrus in the SN. In SMN, there was increased connectivity with the right premotor cortex and decreased connectivity with the left visual cortex. Several areas showed increased (left primary auditory, secondary somatosensory, premotor......, and visual cortices) and decreased (right cerebellum and left frontal lobe) connectivity with DMN. We found no resting-state network changes after VIP (n = 15). CONCLUSIONS: PACAP38-induced migraine attack is associated with altered connectivity of several large-scale functional networks of the brain....

MEK Inhibitors Reverse cAMP-Mediated Anxiety in Zebrafish

DEFF Research Database (Denmark)

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

2015-01-01

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

Molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor

International Nuclear Information System (INIS)

Numa, S.; Fukuda, K.; Kubo, T.; Maeda, A.; Akiba, I.; Bujo, H.; Nakai, J.; Mishina, M.; Higashida, H.

1988-01-01

The muscarinic acetylcholine receptor (mAChR) mediates a variety of cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides, and modulation of potassium channels, through the action of guanine-nucleotide-binding regulatory proteins (G proteins). The question then arises as to whether multiple mAChR species exist that are responsible for the various biochemical and physiological effects. In fact, pharmacologically distinguishable forms of the mAChR occur in different tissues and have been provisionally classified into M 1 (I), M 2 cardiac (II), and M 2 glandular (III) subtypes on the basis of their difference in apparent affinity for antagonists. Here, the authors have made attempts to understand the molecular basis of the functional heterogeneity of the mAChR, using recombinant DNA technology

Enhancement of radioprotective effectiveness of adenosine monophosphate by magnesium aspartate in mice

International Nuclear Information System (INIS)

Pospisil, M.; Netikova, J.; Kozubik, A.; Chertkov, K.S.; Ministry of Health, Moscow

1988-01-01

The enhancing effect of magnesium aspartate on the radioprotective effectiveness of adenosine monophosphate (AMP) administered to whole-body gamma-irradiated mice was studied. Male (CBA x C57BL/10)F 1 hybrid mice of a mean body weight of 32 g were used. 5 mg AMP per mouse was injected i.p. 15 min before and 15 min after irradiation; magnesium aspartate (13.3 mg per mouse) was administered s.c. 35 min before irradiation. The benefical effect of the drug combination used was manifested when investigating hematological indices at the recovery phase of sublethally irradiated animals, as well as when observing the survival of lethally irradiated mice. The synergistic radioprotective effects of AMP and magnesium aspartate are explained by the stimulatory action of both these compounds on the cell adenylate cyclase system. (author)

Immunochemical similarity of GTP-binding proteins from different systems

International Nuclear Information System (INIS)

Kalinina, S.N.

1986-01-01

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

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

Science.gov (United States)

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

2011-11-01

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

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.

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

Science.gov (United States)

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

2012-09-20

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

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

Science.gov (United States)

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

2018-03-28

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

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

Science.gov (United States)

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

2009-11-26

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

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

Science.gov (United States)

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

2010-11-01

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

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

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

Science.gov (United States)

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

2013-09-12

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

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

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

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

Identification of the D-1 dopamine receptor subunit in rat striatum after photoaffinity labeling

Energy Technology Data Exchange (ETDEWEB)

Kuno, T; Tanaka, C [Kobe Univ. (Japan). School of Medicine

1982-12-28

When rat striatal membranes, photolabeled with (/sup 3/H)dopamine under assay conditions similar to those used for dopamine-sensitive adenylate cyclase, were subjected to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, several radioactively labeled bands appeared. Labeling of these bands was reduced in the presence of non-radioactive dopamine during photolysis, but was unaffected by the presence of sulpiride. Haloperidol preferentially reduced the labeling of the main band which had a molecular weight of about 57,000 rather than the other weakly labeled bands. Labeling of this 57,000 dalton protein was not apparent when rat cerebellar membranes were used and was markedly eliminated by kainic acid-induced lesions that destroyed the intrastriatal nerve cell bodies. These results indicate that this 57,000 dalton protein is the binding subunit of the D-1 dopamine receptor.

The Central Metabolism Regulator EIIAGlc Switches Salmonella from Growth Arrest to Acute Virulence through Activation of Virulence Factor Secretion

Directory of Open Access Journals (Sweden)

Alain Mazé

2014-06-01

Full Text Available The ability of Salmonella to cause disease depends on metabolic activities and virulence factors. Here, we show that a key metabolic protein, EIIAGlc, is absolutely essential for acute infection, but not for Salmonella survival, in a mouse typhoid fever model. Surprisingly, phosphorylation-dependent EIIAGlc functions, including carbohydrate transport and activation of adenylate cyclase for global regulation, do not explain this virulence phenotype. Instead, biochemical studies, in vitro secretion and translocation assays, and in vivo genetic epistasis experiments suggest that EIIAGlc binds to the type three secretion system 2 (TTSS-2 involved in systemic virulence, stabilizes its cytoplasmic part including the crucial TTSS-2 ATPase, and activates virulence factor secretion. This unexpected role of EIIAGlc reveals a striking direct link between central Salmonella metabolism and a crucial virulence mechanism.

Effects of activated ACM on expression of signal transducers in cerebral cortical neurons of rats.

Science.gov (United States)

Wang, Xiaojing; Li, Zhengli; Zhu, Changgeng; Li, Zhongyu

2007-06-01

To explore the roles of astrocytes in the epileptogenesis, astrocytes and neurons were isolated, purified and cultured in vitro from cerebral cortex of rats. The astrocytes were activated by ciliary neurotrophic factor (CNTF) and astrocytic conditioned medium (ACM) was collected to treat neurons for 4, 8 and 12 h. By using Western blot, the expression of calmodulin dependent protein kinase II (CaMK II), inducible nitric oxide synthase (iNOS) and adenylate cyclase (AC) was detected in neurons. The results showed that the expression of CaMK II, iNOS and AC was increased significantly in the neurons treated with ACM from 4 h to 12 h (PACM and such signal pathways as NOS-NO-cGMP, Ca2+/CaM-CaMK II and AC-cAMP-PKA might take part in the signal transduction of epileptogenesis.

Emerging migraine treatments and drug targets

DEFF Research Database (Denmark)

Olesen, Jes; Ashina, Messoud

2011-01-01

Migraine has a 1-year prevalence of 10% and high socioeconomic costs. Despite recent drug developments, there is a huge unmet need for better pharmacotherapy. In this review we discuss promising anti-migraine strategies such as calcitonin gene-related peptide (CGRP) receptor antagonists and 5......-hydroxytrypamine (5-HT)(1F) receptor agonists, which are in late-stage development. Nitric oxide antagonists are also in development. New forms of administration of sumatriptan might improve efficacy and reduce side effects. Botulinum toxin A has recently been approved for the prophylaxis of chronic migraine....... Tonabersat, a cortical spreading depression inhibitor, has shown efficacy in the prophylaxis of migraine with aura. Several new drug targets such as nitric oxide synthase, the 5-HT(1D) receptor, the prostanoid receptors EP(2) and EP(4), and the pituitary adenylate cyclase receptor PAC1 await development...

Signalling by CGRP and Adrenomedullin in the Cerebellum and Other Systems

Directory of Open Access Journals (Sweden)

David Poyner

2001-01-01

Full Text Available The best characterised signalling pathway activated by both CGRP and adrenomedullin is stimulation of adenylate cyclase via Gs. However, it is clear that in some circumstances the peptides can activate other signal transduction pathways, e.g., increases in intracellular calcium. Many of these signalling pathways can be observed in cultured cells but it is important also to examine isolated tissues to discover the full repertoire of transduction events. In the rat cerebellum there are receptors that respond to both CGRP and adrenomedullin. These seem to be located postsynaptically on Parallel Fibre nerve terminals and modulate transmission to Purkinje cells. Adrenomedullin acts via cAMP, apparently to augment neurotransmitter release. By contrast, CGRP decreases transmitter release, via a non-cAMP mediated pathway. We are currently examining the role of NO and tyrosine kinases in the responses to these peptides.

Neuropeptides in Lower Urinary Tract (LUT) Function

Science.gov (United States)

Arms, Lauren; Vizzard, Margaret A.

2014-01-01

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 bladder. Neuropeptides have tissue-specific distributions and functions in the LUT and exhibit neuroplastic changes in expression and function with LUT dysfunction following neural injury, inflammation and disease. LUT dysfunction with abnormal voiding including urinary urgency, increased voiding frequency, nocturia, urinary incontinence and pain may reflect a change in the balance of neuropeptides in bladder reflex pathways. LUT neuropeptide/receptor systems may represent potential targets for therapeutic intervention. PMID:21290237

Carbon Catabolite Repression Regulates the Production of the Unique Volatile Sodorifen of Serratia plymuthica 4Rx13

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Nancy Magnus

2017-12-01

Full Text Available Microorganisms are capable of synthesizing a plethora of secondary metabolites including the long-overlooked volatile organic compounds. Little knowledge has been accumulated regarding the regulation of the biosynthesis of such mVOCs. The emission of the unique compound sodorifen of Serratia plymuthica isolates was significantly reduced in minimal medium with glucose, while succinate elevated sodorifen release. The hypothesis of carbon catabolite repression (CCR acting as a major control entity on the synthesis of mVOCs was proven by genetic evidence. Central components of the typical CCR of Gram-negative bacteria such as the adenylate cyclase (CYA, the cAMP binding receptor protein (CRP, and the catabolite responsive element (CRE were removed by insertional mutagenesis. CYA, CRP, CRE1 mutants revealed a lower sodorifen release. Moreover, the emission potential of other S. plymuthica isolates was also evaluated.

The multiplicity of the D-1 dopamine receptor

International Nuclear Information System (INIS)

Mailman, R.B.; Klits, C.D.; Lewis, M.H.; Rollema, H.; Schulz, D.W.; Wyrick, S.

1986-01-01

The authors have sought to address two questions of some neuropharmacological importance in this chapter. First, they examine the nature of mechanisms by which dopamine initiates many psychopharmacological effects and, second, they study the possibility of designing highly specific drugs targeted only at a selected subpopulation of dopamine receptors. Effects of SCH23390 and haloperidol on concentrations of dopamine, DOPAC, and HVA in various rat brain regions are shown. In addition, the effects of SCH23390 on the in vivo binding of dipropyl-5, 6-ADTN are shown. Differential distribution of a dopamine sensitive adenylate cyclase and ( 3 H)-SCH23390 binding sites are examined. A model is presented of D 1 dopamine receptors in membrane, illustrating the lack of identity of some of the ( 3 H)-SCH23390 binding sites with the dopamine receptor linked to stimulation of cAMP synthesis

Differentiation of Nerve Fibers Storing CGRP and CGRP Receptors in the Peripheral Trigeminovascular System

DEFF Research Database (Denmark)

Eftekhari, Sajedeh; Warfvinge, Karin; Blixt, Frank W

2013-01-01

Primary headaches such as migraine are postulated to involve the activation of sensory trigeminal pain neurons that innervate intracranial blood vessels and the dura mater. It is suggested that local activation of these sensory nerves may involve dural mast cells as one factor in local inflammation...... and in human dural vessels. The relative distributions of CGRP, CLR, and RAMP1 were evaluated with respect to each other and in relationship to mast cells, myelin, substance P, neuronal nitric oxide synthase, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal peptide. CGRP expression...... was found in thin unmyelinated fibers, whereas CLR and RAMP1 were expressed in thicker myelinated fibers coexpressed with an A-fiber marker. CLR and RAMP1 immunoreactivity colocalized with mast cell tryptase in rodent; however, expression of both receptor components was not observed in human mast cells...

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

International Nuclear Information System (INIS)

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

1985-01-01

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

Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy

International Nuclear Information System (INIS)

Fry, D.C.; Byler, D.M.; Susi, H.; Brown, M.; Kuby, S.A.; Mildvan, A.S.

1988-01-01

The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme, appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase, with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of β-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% α-helix, 38% β-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possible due to disorder, it can be fit by using methods developed on well-characterized globular proteins. The CD spectrum is best fit by assuming the presence of at most 13% α-helix in the peptide, 24 +/- 2% β-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformation changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assess by CD

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

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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.

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

Science.gov (United States)

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

2017-07-01

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

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

KAUST Repository

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

2010-01-01

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

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.

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

Modulation of the Conformational Dynamics of Apo-Adenylate Kinase through a π-Cation Interaction.

Science.gov (United States)

Halder, Ritaban; Manna, Rabindra Nath; Chakraborty, Sandipan; Jana, Biman

2017-06-15

Large-scale conformational transition from open to closed state of adenylate kinase (ADK) is essential for its catalytic cycle. Apo-ADK undergoes conformational transition in a way that closely resembles an open-to-closed conformational transition. Here, equilibrium simulations, free-energy simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations in combination with several bioinformatics approaches have been used to explore the molecular origin of this conformational transition in apo-ADK. In addition to its conventional open state, Escherichia coli apo-ADK adopts conformations that resemble a closed-like intermediate, the "half-open-half-closed" (HOHC) state, and a π-cation interaction can account for the stability of this HOHC state. Energetics and the electronic properties of this π-cation interaction have been explored using QM/MM calculations. Upon rescinding the π-cation interaction, the conformational landscape of the apo-ADK changes completely. The apo-ADK population is shifted completely toward the open state. This π-cation interaction is highly conserved in bacterial ADK; the cationic guanidinium moiety of a conserved ARG interacts with the delocalized π-electron cloud of either PHE or TYR. Interestingly, this study demonstrates the modulation of a principal protein dynamics by a conserved specific π-cation interaction across different organisms.

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.

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.

The preparation of nucleotides uniformly labelled with carbon-14 by biosynthetic methods. Isolation of adenylic, uridylic, cytidylic,and guanylic acids, from the alkaline hydrolysate of escherichia coli RNA

International Nuclear Information System (INIS)

Garcia Pineda, M. D.; Pacheco Lopez, J.

1978-01-01

A method is described for the preparation and analysis of adenylic, uri dilic, cytidi- 11c and guanylic acids, labelled with 14 C . Escherichia coli cells have been labelled by growing them in a medi dia containing glucose-14 C as their only source of carbon. RNA is isolated from the cells, and after hydrolysis of the molecule the resulting nucleotides are separated by gel filtration and exchange chromatography. Chemical and radiochemical purity of the Isolated nucleotides is determined, and also its specific radioactivity. (Author) 30 refs

Stimulation by parathyroid hormone of sup 45 Ca sup 2+ uptake in osteoblast-like cells: Possible involvement of alkaline phosphatase

Energy Technology Data Exchange (ETDEWEB)

Fukayama, S.; Tashjian, A.H. Jr. (Harvard School of Public Health, Boston, MA (USA))

1990-04-01

We have investigated the actions of human PTH (hPTH-(1-34)) on the association of 45Ca2+ with two human (SaOS-2 and MG-63) and two rat (ROS 17/2.8 and UMR-106) osteoblast-like cell types. In SaOS-2 cells, hPTH-(1-34) binds to specific membrane receptors to activate adenylate cyclase. Treatment of SaOS-2 cells with hPTH-(1-34) resulted in an increase in 45Ca2+ uptake, in a dose-dependent fashion, up to 2- to 4-fold above control values. The increase was first evident at 10 min and persisted for at least 30 min. Treatment with nimodipine, a calcium channel antagonist, was without effect on the stimulatory action of PTH. A similar enhancement of cell-associated 45Ca2+ was observed when the cells were incubated with vasoactive intestinal peptide, which acts via different receptors to activate adenylate cyclase in SaOS-2 cells. Treatment with (Bu)2cAMP also induced an increase in cell-associated 45Ca2+. Pretreatment of SaOS-2 cells with hPTH-(1-34) for 4 h, which induced homologous desensitization to a second challenge with the same peptide for stimulation of cAMP production, did not attenuate the further enhancement of cell-associated 45Ca2+ by a second treatment with hPTH-(1-34). We then examined a possible relationship between alkaline phosphatase (ALPase) and 45Ca2+ uptake. SaOS-2 cells contained high levels of alkaline phosphatase activity and continuously released the enzyme into the medium. Release was enhanced by treatment with hPTH-(1-34) for 10 min. Incubation of cells with levamisole (an inhibitor of the liver/bone/kidney type of ALPase) resulted in a rapid decrease in basal and PTH-stimulated 45Ca2+ uptake, while treatment with L-Phe-Gly-Gly was without effect. Treatment of the cells with ALPase (bovine kidney) enhanced 45Ca2+ uptake. In MG-63 cells, a stimulatory effect of hPTH-(1-34) on cell-associated 45Ca2+ was also observed; however, hPTH-(1-34) did not stimulate cAMP production in MG-63 cells.

Benzazepines: Structure-activity relationships between D1 receptor blockade and selected pharmacological effects

International Nuclear Information System (INIS)

Iorio, L.C.; Billiard, W.; Gold, E.H.

1986-01-01

This chapter describes the displacement of 3 H-23390 and 3 H-spiperone binding by dopamine agonists and antagonists. The authors undertook an evaluation of the ability of selected analogs of SCH 23390 to displace 3 H-SCH 23390 and 3 H-spiperone. Structure-activity relationships of SCH 23390 analogs: 7-position substituents, is shown. It is shown that, in general, benzazepines with a variety of substituents in the 7-position retain their selectivity for D 1 sites. Substituents at the 8-position and at the N-position are also discussed. The authors determine a correlation between displacement of 3 H-SCH 23390 and blockade of dopamine-sensitive adenylate cyclase (DSAC). These effects and inhibition of conditioned avoidance responsing (CAS) in rats was also studied. A detailed evaluation is presented of the effects of SCH 23390 and haloperidol in the Inclined Screen and CAR tests

PACAP-38 infusion causes sustained vasodilation of the middle meningeal artery in the rat

DEFF Research Database (Denmark)

Bhatt, Deepak K; Gupta, Saurabh; Olesen, Jes

2014-01-01

BACKGROUND: In healthy human volunteers and in migraineurs, pituitary adenylate cyclase-activating polypeptide-38 (PACAP-38) infusion caused sustained vasodilation of the middle meningeal artery (MMA) and an immediate as well as a delayed headache. All the study subjects experienced facial flushing....... Mast cells (MCs) might have a role in the long-lasting effect of PACAP-38 infusion. We hypothesized that in mast cell-depleted (MCD) rats the vascular responses to PACAP-38 would be lesser than in control rats because of a lack of vasodilatory products released during MC degranulation. METHODS: MCs...... were depleted by chronic treatment with compound 48/80. The effect of 20 minutes' intravenous (i.v.) infusion of calcitonin gene-related peptide (CGRP), PACAP-38, PACAP(6-38) (PAC-1 receptor antagonist) and PACAP-27 on the diameter of the MMA and on mean arterial blood pressure (MABP) in control...

Improvement of the closed cranial window model in rats by intracarotid infusion of signalling molecules implicated in migraine

DEFF Research Database (Denmark)

Gupta, S; Bhatt, D K; Boni, L J

2010-01-01

required, respectively, compared with i.v. infusion to induce the same dilation in dural artery. Dilating intracarotid (i.c.) doses caused no or a minimal fall in BP, whereas equi-responsive i.v. doses caused a marked BP reduction. The CGRP blocking potential of olcegepant was amplified by > 20 times on i......-related peptide (CGRP) and pituitary adenylate cyclase polypeptide (PACAP)-38. High i.v. doses are required to study their craniovascular pharmacology. Unfortunately, this leads to a drop in blood pressure (BP) that subsequently causes blood vessels to dilate by autoregulation. Hence it is difficult to decipher.......c. infusion. Pial artery responses to CGRP did not change with i.c. infusion, demonstrating that dilations after i.v. CGRP are mediated by autoregulation rather than through specific receptors. We applied CGRP topically, which induced concentration-dependent dural vasodilation, but no effect on pial artery...

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 +/.

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.

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.

Structures of human Golgi-resident glutaminyl cyclase and its complexes with inhibitors reveal a large loop movement upon inhibitor binding.

Science.gov (United States)

Huang, Kai-Fa; Liaw, Su-Sen; Huang, Wei-Lin; Chia, Cho-Yun; Lo, Yan-Chung; Chen, Yi-Ling; Wang, Andrew H-J

2011-04-08

Aberrant pyroglutamate formation at the N terminus of certain peptides and proteins, catalyzed by glutaminyl cyclases (QCs), is linked to some pathological conditions, such as Alzheimer disease. Recently, a glutaminyl cyclase (QC) inhibitor, PBD150, was shown to be able to reduce the deposition of pyroglutamate-modified amyloid-β peptides in brain of transgenic mouse models of Alzheimer disease, leading to a significant improvement of learning and memory in those transgenic animals. Here, we report the 1.05-1.40 Å resolution structures, solved by the sulfur single-wavelength anomalous dispersion phasing method, of the Golgi-luminal catalytic domain of the recently identified Golgi-resident QC (gQC) and its complex with PBD150. We also describe the high-resolution structures of secretory QC (sQC)-PBD150 complex and two other gQC-inhibitor complexes. gQC structure has a scaffold similar to that of sQC but with a relatively wider and negatively charged active site, suggesting a distinct substrate specificity from sQC. Upon binding to PBD150, a large loop movement in gQC allows the inhibitor to be tightly held in its active site primarily by hydrophobic interactions. Further comparisons of the inhibitor-bound structures revealed distinct interactions of the inhibitors with gQC and sQC, which are consistent with the results from our inhibitor assays reported here. Because gQC and sQC may play different biological roles in vivo, the different inhibitor binding modes allow the design of specific inhibitors toward gQC and sQC.

Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

Science.gov (United States)

Banta, Amy B.; Wei, Jeremy H.; Gill, Clare C. C.; Giner, José-Luis; Welander, Paula V.

2017-01-01

Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

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

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.

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.

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

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

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

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.

New Therapeutic Agent against Arterial Thrombosis: An Iridium(III-Derived Organometallic Compound

Directory of Open Access Journals (Sweden)

Chih-Wei Hsia

2017-12-01

Full Text Available Platelet activation plays a major role in cardio and cerebrovascular diseases, and cancer progression. Disruption of platelet activation represents an attractive therapeutic target for reducing the bidirectional cross talk between platelets and tumor cells. Platinum (Pt compounds have been used for treating cancer. Hence, replacing Pt with iridium (Ir is considered a potential alternative. We recently developed an Ir(III-derived complex, [Ir(Cp*1-(2-pyridyl-3-(2-hydroxyphenylimidazo[1,5-a]pyridine Cl]BF4 (Ir-11, which exhibited strong antiplatelet activity; hence, we assessed the therapeutic potential of Ir-11 against arterial thrombosis. In collagen-activated platelets, Ir-11 inhibited platelet aggregation, adenosine triphosphate (ATP release, intracellular Ca2+ mobilization, P-selectin expression, and OH· formation, as well as the phosphorylation of phospholipase Cγ2 (PLCγ2, protein kinase C (PKC, mitogen-activated protein kinases (MAPKs, and Akt. Neither the adenylate cyclase inhibitor nor the guanylate cyclase inhibitor reversed the Ir-11-mediated antiplatelet effects. In experimental mice, Ir-11 prolonged the bleeding time and reduced mortality associated with acute pulmonary thromboembolism. Ir-11 plays a crucial role by inhibiting platelet activation through the inhibition of the PLCγ2–PKC cascade, and the subsequent suppression of Akt and MAPK activation, ultimately inhibiting platelet aggregation. Therefore, Ir-11 can be considered a new therapeutic agent against either arterial thrombosis or the bidirectional cross talk between platelets and tumor cells.

The cytoplasmic domain close to the transmembrane region of the glucagon-like peptide-1 receptor contains sequence elements that regulate agonist-dependent internalisation.

Science.gov (United States)

Vázquez, Patricia; Roncero, Isabel; Blázquez, Enrique; Alvarez, Elvira

2005-07-01

In order to gain better insight into the molecular events involved in the signal transduction generated through glucagon-like peptide-1 (GLP-1) receptors, we tested the effect of deletions and point mutations within the cytoplasmic tail of this receptor with a view to establishing relationships between signal transduction desensitisation and receptor internalisation. Wild-type and truncated (deletion of the last 27 amino acids (GLPR 435R) and deletion of 44 amino acids (GLPR 418R)) GLP-1 receptors bound the agonist with similar affinity. Deletion of the last 27 amino acids decreased the internalisation rate by 78%, while deletion of 44 amino acids containing all the phosphorylation sites hitherto described in this receptor decreased the internalisation rate by only 47%. Binding of the ligand to both receptors stimulated adenylyl cyclase. In contrast, deletion of the region containing amino acids 419 to 435 (GLPR 419delta435) increased the internalisation rate by 268%, and the replacement of EVQ(408-410) by alanine (GLPR A(408-410)) increased this process to 296%. In both receptors, the efficacy in stimulating adenylate cyclase was decreased. All the receptors studied were internalised by coated pits, except for the receptor with a deletion of the last 44 amino acids, which also had a faster resensitisation rate. Our findings indicate that the neighbouring trans-membrane domain of the carboxyl-terminal tail of the GLP-1 receptor contains sequence elements that regulate agonist-dependent internalisation and transmembrane signalling.

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

Science.gov (United States)

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

2014-01-01

Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3′,5′-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 105 GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces “molecule noise.” Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons. PMID:25135936

PACAP decides neuronal laminar fate via PKA signaling in the developing cerebral cortex

International Nuclear Information System (INIS)

Ohtsuka, Masanari; Fukumitsu, Hidefumi; Furukawa, Shoei

2008-01-01

Laminar formation in the developing cerebral cortex requires the precisely regulated generation of phenotype-specified neurons. To test the possible involvement of pituitary adenylate cyclase-activating polypeptide (PACAP) in this formation, we investigated the effects of PACAP administered into the telencephalic ventricular space of 13.5-day-old mouse embryos. PACAP partially inhibited the proliferation of cortical progenitors and altered the position and gene-expression profiles of newly generated neurons otherwise expected for layer IV to those of neurons for the deeper layers, V and VI, of the cerebral cortex. The former and latter effects were seen only when the parent progenitor cells were exposed to PACAP in the later and in earlier G1 phase, respectively; and these effects were suppressed by co-treatment with a protein kinase A (PKA) inhibitor. These observations suggest that PACAP participates in the processes forming the neuronal laminas in the developing cortex via the intracellular PKA pathway

The potential signalling pathways which regulate surface changes induced by phytohormones in the potato cyst nematode (Globodera rostochiensis).

Science.gov (United States)

Akhkha, A; Curtis, R; Kennedy, M; Kusel, J

2004-05-01

It has been demonstrated that the surface lipophilicity of the plant-parasitic nematode Globodera rostochiensis decreases when infective larvae are exposed to the phytohormones indole-3-acetic acid (auxin) or kinetin (cytokinin). In the present study, it was shown that inhibition of phospholipase C (PLC) or phosphatidylinositol 3 kinase (PI3-kinase) reversed the effect of phytohormones on surface lipophilicity. The signalling pathway(s) involved in surface modification were investigated using 'caged' signalling molecules and stimulators or inhibitors of different signalling enzymes. Photolysis of the 'caged' signalling molecules, NPE-caged Ins 1,4,5-P3, NITR-5/AM or caged-cAMP to liberate IP3, Ca2+ or cAMP respectively, decreased the surface lipophilicity. Activation of adenylate cyclase also decreased the surface lipophilicity. In contrast, inhibition of PI3-kinase using Wortmannin, LY-294002 or Quercetin, and inhibition of PLC using U-73122 all increased the surface lipophilicity. Two possible signalling pathways involved in phytohormone-induced surface modification are proposed.

Dual aminergic regulation of central beta adrenoceptors. Effect of atypical antidepressants and 5-hydroxytryptophan

International Nuclear Information System (INIS)

Manier, D.H.; Gillespie, D.D.; Sulser, F.

1989-01-01

Nonlinear regression analysis of agonist competition binding curves reveals that the [ 3 H]-dihydroalprenolol-labeled receptor population with low affinity for isoproterenol is increased by p-chlorophenylalanine (PCPA) and this increase is abolished by 5-hydroxytryptophan (5-HTP) in vivo. Desipramine (DMI) decreased the beta adrenoceptor population with high agonist affinity to the same degree in PCPA-treated animals as in control animals, thus explaining the reported discrepancy between beta adrenoceptor number and responsiveness of the beta adrenoceptor-coupled adenylate cyclase system. Mianserin also selectively reduced the beta adrenoceptor population with high agonist affinity in membrane preparations of normal animals, whereas fluoxetine selectively abolished the upregulation of the low affinity sites in reserpinized animals and had no effect on either receptor population from brain of normal animals. The results emphasize the importance of nonlinear regression analysis of agonist competition binding for the interpretation of drug action and encourage the pursuit of the molecular neurobiology of the serotonin (5-HT)/norepinephrine (NE) link in brain

Heterogeneous Family of Cyclomodulins: Smart Weapons That Allow Bacteria to Hijack the Eukaryotic Cell Cycle and Promote Infections

Directory of Open Access Journals (Sweden)

Rachid A. El-Aouar Filho

2017-05-01

Full Text Available Some bacterial pathogens modulate signaling pathways of eukaryotic cells in order to subvert the host response for their own benefit, leading to successful colonization and invasion. Pathogenic bacteria produce multiple compounds that generate favorable conditions to their survival and growth during infection in eukaryotic hosts. Many bacterial toxins can alter the cell cycle progression of host cells, impairing essential cellular functions and impeding host cell division. This review summarizes current knowledge regarding cyclomodulins, a heterogeneous family of bacterial effectors that induce eukaryotic cell cycle alterations. We discuss the mechanisms of actions of cyclomodulins according to their biochemical properties, providing examples of various cyclomodulins such as cycle inhibiting factor, γ-glutamyltranspeptidase, cytolethal distending toxins, shiga toxin, subtilase toxin, anthrax toxin, cholera toxin, adenylate cyclase toxins, vacuolating cytotoxin, cytotoxic necrotizing factor, Panton-Valentine leukocidin, phenol soluble modulins, and mycolactone. Special attention is paid to the benefit provided by cyclomodulins to bacteria during colonization of the host.

Dual aminergic regulation of central beta adrenoceptors. Effect of atypical antidepressants and 5-hydroxytryptophan

Energy Technology Data Exchange (ETDEWEB)

Manier, D.H.; Gillespie, D.D.; Sulser, F.

1989-06-01

Nonlinear regression analysis of agonist competition binding curves reveals that the (/sup 3/H)-dihydroalprenolol-labeled receptor population with low affinity for isoproterenol is increased by p-chlorophenylalanine (PCPA) and this increase is abolished by 5-hydroxytryptophan (5-HTP) in vivo. Desipramine (DMI) decreased the beta adrenoceptor population with high agonist affinity to the same degree in PCPA-treated animals as in control animals, thus explaining the reported discrepancy between beta adrenoceptor number and responsiveness of the beta adrenoceptor-coupled adenylate cyclase system. Mianserin also selectively reduced the beta adrenoceptor population with high agonist affinity in membrane preparations of normal animals, whereas fluoxetine selectively abolished the upregulation of the low affinity sites in reserpinized animals and had no effect on either receptor population from brain of normal animals. The results emphasize the importance of nonlinear regression analysis of agonist competition binding for the interpretation of drug action and encourage the pursuit of the molecular neurobiology of the serotonin (5-HT)/norepinephrine (NE) link in brain.

Human cDNA clones for an α subunit of G/sub i/ signal-transduction protein

International Nuclear Information System (INIS)

Bray, P.; Carter, A.; Guo, V.; Puckett, C.; Kamholz, J.; Spiegel, A.; Nirenberg, M.

1987-01-01

Two cDNA clones were obtained from a λgt11 cDNA human brain library that correspond to α/sub i/ subunits of G signal-transduction proteins (where α/sub i/ subunits refer to the α subunits of G proteins that inhibit adenylate cyclase). The nucleotide sequence of human brain α/sub i/ is highly homologous to that of bovine brain α/sub i/ and the predicted amino acid sequences are identical. However, human and bovine brain α/sub i/ cDNAs differ significantly from α/sub i/ cDNAs from human monocytes, rat glioma, and mouse macrophages in amino acid (88% homology) and nucleotide (71-75% homology) sequences. In addition, the nucleotide sequences of the 3' untranslated regions of human and bovine brain α/sub i/ cDNAs differ markedly from the sequences of human monocyte, rat glioma, and mouse macrophage α/sub i/ cDNAs. These results suggest there are at least two classes of α/sub i/ mRNA

Anti-Melanogenic Property of Geoditin A in Murine B16 Melanoma Cells

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Chun-Tao Che

2012-02-01

Full Text Available Geoditin A, an isomalabaricane triterpene isolated from marine sponge Geodia japonica, has been demonstrated to induce apoptosis in leukemia HL60 cells and human colon HT29 cancer cells through an oxidative stress, a process also interfering with normal melanogenesis in pigment cells. Treatment of murine melanoma B16 cells with geoditin A decreased expression of melanogenic proteins and cell melanogenesis which was aggravated with adenylate cyclase inhibitor SQ22536, indicating melanogenic inhibition was mediated through a cAMP-dependent signaling pathway. Immunofluorescence microscopy and glycosylation studies revealed abnormal glycosylation patterns of melanogenic proteins (tyrosinase and tyrosinase-related protein 1, and a co-localization of tyrosinase with calnexin (CNX and lysosome-associated membrane protein 1 (LAMP-1, implicating a post-translational modification in the ER and a degradation of tyrosinase in the lysosome. Taken together, potent anti-melanogenic property and the relatively low cytotoxicity of geoditin A have demonstrated its therapeutic potential as a skin lightening agent.

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.

Measurement of cAMP in an undergraduate teaching laboratory, using ALPHAscreen technology.

Science.gov (United States)

Bartho, Joseph D; Ly, Kien; Hay, Debbie L

2012-02-14

Adenosine 3',5'-monophosphate (cAMP) is a cellular second messenger with central relevance to pharmacology, cell biology, and biochemistry teaching programs. cAMP is produced from adenosine triphosphate by adenylate cyclase, and its production is reduced or enhanced upon activation of many G protein-coupled receptors. Therefore, the measurement of cAMP serves as an indicator of receptor activity. Although there are many assays available for measuring cAMP, few are suitable for large class teaching, and even fewer seem to have been adapted for this purpose. Here, we describe the use of bead-based ALPHAscreen (Amplified Luminescent Proximity Homogenous Assay) technology for teaching a class of more than 300 students the practical aspects of detecting signal transduction. This technology is applicable to the measurement of many different signaling pathways. This resource is designed to provide a practical guide for instructors and a useful model for developing other classes using similar technologies.

Distribution in rat tissues of modulator-binding protein of particulate nature

International Nuclear Information System (INIS)

Sobue, K.; Muramoto, Y.; Kakiuchi, S.; Yamazaki, R.

1979-01-01

Studies on Ca 2+ -activatable cyclic nucleotide phosphodiesterase led to the discovery of a protein modulator that is required for the activation of this enzyme by Ca 2+ . Later, this protein has been shown to cause the Ca 2+ -dependent activation of several enzymes that include phosphodiesterase, adenylate cyclase, a protein kinase from muscles, phosphorylase b kinase, actomyosin ATPase, membranous ATPase from erythrocytes and nerve synapses. Thus, modulator protein appears to be an intracellular mediator of actions of Ca 2+ . The present work shows the distribution of this particulate modulator-binding component in rat tissues. This paper also describes the labeling of modulator protein with tritium without deteriorating its biological activities and application of this 3 H-modulator protein to the determination of the Ca ++ dependent binding of modulator protein with membranous protein. This technique proves to be useful in studying enzymes or proteins whose functions are regulated by Ca ++ /modulator protein system. (Auth.)

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

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

Science.gov (United States)

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

2012-04-01

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

Dural mast cell degranulation is a putative mechanism for headache induced by PACAP-38

DEFF Research Database (Denmark)

Baun, Michael; Pedersen, Martin Holst Friborg; Olesen, Jes

2012-01-01

but not VIP cause degranulation of mast cells in peritoneum and in dura mater. METHODS: The degranulatory effects of PACAP-38, PACAP-27 and VIP were investigated by measuring the amount of N-acetyl-β-hexosaminidase released from isolated peritoneal mast cells and from dura mater attached to the skull...... of the rat in vitro. In peritoneal mast cells N-truncated fragments of PACAP-38 (PACAP(6–38), PACAP(16–38) and PACAP(28–38)) were also studied. To investigate transduction pathways involved in mast cell degranulation induced by PACAP-38, PACAP-27 and VIP, the phospholipase C inhibitor U-73122...... and the adenylate cyclase inhibitor SQ 22536 were used. RESULTS: The peptides induced degranulation of isolated peritoneal mast cells of the rat with the following order of potency: PACAP-38 = PACAP(6–38) = PACAP(16–38) » PACAP-27 = VIP = PACAP(28–38). In the dura mater we found that 10–5 M PACAP-38...

Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

Science.gov (United States)

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

2012-06-01

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

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.

Effects of Catalytic Action and Ligand Binding on Conformational Ensembles of Adenylate Kinase.

Science.gov (United States)

Onuk, Emre; Badger, John; Wang, Yu Jing; Bardhan, Jaydeep; Chishti, Yasmin; Akcakaya, Murat; Brooks, Dana H; Erdogmus, Deniz; Minh, David D L; Makowski, Lee

2017-08-29

Crystal structures of adenylate kinase (AdK) from Escherichia coli capture two states: an "open" conformation (apo) obtained in the absence of ligands and a "closed" conformation in which ligands are bound. Other AdK crystal structures suggest intermediate conformations that may lie on the transition pathway between these two states. To characterize the transition from open to closed states in solution, X-ray solution scattering data were collected from AdK in the apo form and with progressively increasing concentrations of five different ligands. Scattering data from apo AdK are consistent with scattering predicted from the crystal structure of AdK in the open conformation. In contrast, data from AdK samples saturated with Ap5A do not agree with that calculated from AdK in the closed conformation. Using cluster analysis of available structures, we selected representative structures in five conformational states: open, partially open, intermediate, partially closed, and closed. We used these structures to estimate the relative abundances of these states for each experimental condition. X-ray solution scattering data obtained from AdK with AMP are dominated by scattering from AdK in the open conformation. For AdK in the presence of high concentrations of ATP and ADP, the conformational ensemble shifts to a mixture of partially open and closed states. Even when AdK is saturated with Ap5A, a significant proportion of AdK remains in a partially open conformation. These results are consistent with an induced-fit model in which the transition of AdK from an open state to a closed state is initiated by ATP binding.

Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

Science.gov (United States)

Itoh, Takayuki; Tokunaga, Kinya; Matsuda, Yudai; Fujii, Isao; Abe, Ikuro; Ebizuka, Yutaka; Kushiro, Tetsuo

2010-10-01

Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

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

KAUST Repository

Al-Younis, Inas; Wong, Aloysius Tze; Gehring, Christoph A

2015-01-01

KUP7) 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

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

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

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...

Characterization of CYP264B1 and a terpene cyclase of a terpene biosynthesis gene cluster from the myxobacterium Sorangium cellulosum So ce56

OpenAIRE

Ly, Thuy Thi Bich

2011-01-01

In the work presented here, CYP264B1 and the terpene cyclase GeoA of Sorangium cellulosum So ce56 have been characterized. CYP264B1 is able to convert norisoprenoids (a-ionone and b-ionone) and diverse sesquiterpene compounds, including nootkatone. Three products, 3-hydroxy-a-ionone, 3-hydroxy-b-ionone and 13-hydroxy-nootkatone were characterized using HPLC and 1H and 13C NMR. CYP264B1 is the first enzyme reported to be capable to hydroxylate regioselectively both norisoprenoids at the positi...

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.

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

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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.

PTHrP regulates water absorption and aquaporin expression in the intestine of the marine sea bream (Sparus aurata, L.).

Science.gov (United States)

Carvalho, Edison S M; Gregório, Sílvia F; Canário, Adelino V M; Power, Deborah M; Fuentes, Juan

2015-03-01

Water ingestion by drinking is fundamental for ion homeostasis in marine fish. However, the fluid ingested requires processing to allow net water absorption in the intestine. The formation of luminal carbonate aggregates impacts on calcium homeostasis and requires epithelial HCO3(-) secretion to enable water absorption. In light of its endocrine importance in calcium handling and the indication of involvement in HCO3(-) secretion the present study was designed to expose the role of the parathyroid hormone-related protein (PTHrP) in HCO3(-) secretion, water absorption and the regulation of aqp1 gene expression in the anterior intestine of the sea bream. HCO3(-) secretion rapidly decreased when PTHrP(1-34) was added to anterior intestine of the sea bream mounted in Ussing chambers. The effect achieved a maximum inhibition of 60% of basal secretion rates, showing a threshold effective dose of 0.1 ng ml(-1) compatible with reported plasma values of PTHrP. When applied in combination with the adenylate cyclase inhibitor (SQ 22.536, 100 μmol l(-1)) or the phospholipase C inhibitor (U73122, 10 μmol l(-1)) the effect of PTHrP(1-34) on HCO3(-) secretion was reduced by about 50% in both cases. In parallel, bulk water absorption measured in intestinal sacs was sensitive to inhibition by PTHrP. The inhibitory action conforms to a typical dose-response curve in the range of 0.1-1000 ng ml(-1), achieves a maximal effect of 60-65% inhibition from basal rates and shows threshold significant effects at hormone levels of 0.1 ng ml(-1). The action of PTHrP in water absorption was completely abolished in the presence of the adenylate cyclase inhibitor (SQ 22.536, 100 μmol l(-1)) and was insensitive to the phospholipase C inhibitor (U73122, 10 μmol l(-1)). In vivo injections of PTHrP(1-34) or the PTH/PTHrP receptor antagonist PTHrP(7-34) evoked respectively, a significant decrease or increase of aqp1ab, but not aqp1a. Overall the present results suggest that PTHrP acts as a key

A new knock-in mouse model of l-DOPA-responsive dystonia.

Science.gov (United States)

Rose, Samuel J; Yu, Xin Y; Heinzer, Ann K; Harrast, Porter; Fan, Xueliang; Raike, Robert S; Thompson, Valerie B; Pare, Jean-Francois; Weinshenker, David; Smith, Yoland; Jinnah, Hyder A; Hess, Ellen J

2015-10-01

Abnormal dopamine neurotransmission is associated with many different genetic and acquired dystonic disorders. For instance, mutations in genes critical for the synthesis of dopamine, including GCH1 and TH cause l-DOPA-responsive dystonia. Despite evidence that implicates abnormal dopamine neurotransmission in dystonia, the precise nature of the pre- and postsynaptic defects that result in dystonia are not known. To better understand these defects, we generated a knock-in mouse model of l-DOPA-responsive dystonia (DRD) mice that recapitulates the human p.381Q>K TH mutation (c.1141C>A). Mice homozygous for this mutation displayed the core features of the human disorder, including reduced TH activity, dystonia that worsened throughout the course of the active phase, and improvement in the dystonia in response to both l-DOPA and trihexyphenidyl. Although the gross anatomy of the nigrostriatal dopaminergic neurons was normal in DRD mice, the microstructure of striatal synapses was affected whereby the ratio of axo-spinous to axo-dendritic corticostriatal synaptic contacts was reduced. Microinjection of l-DOPA directly into the striatum ameliorated the dystonic movements but cerebellar microinjections of l-DOPA had no effect. Surprisingly, the striatal dopamine concentration was reduced to ∼1% of normal, a concentration more typically associated with akinesia, suggesting that (mal)adaptive postsynaptic responses may also play a role in the development of dystonia. Administration of D1- or D2-like dopamine receptor agonists to enhance dopamine signalling reduced the dystonic movements, whereas administration of D1- or D2-like dopamine receptor antagonists to further reduce dopamine signalling worsened the dystonia, suggesting that both receptors mediate the abnormal movements. Further, D1-dopamine receptors were supersensitive; adenylate cyclase activity, locomotor activity and stereotypy were exaggerated in DRD mice in response to the D1-dopamine receptor agonist SKF

Expression of adenylyl cyclase types III and VI in human hyperfunctioning thyroid nodules.

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Celano, M; Arturi, F; Presta, I; Bruno, R; Scarpelli, D; Calvagno, M G; Cristofaro, C; Bulotta, S; Giannasio, P; Sacco, R; Filetti, S; Russo, D

2003-05-30

Hyperfunctioning thyroid nodules are characterized by the presence of spontaneous somatic mutations responsible for constitutive activation of the cAMP pathway. However, alterations affecting other elements of the cAMP signaling system may counteract the effects of the mutations. In this study, the expression of the adenylyl cyclase (AC) types III and VI was investigated by Western blot in 18 hyperfunctioning thyroid nodules; in 12 samples, we also assessed the presence of TSH receptor (TSHR) or gsp mutations and levels of AC VI and III mRNA. We found that the expression of nodular AC VI (but not AC III) was significantly lower (85.1% of normal, P=0.014) than the expression of both adenylyl cycles types of perinodular tissue from the same patients. Slightly, but not significant differences were detected in nodules with or without mutations and AC protein levels generally showed correlation with the levels of the transcripts detected by RT-PCR. In addition, AC III and AC VI expression levels within a given nodule were characterized by a significant positive correlation. These findings indicate that a diminished expression of AC type VI may be part of the mechanisms occurring in the hyperfunctioning nodules, independently of the presence of TSHR or gsp mutations, which influence the resulting phenotype.

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

Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

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Löw, Hans; Crane, Frederick L; Morré, D James

2012-11-01

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

Role of water in the enzymatic catalysis: study of ATP + AMP → 2ADP conversion by adenylate kinase.

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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.

Complexes of Escherichia coli adenylate kinase and nucleotides: 1H NMR studies of the nucleotide sites in solution

International Nuclear Information System (INIS)

Vetter, I.R.; Reinstein, J.; Roesch, P.

1990-01-01

One- and two-dimensional nuclear magnetic resonance (NMR) studies, in particular substrate-protein nuclear Overhauser effect (NOESY) measurements, as well as nucleotide and P 1 ,P 5 -bis-(5'-adenosyl) pentaphosphate (AP 5 A) titrations and studies of the temperature-dependent unfolding of the tertiary structure of Escherichia coli adenylate kinase (AK EC ) were performed. These experiments and comparison with the same type of experiments performed with the porcine enzyme led them to the following conclusions: (1) at pH 8 and concentrations of approximately 2.5-3 mM, AK EC is partially unfolded at 318 K; (2) ATP·Mg 2+ binds to the ATP site with a dissociation constant of approximately 40 μM under the assumption that ATP binds to one nucleotide site only; (3) AP 5 A·Mg 2+ binds to both nucleotide sites and thus simulates the active complex; (4) the ATP·Mg 2+ adenine in the AK EC ·AP 5 A·Mg 2+ complex is located close to His 134 and Phe 19 ; (5) the AK EC G-loop with bound ATP·Mg 2+ is structurally highly homologous to the loop region in the oncogene product p21 with bound GTP·Mg 2+

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon.

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Natalia López-del Hoyo

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

The adenylate energy charge as a new and useful indicator of capture stress in chondrichthyans.

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Guida, Leonardo; Walker, Terence I; Reina, Richard D

2016-02-01

Quantifying the physiological stress response of chondrichthyans to capture has assisted the development of fishing practices conducive to their survival. However, currently used indicators of stress show significant interspecific and intraspecific variation in species' physiological responses and tolerances to capture. To improve our understanding of chondrichthyan stress physiology and potentially reduce variation when quantifying the stress response, we investigated the use of the adenylate energy charge (AEC); a measure of available metabolic energy. To determine tissues sensitive to metabolic stress, we extracted samples of the brain, heart, liver, white muscle and blood from gummy sharks (Mustelus antarcticus) immediately following gillnet capture and after 3 h recovery under laboratory conditions. Capture caused significant declines in liver, white muscle and blood AEC, whereas no decline was detected in the heart and brain AEC. Following 3 h of recovery from capture, the AEC of the liver and blood returned to "unstressed" levels (control values) whereas white muscle AEC was not significantly different to that immediately after capture. Our results show that the liver is most sensitive to metabolic stress and white muscle offers a practical method to sample animals non-lethally for determination of the AEC. The AEC is a highly informative indicator of stress and unlike current indicators, it can directly measure the change in available energy and thus the metabolic stress experienced by a given tissue. Cellular metabolism is highly conserved across organisms and, therefore, we think the AEC can also provide a standardised form of measuring capture stress in many chondrichthyan species.

Mechanism of MenE inhibition by acyl-adenylate analogues and discovery of novel antibacterial agents.

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Matarlo, Joe S; Evans, Christopher E; Sharma, Indrajeet; Lavaud, Lubens J; Ngo, Stephen C; Shek, Roger; Rajashankar, Kanagalaghatta R; French, Jarrod B; Tan, Derek S; Tonge, Peter J

2015-10-27

MenE is an o-succinylbenzoyl-CoA (OSB-CoA) synthetase in the bacterial menaquinone biosynthesis pathway and is a promising target for the development of novel antibacterial agents. The enzyme catalyzes CoA ligation via an acyl-adenylate intermediate, and we have previously reported tight-binding inhibitors of MenE based on stable acyl-sulfonyladenosine analogues of this intermediate, including OSB-AMS (1), which has an IC50 value of ≤25 nM for Escherichia coli MenE. Herein, we show that OSB-AMS reduces menaquinone levels in Staphylococcus aureus, consistent with its proposed mechanism of action, despite the observation that the antibacterial activity of OSB-AMS is ∼1000-fold lower than the IC50 for enzyme inhibition. To inform the synthesis of MenE inhibitors with improved antibacterial activity, we have undertaken a structure-activity relationship (SAR) study stimulated by the knowledge that OSB-AMS can adopt two isomeric forms in which the OSB side chain exists either as an open-chain keto acid or a cyclic lactol. These studies revealed that negatively charged analogues of the keto acid form bind, while neutral analogues do not, consistent with the hypothesis that the negatively charged keto acid form of OSB-AMS is the active isomer. X-ray crystallography and site-directed mutagenesis confirm the importance of a conserved arginine for binding the OSB carboxylate. Although most lactol isomers tested were inactive, a novel difluoroindanediol inhibitor (11) with improved antibacterial activity was discovered, providing a pathway toward the development of optimized MenE inhibitors in the future.

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

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

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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.

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

Microglia PACAP and glutamate: Friends or foes in seizure-induced autonomic dysfunction and SUDEP?

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Bhandare, Amol M; Kapoor, Komal; Farnham, Melissa M J; Pilowsky, Paul M

2016-06-01

Seizure-induced cardiorespiratory autonomic dysfunction is a major cause of sudden unexpected death in epilepsy (SUDEP), and the underlying mechanism is unclear. Seizures lead to increased synthesis, and release of glutamate, pituitary adenylate cyclase activating polypeptide (PACAP), and other neurotransmitters, and cause extensive activation of microglia at multiple regions in the brain including central autonomic cardiorespiratory brainstem nuclei. Glutamate contributes to neurodegeneration, and inflammation in epilepsy. PACAP has neuroprotective, and anti-inflammatory properties, whereas microglia are key players in inflammatory responses in CNS. Seizure-induced increase in PACAP is neuroprotective. PACAP produces neuroprotective effects acting on microglial PAC1 and VPAC1 receptors. Microglia also express glutamate transporters, and their expression can be increased by PACAP in response to harmful or stressful situations such as seizures. Here we discuss the mechanism of autonomic cardiorespiratory dysfunction in seizure, and the role of PACAP, glutamate and microglia in regulating cardiorespiratory brainstem neurons in their physiological state that could provide future therapeutic options for SUDEP. Copyright © 2016 Elsevier B.V. All rights reserved.

Central Pathways Integrating Metabolism and Reproduction in Teleosts

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Shahjahan, Md.; Kitahashi, Takashi; Parhar, Ishwar S.

2014-01-01

Energy balance plays an important role in the control of reproduction. However, the cellular and molecular mechanisms connecting the two systems are not well understood especially in teleosts. The hypothalamus plays a crucial role in the regulation of both energy balance and reproduction, and contains a number of neuropeptides, including gonadotropin-releasing hormone (GnRH), orexin, neuropeptide-Y, ghrelin, pituitary adenylate cyclase-activating polypeptide, α-melanocyte stimulating hormone, melanin-concentrating hormone, cholecystokinin, 26RFamide, nesfatin, kisspeptin, and gonadotropin-inhibitory hormone. These neuropeptides are involved in the control of energy balance and reproduction either directly or indirectly. On the other hand, synthesis and release of these hypothalamic neuropeptides are regulated by metabolic signals from the gut and the adipose tissue. Furthermore, neurons producing these neuropeptides interact with each other, providing neuronal basis of the link between energy balance and reproduction. This review summarizes the advances made in our understanding of the physiological roles of the hypothalamic neuropeptides in energy balance and reproduction in teleosts, and discusses how they interact with GnRH, kisspeptin, and pituitary gonadotropins to control reproduction in teleosts. PMID:24723910

Activation of β-adrenergic receptors is required for elevated α1A-adrenoreceptors expression and signaling in mesenchymal stromal cells

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Tyurin-Kuzmin, Pyotr A.; Fadeeva, Julia I.; Kanareikina, Margarita A.; Kalinina, Natalia I.; Sysoeva, Veronika Yu.; Dyikanov, Daniyar T.; Stambolsky, Dmitriy V.; Tkachuk, Vsevolod A.

2016-01-01

Sympathetic neurons are important components of mesenchymal stem cells (MSCs) niche and noradrenaline regulates biological activities of these cells. Here we examined the mechanisms of regulation of MSCs responsiveness to noradrenaline. Using flow cytometry, we demonstrated that α1A adrenergic receptors isoform was the most abundant in adipose tissue-derived MSCs. Using calcium imaging in single cells, we demonstrated that only 6.9 ± 0.8% of MSCs responded to noradrenaline by intracellular calcium release. Noradrenaline increases MSCs sensitivity to catecholamines in a transitory mode. Within 6 hrs after incubation with noradrenaline the proportion of cells responding by Ca2+ release to the fresh noradrenaline addition has doubled but declined to the baseline after 24 hrs. Increased sensitivity was due to the elevated quantities of α1A-adrenergic receptors on MSCs. Such elevation depended on the stimulation of β-adrenergic receptors and adenylate cyclase activation. The data for the first time clarify mechanisms of regulation of MSCs sensitivity to noradrenaline. PMID:27596381

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

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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.

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

International Nuclear Information System (INIS)

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

1988-01-01

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