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.

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.

Pituitary adenylate cyclase activating polypeptide and migraine

DEFF Research Database (Denmark)

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

2014-01-01

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

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

Science.gov (United States)

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

1978-06-01

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

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

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.

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

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

Science.gov (United States)

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

2017-01-01

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

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

International Nuclear Information System (INIS)

Borgundvaag, B.; George, S.R.

1985-01-01

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

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

International Nuclear Information System (INIS)

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

1987-01-01

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

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.

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

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Sarah J. Casey

2014-03-01

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

Adenylate cyclase toxin-hemolysin relevance for pertussis vaccines

Czech Academy of Sciences Publication Activity Database

Šebo, Peter; Osička, Radim; Mašín, Jiří

2014-01-01

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

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

International Nuclear Information System (INIS)

Niles, L.P.; Hashemi, F.

1990-01-01

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [ 125 I]iodomelatonin, was examined using an incubation temperature (30 degree C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [ 125 I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus

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

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

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.

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

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

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

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

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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.

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

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

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.

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.

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.

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...

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1986-05-01

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

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

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

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

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.

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

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 ...

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

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.

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

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.

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

Directory of Open Access Journals (Sweden)

Giorgio Fedele

2017-09-01

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

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.

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.

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

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

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.

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.

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

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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.

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

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

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.

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

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

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 -

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

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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.

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.

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

Science.gov (United States)

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

2013-03-06

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

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.

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.

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

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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

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

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.

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

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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

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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.

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

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

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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

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

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

Science.gov (United States)

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.

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

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)

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.

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

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

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

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

Directory of Open Access Journals (Sweden)

Mounira Tlili

2015-01-01

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

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

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.

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...

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

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.

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.

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.

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.

Science.gov (United States)

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

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

Science.gov (United States)

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.

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

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

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.

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

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

Science.gov (United States)

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

2010-01-01

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

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

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.)

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.

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.

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.

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.

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.

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

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

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

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

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...

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

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

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.

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

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

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

Science.gov (United States)

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

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.

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.

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

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

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.

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

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

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

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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.

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...

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

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

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...

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

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...

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

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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.

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

Science.gov (United States)

Roa, Jinae N; Tresguerres, Martin

2016-08-01

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

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

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.

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.

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.

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

Science.gov (United States)

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

2010-11-01

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

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.

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

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

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

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

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

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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.

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...

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...

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.

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

Science.gov (United States)

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.

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...

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

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....

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

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

[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.

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

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.

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

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.

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

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.

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

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.

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.

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

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

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.

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

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...

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.

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

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)

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)

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.

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

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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.

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.

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.

Functional somatostatin receptors on a rat pancreatic acinar cell line

International Nuclear Information System (INIS)

Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.; Clerc, P.; Logsdon, C.; Svoboda, M.; Susini, C.; Vaysse, N.; Ribet, A.

1988-01-01

Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125 I-[Tyr 11 ]Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 ± 20 fmol/10 6 cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125 I-[Tyr 11 ]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N i to inhibit adenylate cyclase

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.

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.

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 ...

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

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.

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

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,.

Molecular Mechanisms Elicited by d-Aspartate in Leydig Cells and Spermatogonia

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Maria Maddalena Di Fiore

2016-07-01

Full Text Available A bulk of evidence suggests that d-aspartate (d-Asp regulates steroidogenesis and spermatogenesis in vertebrate testes. This review article focuses on intracellular signaling mechanisms elicited by d-Asp possibly via binding to the N-methyl-d-aspartate receptor (NMDAR in both Leydig cells, and spermatogonia. In Leydig cells, the amino acid upregulates androgen production by eliciting the adenylate cyclase-cAMP and/or mitogen-activated protein kinase (MAPK pathways. d-Asp treatment enhances gene and protein expression of enzymes involved in the steroidogenic cascade. d-Asp also directly affects spermatogonial mitotic activity. In spermatogonial GC-1 cells, d-Asp induces phosphorylation of MAPK and AKT serine-threonine kinase proteins, and stimulates expression of proliferating cell nuclear antigen (PCNA and aurora kinase B (AURKB. Further stimulation of spermatogonial GC-1 cell proliferation might come from estradiol/estrogen receptor β (ESR2 interaction. d-Asp modulates androgen and estrogen levels as well as the expression of their receptors in the rat epididymis by acting on mRNA levels of Srd5a1 and Cyp19a1 enzymes, hence suggesting involvement in spermatozoa maturation.

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

Science.gov (United States)

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

2017-08-31

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

The M1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

International Nuclear Information System (INIS)

Mei, Lin.

1989-01-01

The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated [ 3 H]IP 1 accumulation in the SH-SY5Y cells was decreased in the presence of 1μg/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M 1 mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m 1 gene. The transfected B82 cells (cTB10) showed specific [ 3 H](-)QNB binding activity. The mAChRs in these cells are of the M 1 type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M 1 mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M 1 mAChR densities in these cells characterized by [ 3 H](-)MQNB binding ranged from 12 fmol/10 6 cells in LK3-1 cells to 260 fmol/10 6 cells in the LK3-8 cells

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.

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

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

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.

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

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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.

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

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

Induction of autocrine factor inhibiting cell motility from murine B16-BL6 melanoma cells by alpha-melanocyte stimulating hormone.

Science.gov (United States)

Murata, J; Ayukawa, K; Ogasawara, M; Watanabe, H; Saiki, I

1999-03-15

We have previously reported that neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH) successfully inhibited Matrigel invasion and haptotactic migration of B16-BL6 melanoma cells towards both fibronectin and laminin without affecting their growth. In the present study, we investigated the inhibitory mechanism of tumor cell motility by alpha-MSH. Alpha-MSH significantly blocked the autocrine motility factor (AMF)-enhanced cell motility. However, alpha-MSH did neither prevent the secretion of AMF from B16-BL6 cells nor alter the expression level of AMF receptor (gp78). On the other hand, alpha-MSH induced the secretion of the motility inhibitory factor(s) from B16-BL6 cells in a concentration- and time-dependent manner. The induction of the motility inhibitor(s) was proportional to increasing levels of intracellular cAMP induced by alpha-MSH as well as forskolin, and the activity was abolished by an adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (DDA). The motility-inhibiting activity in conditioned medium (CM) from alpha-MSH-treated B16-BL6 cells was found to have a m.w. below 3 kDa after fractionation. This activity was abolished by boiling but insensitive to trypsin. The treatment of tumor cells with cycloheximide reduced the activity in alpha-MSH-stimulated CM. Our results suggest that alpha-MSH inhibited the motility of B16-BL6 cells through induction of autocrine factor(s).

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

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.

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

Science.gov (United States)

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

Neuronal localization of pituitary adenylate cyclase-activating polypeptide 38 in the adrenal medulla and growth-inhibitory effect on chromaffin cells

DEFF Research Database (Denmark)

Frödin, M; Hannibal, J; Wulff, B S

1995-01-01

medulla showed PACAP38 immunoreactivity in a widely distributed network of delicate nerve fibers surrounding the chromaffin cells. In a primary culture system, PACAP38 inhibited growth factor-stimulated DNA synthesis by 90% in neonatal and adult rat chromaffin cells with half-maximal inhibition at 4 and 0.......5 nM, respectively, as demonstrated by bromodeoxyuridine pulse-labeling and immunocytochemical staining of cell nuclei. In comparison, corticosterone inhibited neonatal and adult chromaffin cell proliferation by 70% and 95%, respectively, with half-maximal effect at 100 nM. In neonatal chromaffin...

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

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

International Nuclear Information System (INIS)

Mukai, Atsushi; Hashimoto, Naohiro

2008-01-01

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

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

Directory of Open Access Journals (Sweden)

Chen Yun-Ru

2012-09-01

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

Effect of beta-carotene-rich tomato lycopene beta-cyclase ( tlcy-b) on cell growth inhibition in HT-29 colon adenocarcinoma cells.

Science.gov (United States)

Palozza, Paola; Bellovino, Diana; Simone, Rossella; Boninsegna, Alma; Cellini, Francesco; Monastra, Giovanni; Gaetani, Sancia

2009-07-01

Lycopene beta-cyclase (tlcy-b) tomatoes, obtained by modulating carotenogenesis via genetic engineering, contain a large amount of beta-carotene, as clearly visible by their intense orange colour. In the present study we have subjected tlcy-b tomatoes to an in vitro simulated digestion and analysed the effects of digestate on cell proliferation. To this aim we used HT-29 human colon adenocarcinoma cells, grown in monolayers, as a model. Digested tomatoes were diluted (20 ml, 50 ml and 100 ml/l) in culture medium and added to the cells for different incubation times (24 h, 48 h and 72 h). Inhibition of cell growth by tomato digestate was dose-dependent and resulted from an arrest of cell cycle progression at the G0/G1 and G2/M phase and by apoptosis induction. A down-regulation of cyclin D1, Bcl-2 and Bcl-xl expression was observed. We also found that heat treatment of samples before digestion enhanced beta-carotene release and therefore cell growth inhibition. To induce with purified beta-carotene solubilised in tetrahydrofuran the same cell growth inhibition obtained with the tomato digestate, a higher amount of the carotenoid was necessary, suggesting that beta-carotene micellarised during digestion is utilised more efficiently by the cells, but also that other tomato molecules, reasonably made available during digestion, may be present and cooperate with beta-carotene in promoting cell growth arrest.

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

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)....

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.

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...

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)

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.

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

Adenylyl cyclase-associated protein 1 in metastasis of squamous cell carcinoma of the head and neck and non-small cell lung cancer

Science.gov (United States)

Kakurina, G. V.; Kolegova, E. S.; Cheremisina, O. V.; Zavyalov, A. A.; Shishkin, D. A.; Kondakova, I. V.; Choinzonov, E. L.

2016-08-01

Progression of tumors and metastasis in particular is one of the main reasons of the high mortality rate among cancer patients. The primary role in developing metastases plays cell locomotion which requires remodeling of the actin cytoskeleton. Form, dynamics, localization and mechanical properties of the actin cytoskeleton are regulated by a variety of actin-binding proteins, which include the adenylyl cyclase-associated protein 1 (CAP1). The study is devoted to the investigation of CAP1 level depending on the presence or absence of metastases in patients with squamous cell carcinoma of the head and neck (SCCHN) and non-small cell lung cancer (NSCLC). The results show the contribution of CAP1 to SCCHN and NSCLC progression. We detected the connection between the tissue protein CAP1 level and the stage of NSCLC and SCCHN disease. Also the levels of the CAP1 protein in tissues of primary tumors and metastases in lung cancer were different. Our data showed that CAP is important in the development of metastases, which suggests further perspectives in the study of this protein for projecting metastasis of NSCLC and SCCHN.

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

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...

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

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

Directory of Open Access Journals (Sweden)

Sugimoto Naotoshi

2016-01-01

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

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

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

Science.gov (United States)

Lauzeral, J; Halloy, J; Goldbeter, A

1997-08-19

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 observed during the hours that follow starvation. These changes bring the cells successively from a nonexcitable state to an excitable state in which they relay suprathreshold cAMP pulses, and then to autonomous oscillations of cAMP, before the system returns to an excitable state. By analyzing a model for cAMP signaling based on receptor desensitization, we show that the desynchronization of cells on this developmental path triggers the formation of fully developed spirals of cAMP. Developmental paths that do not correspond to the sequence of dynamic transitions no relay-relay-oscillations-relay are less able or fail to give rise to the formation of spirals.

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...

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...

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.

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

Directory of Open Access Journals (Sweden)

Ravikrishna Ramanujam

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

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.

Influence of CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase release of human gingival fibroblasts and oral keratinocytes.

Science.gov (United States)

Pabst, A M; Walter, C; Grassmann, L; Weyhrauch, M; Brüllmann, D D; Ziebart, T; Scheller, H; Lehmann, K M

2014-05-01

The aim of this study was to analyze the influence of four CAD/CAM all-ceramic materials on cell viability, migration ability and adenylate kinase (ADK) release of human gingival fibroblasts (HGF) and oral keratinocytes (HOK). HGF and HOK were cultured on disc-shaped CAD/CAM all-ceramic materials (e.max CAD LT, e.max CAD HT, Empress CAD and Mark II) and on discs made of tissue culture polystyrene surface (TCPS) serving as control. Cell viability was analyzed by using an MTT assay, and migration ability was investigated by a scratch assay. A ToxiLight assay has been performed to analyze the effect of all-ceramic materials on ADK release and cell apoptosis. At MTT assay for HGF, no significant decrease of cell viability could be detected at all points of measurement (p each > 0.05), while HOK demonstrated a significant decrease in cell viability especially on Empress CAD and Mark II at each point of measurement (p each materials at all points of measurement (between -36 % and -71 %; p each ceramic materials could be investigated. This study disclosed significant differences in cell viability and migration ability of HGF and HOK on CAD/CAM all-ceramic materials. CAD/CAM all-ceramic materials can influence oral cell lines responsible for soft tissue creation which may affect the esthetic outcome.

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.

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...

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.

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.

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

Science.gov (United States)

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

2015-09-08

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

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...

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

Directory of Open Access Journals (Sweden)

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.

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

Science.gov (United States)

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

2017-02-01

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

G/sub o/ protein of fat cells: role in hormonal regulation of agonist-stimulated phosphatidyl inositol breakdown

International Nuclear Information System (INIS)

Rapiejko, P.J.; Northup, J.K.; Malbon, C.C.

1986-01-01

Incubating rat fat cell membranes in the presence of [ 32 P]NAD + and pertussis toxin (PT) results in the ADP-ribosylation of two peptides (M/sub r/ = 41,000 and 40,000). The 41,000-M/sub r/ peptide is the inhibitory G-protein of adenylate cyclase (G/sub i/). The 40,000-M/sub r/ peptide radiolabeled in the presence of [ 32 P]NAD + and PT has been purified from rabbit heart and bovine brain, but has not been identified uniformly in membranes of fat cells. Two rabbit polyclonal antisera raised against the alpha-subunit of bovine brain G/sub o/ were used to probe the nature of the 40,000-M/sub r/ peptide in rat fat cell membranes that had been separated by gel electrophoresis in the presence of sodium dodecyl sulfate and transferred electrophoretically to nitrocellulose. Both antisera specific for the alpha-subunit of G/sub o/ recognized the M/sub r/ = 40,000 peptide of fat cells that is ADP-ribosylated in the presence of PT. PT treatment of rat fat cells blocks epinephrine-stimulated inositol 1,4,5 trisphosphate (IP 3 ) generation. The inhibition of IP 3 generation by PT suggests a role for either G/sub i/ or G/sub o/ in receptor-mediated phosphatidyl inositol breakdown in the rat fat cell

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

Science.gov (United States)

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

2017-10-01

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

[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.

Cell-Penetrating Ability of Peptide Hormones: Key Role of Glycosaminoglycans Clustering

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Armelle Tchoumi Neree

2015-11-01

Full Text Available Over the last two decades, the potential usage of cell-penetrating peptides (CPPs for the intracellular delivery of various molecules has prompted the identification of novel peptidic identities. However, cytotoxic effects and unpredicted immunological responses have often limited the use of various CPP sequences in the clinic. To overcome these issues, the usage of endogenous peptides appears as an appropriate alternative approach. The hormone pituitary adenylate-cyclase-activating polypeptide (PACAP38 has been recently identified as a novel and very efficient CPP. This 38-residue polycationic peptide is a member of the secretin/glucagon/growth hormone-releasing hormone (GHRH superfamily, with which PACAP38 shares high structural and conformational homologies. In this study, we evaluated the cell-penetrating ability of cationic peptide hormones in the context of the expression of cell surface glycosaminoglycans (GAGs. Our results indicated that among all peptides evaluated, PACAP38 was unique for its potent efficiency of cellular uptake. Interestingly, the abilities of the peptides to reach the intracellular space did not correlate with their binding affinities to sulfated GAGs, but rather to their capacity to clustered heparin in vitro. This study demonstrates that the uptake efficiency of a given cationic CPP does not necessarily correlate with its affinity to sulfated GAGs and that its ability to cluster GAGs should be considered for the identification of novel peptidic sequences with potent cellular penetrating properties.

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.

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

Czech Academy of Sciences Publication Activity Database

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

2017-01-01

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

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

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 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

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

Science.gov (United States)

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 observed during the hours that follow starvation. These changes bring the cells successively from a nonexcitable state to an excitable state in which they relay suprathreshold cAMP pulses, and then to autonomous oscillations of cAMP, before the system returns to an excitable state. By analyzing a model for cAMP signaling based on receptor desensitization, we show that the desynchronization of cells on this developmental path triggers the formation of fully developed spirals of cAMP. Developmental paths that do not correspond to the sequence of dynamic transitions no relay-relay-oscillations-relay are less able or fail to give rise to the formation of spirals. PMID:9256451

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

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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.

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

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

Substantial gaps in knowledge of Bordetella pertussis antibody and T cell epitopes relevant for natural immunity and vaccine efficacy

Science.gov (United States)

Vaughan, Kerrie; Seymour, Emily; Peters, Bjoern; Sette, Alessandro

2016-01-01

The recent increase in whooping cough in vaccinated populations has been attributed to waning immunity associated with the acellular vaccine. The Immune Epitope Database (IEDB) is a repository of immune epitope data from the published literature and includes T cell and antibody epitopes for human pathogens. The IEDB conducted a review of the epitope literature, which revealed 300 Bordetella pertussis-related epitopes from 39 references. Epitope data are currently available for six virulence factors of B. pertussis: pertussis toxin, pertactin, fimbrial 2, fimbrial 3, adenylate cyclase and filamentous hemagglutinin. The majority of epitopes were defined for antibody reactivity; fewer T cell determinants were reported. Analysis of available protective correlates data revealed a number of candidate epitopes; however few are defined in humans and few have been shown to be protective. Moreover, there are a limited number of studies defining epitopes from natural infection versus whole cell or acellular/subunit vaccines. The relationship between epitope location and structural features, as well as antigenic drift (SNP analysis) was also investigated. We conclude that the cumulative data is yet insufficient to address many fundamental questions related to vaccine failure and this underscores the need for further investigation of B. pertussis immunity at the molecular level. PMID:24530743

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.

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...

Identification of 5'-adenylylimidodiphosphate-hydrolyzing enzyme activity in rabbit taste bud cells using X-ray microanalysis

International Nuclear Information System (INIS)

Asanuma, N.

1990-01-01

X-ray microanalysis has been used to characterize the enzyme activity hydrolyzing the ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) in taste bud cells. Rabbit foliate papillae fixed with paraformaldehyde and glutaraldehyde were incubated cytochemically with AMP-PNP as the substrate and lead ion as capture agent. The reaction product which appeared on the microvilli of taste bud cells was examined using an energy dispersive X-ray microanalyzer connected to an analytical electron microscope. The X-ray spectrum thus obtained was compared with that obtained from the product obtained from the demonstration of ATPase activity. Comparison of the phosphorus/lead ratios in the two products showed that twice as much phosphorus was released from an AMP-PNP molecule by the activity in question compared with that released from an ATP molecule by ATPase activity. This indicates that the enzyme hydrolyzes AMP-PNP into AMP and imidodiphosphate and that the enzyme is adenylate cyclase or ATP pyrophosphohydrolase, which possesses a similar hydrolytic property, but not ATPase or alkaline phosphatase, which hydrolyzes AMP-PNP into ADP-NH2 and orthophosphate. This paper provides an example of the use of X-ray microanalysis as a tool for enzyme distinction. The method is applicable to a variety of enzymes and tissues

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.

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....

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

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

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

Directory of Open Access Journals (Sweden)

Bruno P. Carreira

2012-02-01

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

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.

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.

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.

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

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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.

Purine-Metabolizing Ectoenzymes Control IL-8 Production in Human Colon HT-29 Cells

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Fariborz Bahrami

2014-01-01

Full Text Available Interleukin-8 (IL-8 plays key roles in both chronic inflammatory diseases and tumor modulation. We previously observed that IL-8 secretion and function can be modulated by nucleotide (P2 receptors. Here we investigated whether IL-8 release by intestinal epithelial HT-29 cells, a cancer cell line, is modulated by extracellular nucleotide metabolism. We first identified that HT-29 cells regulated adenosine and adenine nucleotide concentration at their surface by the expression of the ectoenzymes NTPDase2, ecto-5′-nucleotidase, and adenylate kinase. The expression of the ectoenzymes was evaluated by RT-PCR, qPCR, and immunoblotting, and their activity was analyzed by RP-HPLC of the products and by detection of Pi produced from the hydrolysis of ATP, ADP, and AMP. In response to poly (I:C, with or without ATP and/or ADP, HT-29 cells released IL-8 and this secretion was modulated by the presence of NTPDase2 and adenylate kinase. Taken together, these results demonstrate the presence of 3 ectoenzymes at the surface of HT-29 cells that control nucleotide levels and adenosine production (NTPDase2, ecto-5′-nucleotidase and adenylate kinase and that P2 receptor-mediated signaling controls IL-8 release in HT-29 cells which is modulated by the presence of NTPDase2 and adenylate kinase.

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

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.

Characterization of muscarinic receptor subtypes in primary cultures of cerebellar granule cells using specific muscarinic receptor antagonists

International Nuclear Information System (INIS)

McLeskey, S.W.

1989-01-01

In cerebellar granule cell cultures, two muscarinic receptor mediated responses were observed: inhibition of adenylate cyclase (M-AC) and stimulation of phosphoinositide hydrolysis (M-PI). These responses were antagonized by three purported specific muscarinic antagonists: pirenzipine and (-)QNX (specific for M-PI) and methoctramine (specific for M-AC). However, the specificity for the three antagonists in blocking these responses is not comparable to the specificity observed in binding studies on these cells or to that quoted in the literature. Two peaks of molecular sizes were found in these cells corresponding to the two molecular sizes of muscarinic receptive proteins reported in the literature. Muscarinic receptive proteins were alkylated with 3 H-propylbenzilylcholine mustard followed by sodium dodecylsulfate polyacrylamide gel electrophoresis. Pirenzipine and (-)QNX were able to block alkylation of the high molecular size peak, which corresponds to the receptive protein m 3 reported in the literature. Methoctramine was able to block alkylation of a portion of the lower molecular size peak, possibly corresponding to the m 2 and/or m 4 receptive proteins reported in the literature. Studies attempting to show the presence of receptor reserve for either of the two biochemical responses present in these cells by alkylation of the receptive protein with nonradiolabeled propylbenzilylcholine mustard (PBCM) were confounded by specificity of this agent for the lower molecular weight peak of muscarinic receptive protein. Thus the muscarinic receptive proteins coupled to M-AC were alkylated preferentially over the ones coupled to M-PI

Human fat cell alpha-2 adrenoceptors. I. Functional exploration and pharmacological definition with selected alpha-2 agonists and antagonists

International Nuclear Information System (INIS)

Galitzky, J.; Mauriege, P.; Berlan, M.; Lafontan, M.

1989-01-01

This study was undertaken to investigate more fully the pharmacological characteristics of the human fat cell alpha-2 adrenoceptor. Biological assays were performed on intact isolated fat cells while radioligand binding studies were carried out with [ 3 H]yohimbine in membranes. These pharmacological studies brought: (1) a critical definition of the limits of the experimental conditions required for the exploration of alpha-2 adrenergic responsiveness on human fat cells and membranes; (2) an improvement in the pharmacological definition of the human fat cell postsynaptic alpha-2 adrenoceptor. Among alpha-2 agonists, UK-14,304 was the most potent and the relative order of potency was: UK-14,304 greater than p-aminoclonidine greater than clonidine = B-HT 920 greater than rilmenidine. For alpha-2 antagonists, the potency order was: yohimbine greater than idazoxan greater than SK ampersand F-86,466 much greater than benextramine; (3) a description of the impact of benextramine (irreversible alpha-1/alpha-2 antagonist) on human fat cell alpha-2 adrenergic receptors and on human fat cell function; the drug inactivates the alpha-2 adrenergic receptors with a minor impact on beta adrenergic receptors and without noticeable alterations of fat cell function as assessed by preservation of beta adrenergic and Al-adenosine receptor-mediated lipolytic responses; and (4) a definition of the relationship existing between alpha-2 adrenergic receptor occupancy, inhibition of adenylate cyclase activity and antilipolysis with full and partial agonists. The existence of a receptor reserve must be taken into account when evaluating alpha-2 adrenergic receptor distribution and regulation of human fat cells

Guanine nucleotide regulation of α1-adrenergic receptors of muscle and kidney eptihelial cells

International Nuclear Information System (INIS)

Terman, B.I.; Hughes, R.J.; Slivka, S.R.; Insel, P.A.

1986-01-01

The authors have examined the effect of guanine nucleotides on the interaction of adrenergic agents with α 1 -adrenergic receptors of two cell lines, the Madin-Darby Canine Kidney (MDCK) and BC3H-1 muscle cells. While gaunylylimidodiphosphoate (Gpp(NH)p) had no effect on the affinity or the total number of [ -3 H]prazosin binding sites in membranes prepared from these cells, the nucleotide decreased the apparent affinity of the agonist epinephrine in competing for [ 3 H]prazosin binding sites in both cell types. The EC 50 of Gpp(NH)p was ∼100 μM, and a maximal effect was seen at 500 μM. In contrast, 100 μM Gpp(NH)p yielding maximal shifts in binding of epinephrine to β-adrenergic receptors in BC3H-1 cell membranes. Guanine nucleotides were significantly more effective than adenine nucleotides in shifting agonist affinity for the α 1 -receptor and Mg ++ was required to observe a maximal effect. α 1 -receptor agonists activated phosphatidylinositol (PI) hydrolysis in both cell types, but have no direct effect on membrane adenylate cyclase activity. In intact BC3H-1 cells, α 1 -agonists inhibited β-adrenergic cAMP production, an effect which appears in preliminary studies not to result from enhanced phosphodieterase activity. These results show that agonist binding to α 1 -adrenergic receptors in mammalian kidney and muscle cells is regulated by guanine nucleotides. This regulation and inturn transmembrane signalling (PI hydrolysis) by these receptors appear to involve a guanine nucleotide binding (G) protein, which may be different than G/sub s/ and G/sub i/

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

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

Science.gov (United States)

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

1997-01-01

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

Glutamatergic neurotransmission from melanopsin retinal ganglion cells is required for neonatal photoaversion but not adult pupillary light reflex.

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Anton Delwig

Full Text Available Melanopsin-expressing retinal ganglion cells (mRGCs in the eye play an important role in many light-activated non-image-forming functions including neonatal photoaversion and the adult pupillary light reflex (PLR. MRGCs rely on glutamate and possibly PACAP (pituitary adenylate cyclase-activating polypeptide to relay visual signals to the brain. However, the role of these neurotransmitters for individual non-image-forming responses remains poorly understood. To clarify the role of glutamatergic signaling from mRGCs in neonatal aversion to light and in adult PLR, we conditionally deleted vesicular glutamate transporter (VGLUT2 selectively from mRGCs in mice. We found that deletion of VGLUT2 in mRGCs abolished negative phototaxis and light-induced distress vocalizations in neonatal mice, underscoring a necessary role for glutamatergic signaling. In adult mice, loss of VGLUT2 in mRGCs resulted in a slow and an incomplete PLR. We conclude that glutamatergic neurotransmission from mRGCs is required for neonatal photoaversion but is complemented by another non-glutamatergic signaling mechanism for the pupillary light reflex in adult mice. We speculate that this complementary signaling might be due to PACAP neurotransmission from mRGCs.

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

Science.gov (United States)

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

2016-12-15

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

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

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

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.

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

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

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.

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

Directory of Open Access Journals (Sweden)

Grazia eMaugeri

2016-05-01

Full Text Available 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 is linked to epidermal growth factor receptor (EGFR overexpression. Previous studies have shown that VIP interferes with the invasive nature of gliomas by regulating cell migration. However, the role of VIP family members in GBM infiltration under low oxygen tension has not been clarified yet. Therefore, in the present study we have investigated, for the first time, the molecular mechanisms involved in the anti-invasive effect of PACAP or VIP in U87MG glioblastoma cells exposed to hypoxia induced by treatment with desferrioxamine (DFX. The results suggest that either PACAP or VIP exert an anti-infiltrative effect under low oxygen tension by modulating HIFs and EGFR expression, key elements involved in cell migration and angiogenesis. These peptides act through the inhibition of PI3K/Akt and MAPK/ERK signaling pathways, which are known to have a crucial role in HIFs regulation. In conclusion, the modulation of hypoxic event and the anti-invasive effect exerted by some VIP family members might open new insights in the therapeutic approach to GBM.

The effect of prefreeze rejuvenation on postthaw storage of red blood cells in AS-3 and SAGM.

Science.gov (United States)

Lelkens, Charles C M; Lagerberg, Johan W M; de Korte, Dirk

2017-06-01

We investigated whether improving the metabolic status of red blood cell concentrates before freezing could extend the postthaw shelf life beyond 14 days while still meeting the requirements for hemolysis (0.8%) and total adenylate (>82% of original values). At Day 8 after collection, four leukoreduced red blood cell concentrates in saline-adenine-glucose-mannitol (SAGM) were pooled, mixed, and split (n = 4). Of these concentrates, two were rejuvenated in Rejuvesol. In addition, two leukoreduced red blood cell concentrates in phosphate-adenine-glucose-guanosine-gluconate-mannitol (PAGGGM) were pooled, mixed, and split at Day 8 after collection (n = 4). All concentrates were glycerolized, frozen, and stored for at least 2 weeks at -80°C. After thawing and deglycerolization, from each pair, one red blood cell concentrate was resuspended in SAGM, and one was suspended in AS-3. During postthaw storage at 2 to 6°C for 35 days, all concentrates were sampled weekly and analyzed for hematologic, metabolic, and morphologic parameters. Both Rejuvesol and PAGGGM treatment produced increased adenosine triphosphate and total adenylate and 2,3-diphosphoglycerate levels compared with untreated red blood cell concentrates. Regardless of prefreeze Rejuvesol or PAGGGM treatment, postthaw hemolysis remained below 0.8% during 7 days in SAGM and during 35 days in AS-3. At Day 35 of postthaw storage in AS-3, total adenylate in nonrejuvenated red blood cell concentrates had decreased to 72% of the original values; whereas, in prefreeze Rejuvesol-treated and PAGGGM-treated concentrates, adenylate values were still were at 101% and 98%, respectively. Based on maximum allowable hemolysis of 0.8% and total adenylate content greater than 82% of the original value, thawed, prefreeze Rejuvesol-treated or PAGGGM-treated red blood cell concentrates can be stored for 35 days at 2 to 6ºC in AS-3. © 2017 AABB.

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.

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

Directory of Open Access Journals (Sweden)

Sonya Marshall-Gradisnik

2008-10-01

contribute to the BBB and BSB integrity and contain PACAP and VIP receptors. Autoimmunity of these receptors would likely affect BBB and VRS function and therefore may contribute to the etiology of these conditions by affecting CNS and immunological homeostasis, including promoting neuropsychological symptomatology. PACAP and VIP, as potent activators of adenylate cyclase (AC, have a key role in cyclic adenosine monophosphate (cAMP production affecting regulatory T cell (Treg and other immune functions. Phosphodiesterase enzymes (PDEs catalyze cAMP and PDE inhibitors (PDEIs maintain cAMP levels and have proven and well known therapeutic benefit in animal models such as experimental allergic encephalomyelitis (EAE. Therefore PDEIs may have a role in therapy for certain neuropsychiatric fatigue-related conditions.Keywords: vasoactive neuropeptides, multiple sclerosis, Parkinson’s disease, chronic fatigue syndrome, phosphodiesterase inhibitors, cyclic AMP, adenylate cyclase, Virchow–Robin spaces

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

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Lauren Forbes

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

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

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.

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.

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells.

Science.gov (United States)

Algarni, Alanood S; Hargreaves, Alan J; Dickenson, John M

2017-03-15

The PAC 1 receptor and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 activity by the PAC 1 receptor in retinoic acid-induced differentiating N2a neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. The role of TG2 in PAC 1 receptor-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27). PACAP-27 mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON and R283 and by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), protein kinase C (Ro 31-8220), MEK1/2 (PD 98059), and removal of extracellular Ca 2+ . Fluorescence microscopy demonstrated PACAP-27 induced in situ TG2 activity. TG2 inhibition blocked PACAP-27 induced attenuation of hypoxia-induced cell death and outgrowth of axon-like processes. TG2 activation and cytoprotection were also observed in human SH-SY5Y cells. Together, these results demonstrate that TG2 activity was stimulated downstream of the PAC 1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC 1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC 1 receptor. Copyright © 2017 Elsevier Inc. All rights reserved.

Cyclic guanosine monophosphate in the regulation of the cell function

Directory of Open Access Journals (Sweden)

Małgorzata Zbrojkiewicz

2016-12-01

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

Regulation of human renin expression in chorion cell primary cultures

International Nuclear Information System (INIS)

Duncan, K.G.; Haidar, M.A.; Baxter, J.D.; Reudelhuber, T.L.

1990-01-01

The human renin gene is expressed in the kidney, placenta, and several other sites. The release of renin or its precursor, prorenin, can be affected by several regulatory agents. In this study, primary cultures of human placental cells were used to examine the regulation of prorenin release and renin mRNA levels and of the transfected human renin promoter linked to chloramphenicol acetyltransferase reporter sequences. Treatment of the cultures with a calcium ionophore alone, calcium ionophore plus forskolin (that activates adenylate cyclase), or forskolin plus a phorbol ester increased prorenin release and renin mRNA levels 1.3 endash to 6 endash fold, but several classes of steroids did not affect prorenin secretion or renin RNA levels. These results suggest that (i) the first 584 base pairs of the renin gene 5'endash flanking DNA do not contain functional glucocorticoid or estrogen response elements, (ii) placental prorenin release and renin mRNA are regulated by calcium ion and by the combinations of cAMP with either C kinase or calcium ion, and (iii) the first 100 base pairs of the human renin 5'endash flanking DNA direct accurate initiation of transcription and can be regulated by cAMP. Thus, some control of renin release in the placenta (and by inference in other tissues) occurs via transcriptional influences on its promoter

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.

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.

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...

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

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

Directory of Open Access Journals (Sweden)

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.

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

PACAP Protects Against Ethanol and Nicotine Toxicity in SH-SY5Y Cells: Implications for Drinking-Smoking Co-morbidity.

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Manavalan, Sridharan; Getachew, Bruk; Manaye, Kebreten F; Khundmiri, Syed J; Csoka, Antonei B; McKinley, Raechel; Tamas, Andrea; Reglodi, Dora; Tizabi, Yousef

2017-07-01

The detrimental effects of heavy drinking and smoking are multiplied when the two are combined. Treatment modalities for each and especially for the combination are very limited. Although in low concentration, alcohol and nicotine, each may have beneficial effects including neuroprotection, their combination, instead of providing additive protection, may actually lead to toxicity in cell cultures. Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous 38 amino-acid peptide with demonstrated protection against neuronal injury, trauma as well as various endogenous and exogenous toxic agents. The aim of this study was to investigate whether PACAP may also protect against toxicity induced by high alcohol, high nicotine, or the combination of low alcohol and nicotine concentrations, and if so, whether this effect was mediated via PAC1 receptor. We used the neuroblastoma-derived SH-SY5Y cells and applied various colorimetric assays for determination of cell viability or toxicity. Results indicate that PACAP blocks toxicity induced by high alcohol and high nicotine as well as their combination at low concentrations. The effects of PACAP in turn were blocked by the PACAP antagonist (PACAP 6-38), indicating involvement of the PACAP receptor PAC1 and possibly vasoactive intestinal peptide (VIP) receptors in PACAP's protection. Moreover, no combined toxicity of low alcohol and low nicotine could be detected in calcium-free medium. These findings suggest possible beneficial effects of PACAP in preventing alcohol and nicotine toxicity and that calcium contributes to the damage induced by combination of low alcohol and nicotine in SH-SY5Y cells.

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)

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.

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

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

Science.gov (United States)

Lim, C J; Spiegelman, G B; Weeks, G

2001-08-15

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

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

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Siou-Huei Wang

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

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

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

Effects of transforming growth factor-beta on growth and differentiation of the continuous rat thyroid follicular cell line, FRTL-5

International Nuclear Information System (INIS)

Morris, J.C. III; Ranganathan, G.; Hay, I.D.; Nelson, R.E.; Jiang, N.S.

1988-01-01

Transforming growth factor-beta (TGF beta) has been shown to influence the growth and differentiation of many widely varied cell types in vitro, including some that are endocrinologically active. We have investigated the previously unknown effects of this unique growth factor in the differentiated rat thyroid follicular cell line FRTL-5. The cells demonstrated specific, high affinity binding of TGF beta, and as with other epithelial cells, the growth of these thyroid follicular cells was potently inhibited by addition of TGF beta to the culture medium. TGF beta caused a significant reduction in TSH-sensitive adenylate cyclase activity in the cells. The addition of (Bu)2cAMP along with the growth factor to cultures partially reversed the characteristic morphological changes seen with TGF beta, but did not reverse the growth inhibition. To further investigate the possible mechanisms of the effects of TGF beta on the cells, we measured the influence of the growth factor on [125I]TSH binding. TGF beta did not compete for specific TSH-binding sites; however, exposure of the cells to TGF beta for 12 or more h resulted in a dose-dependent down-regulation of TSH receptors that was fully reversible. While cellular proliferation was potently inhibited by TGF beta, differentiated function, as manifest by iodine-trapping ability, was stimulated by the growth factor. This stimulation of iodine uptake was independent of, and additive to, the stimulatory effects of TSH. Finally, FRTL-5 cells in serum-free medium and in response to TSH were shown to secrete TGF beta-like activity that competed for [125I]TGF beta in a RRA. These studies suggest that TGF beta may represent an autocrine mechanism of controlling the growth response to TSH in thyroid follicular cells, while allowing the continuance of differentiated function

An Improved Targeted cAMP Sensor to Study the Regulation of Adenylyl Cyclase 8 by Ca2+ Entry through Voltage-Gated Channels

Science.gov (United States)

Everett, Katy L.; Cooper, Dermot M. F.

2013-01-01

Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes. PMID:24086669

An improved targeted cAMP sensor to study the regulation of adenylyl cyclase 8 by Ca2+ entry through voltage-gated channels.

Directory of Open Access Journals (Sweden)

Katy L Everett

Full Text Available Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca(2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca(2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes.

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.

In vitro study of alpha 2-adrenoceptor turnover and metabolism using the adenocarcinoma cell line HT29

International Nuclear Information System (INIS)

Paris, H.; Taouis, M.; Galitzky, J.

1987-01-01

The biosynthesis rate of the receptor was studied in postconfluent HT29 cells, when its density expressed as fmol/mg of cell membrane protein is constant, by following the recovery of the receptor binding capacity after blockade with the non-reversible alpha-adrenergic antagonist benextramine. Study of the inhibition of [ 3 H]yohimbine and [ 3 H]UK-14,304 binding showed that benextramine was a more potent antagonist at alpha 2-adrenoceptor than phenoxybenzamine. The incubation of intact HT29 cells for 30 min in the presence of 10(-5) M benextramine irreversibly blocked more than 95% of the alpha 2-adrenoceptors and totally suppressed the inhibitory effect of UK-14,304 on cyclic AMP production. The blockade appeared specific, since benextramine effects were prevented by alpha 2-adrenergic agents. Moreover, neither vasoactive intestinal polypeptide responsiveness nor other tested aspects of the regulation of the adenylate cyclase was altered by the treatment. Study of the time course of receptor recovery after irreversible blockade indicated that alpha 2-adrenoceptors reappeared in the cells with a monoexponential kinetic. The linearization of the repopulation curve obtained with the labeled antagonist [ 3 H]yohimbine allowed the determination of the rate constant for receptor degradation (k = 0.0268 +/- 0.0025 hr-1) and the rate of receptor synthesis (6.91 +/- 0.64 fmol/mg of cell membrane protein/hr) corresponding to the synthesis of about 500 receptors/cell/hr. The alpha 2-adrenoceptor half-life was 26 +/- 3 hr. Measurement of the biological effects associated to the alpha-adrenoceptor stimulation during the course of receptor recovery indicated a relationship between the number of cell receptors and the percentage of inhibition of the cyclic AMP accumulation induced by forskolin

Pertussis toxin treatment does not block inhibition by atrial natriuretic factor of aldosterone secretion in cultured bovine zona glomerulosa cells

International Nuclear Information System (INIS)

De Lean, A.; Cantin, M.

1986-01-01

The authors have previously reported that atrial natriuretic factor (ANF) potently inhibits PGE or forskolin-stimulation aldosterone secretion in bovine zona glomerulosa (ZG) by acting through specific high affinity receptors. In order to evaluate the functional role of the regulatory protein N/sub i/ and the inhibition of adenylate cyclase activity (AC) in ZG, the authors have studied the effect of treatment with PT on inhibition by ANF of aldosterone production. Primary cultures of ZG were treated for 18 hours in serum-free F12 medium with (0-100 ng/ml PT). No effect of PT pretreatment was observed either on basal, PGE-stimulated or ANF-inhibited levels of steroidogenesis. When membranes prepared from control ZG were ADP-ribosylated with [ 32 P] NAD in the presence of PT, two toxin-specific bands with 39 Kd and 41 Kd were documented on SDS gel. Cell pretreatment with as low as 1 ng/ml drastically reduced further labelling of these two bands while higher doses completely abolished them. Since PT treatment covalently modifies completely the toxin substrate without altering ANF inhibition of adrenal steroidogenesis, the authors conclude that N/sub i/ is not involved in the mode of action of ANF on aldosterone production

Release of ATP from Marginal Cells in the Cochlea of Neonatal Rats Can Be Induced by Changes in Extracellular and Intracellular Ion Concentrations

Science.gov (United States)

Peng, Yating; Chen, Jie; He, Shan; Yang, Jun; Wu, Hao

2012-01-01

Background Adenosine triphosphate (ATP) plays an important role in the cochlea. However, the source of ATP and the mechanism by which it is released remain unclear. This study investigates the presence and release mechanism of ATP in vitro cultured marginal cells isolated from the stria vascularis of the cochlea in neonatal rats. Methods Sprague-Dawley rats aged 1–3 days old were used for isolation, in vitro culture, and purification of marginal cells. Cultured marginal cells were verified by flow cytometry. Vesicles containing ATP in these cells were identified by fluorescence staining. The bioluminescence assay was used for determination of ATP concentration in the extracellular fluid released by marginal cells. Assays for ATP concentration were performed when the ATP metabolism of cells was influenced, and ionic concentrations in intracellular and extracellular fluid were found to change. Results Evaluation of cultured marginal cells with flow cytometry revealed the percentage of fluorescently-labeled cells as 92.9% and 81.9%, for cytokeratin and vimentin, respectively. Quinacrine staining under fluorescence microscopy revealed numerous green, star-like spots in the cytoplasm of these cells. The release of ATP from marginal cells was influenced by changes in the concentration of intracellular and extracellular ions, namely extracellular K+ and intra- and extracellular Ca2+. Furthermore, changes in the concentration of intracellular Ca2+ induced by the inhibition of the phospholipase signaling pathway also influence the release of ATP from marginal cells. Conclusion We confirmed the presence and release of ATP from marginal cells of the stria vascularis. This is the first study to demonstrate that the release of ATP from such cells is associated with the state of the calcium pump, K+ channel, and activity of enzymes related to the phosphoinositide signaling pathway, such as adenylate cyclase, phospholipase C, and phospholipase A2. PMID:23071731

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....

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.

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

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...

Two skin cell lines from wild-type and albino Japanese flounder (Paralichthys olivaceus): establishment, characterization, virus susceptibility, efficient transfection, and application to albinism study.

Science.gov (United States)

Wang, Ruoqing; Zhang, Nianwei; Wang, Renkai; Wang, Shengpeng; Wang, Na

2017-12-01

In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300 days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24 °C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.

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

Induction of Pro-Apoptotic Endoplasmic Reticulum Stress in Multiple Myeloma Cells by NEO214, Perillyl Alcohol Conjugated to Rolipram.

Science.gov (United States)

Chen, Thomas C; Chan, Nymph; Labib, Shirin; Yu, Jiali; Cho, Hee-Yeon; Hofman, Florence M; Schönthal, Axel H

2018-01-17

Despite the introduction of new therapies for multiple myeloma (MM), many patients are still dying from this disease and novel treatments are urgently needed. We have designed a novel hybrid molecule, called NEO214, that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH), an inducer of endoplasmic reticulum (ER) stress, to rolipram (Rp), an inhibitor of phosphodiesterase-4 (PDE4). Its potential anticancer effects were investigated in a panel of MM cell lines. We found that NEO214 effectively killed MM cells in vitro with a potency that was over an order of magnitude stronger than that of its individual components, either alone or in combination. The cytotoxic mechanism of NEO214 involved severe ER stress and prolonged induction of CCAAT/enhancer-binding protein homologous protein (CHOP), a key pro-apoptotic component of the ER stress response. These effects were prevented by salubrinal, a pharmacologic inhibitor of ER stress, and by CHOP gene knockout. Conversely, combination of NEO214 with bortezomib, a drug in clinical use for patients with MM, resulted in synergistic enhancement of MM cell death. Combination with the adenylate cyclase stimulant forskolin did not enhance NEO214 impact, indicating that cyclic adenosine 3',5'-monophosphate (AMP) pathways might play a lesser role. Our study introduces the novel agent NEO214 as a potent inducer of ER stress with significant anti-MM activity in vitro. It should be further investigated as a potential MM therapy aimed at exploiting this tumor's distinct sensitivity to ER stress.

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

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.

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

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.

Cannabis Users Show Enhanced Expression of CB1-5HT2A Receptor Heteromers in Olfactory Neuroepithelium Cells.

Science.gov (United States)

Galindo, Liliana; Moreno, Estefanía; López-Armenta, Fernando; Guinart, Daniel; Cuenca-Royo, Aida; Izquierdo-Serra, Mercè; Xicota, Laura; Fernandez, Cristina; Menoyo, Esther; Fernández-Fernández, José M; Benítez-King, Gloria; Canela, Enric I; Casadó, Vicent; Pérez, Víctor; de la Torre, Rafael; Robledo, Patricia

2018-01-02

Cannabinoid CB1 receptors (CB 1 R) and serotonergic 2A receptors (5HT 2A R) form heteromers in the brain of mice where they mediate the cognitive deficits produced by delta-9-tetrahydrocannabinol. However, it is still unknown whether the expression of this heterodimer is modulated by chronic cannabis use in humans. In this study, we investigated the expression levels and functionality of CB 1 R-5HT 2A R heteromers in human olfactory neuroepithelium (ON) cells of cannabis users and control subjects, and determined their molecular characteristics through adenylate cyclase and the ERK 1/2 pathway signaling studies. We also assessed whether heteromer expression levels correlated with cannabis consumption and cognitive performance in neuropsychological tests. ON cells from controls and cannabis users expressed neuronal markers such as βIII-tubulin and nestin, displayed similar expression levels of genes related to cellular self-renewal, stem cell differentiation, and generation of neural crest cells, and showed comparable Na + currents in patch clamp recordings. Interestingly, CB 1 R-5HT 2A R heteromer expression was significantly increased in cannabis users and positively correlated with the amount of cannabis consumed, and negatively with age of onset of cannabis use. In addition, a negative correlation was found between heteromer expression levels and attention and working memory performance in cannabis users and control subjects. Our findings suggest that cannabis consumption regulates the formation of CB 1 R-5HT 2A R heteromers, and may have a key role in cognitive processing. These heterodimers could be potential new targets to develop treatment alternatives for cognitive impairments.

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.

Neuroprotection of a novel cyclopeptide C*HSDGIC* from the cyclization of PACAP (1-5 in cellular and rodent models of retinal ganglion cell apoptosis.

Directory of Open Access Journals (Sweden)

Huanhuan Cheng

Full Text Available To investigate the protective effects of a novel cyclopeptide C*HSDGIC* (CHC from the cyclization of Pituitary adenylate cyclase-activating polypeptide (PACAP (1-5 in cellular and rodent models of retinal ganglion cell apoptosis.Double-labeling immunohistochemistry was used to detect the expression of Thy-1 and PACAP receptor type 1 in a retinal ganglion cell line RGC-5. The apoptosis of RGC-5 cells was induced by 0.02 J/cm(2 Ultraviolet B irradiation. MTT assay, flow cytometry, fluorescence microscopy were used to investigate the viability, the level of reactive oxygen species (ROS and apoptosis of RGC-5 cells respectively. CHC attenuated apoptotic cell death induced by Ultraviolet B irradiation and inhibited the excessive generation of ROS. Moreover, CHC treatment resulted in decreased expression of Bax and concomitant increase of Bcl-2, as was revealed by western-blot analysis. The in vivo apoptosis of retinal ganglion cells was induced by injecting 50 mM N-methyl-D-aspartate (NMDA (100 nmol in a 2 µL saline solution intravitreally, and different dosages of CHC were administered. At day 7, rats in CHC+ NMDA-treated groups showed obvious aversion to light when compared to NMDA rats. Electroretinogram recordings revealed a marked decrease in the amplitudes of a-wave, b-wave, and photopic negative response due to NMDA damage. In retina receiving intravitreal NMDA and CHC co-treatment, these values were significantly increased. CHC treatment also resulted in less NMDA-induced cell loss and a decrease in the proportion of dUTP end-labeling-positive cells in ganglion cell line.C*HSDGIC*, a novel cyclopeptide from PACAP (1-5 attenuates apoptosis in RGC-5 cells and inhibits NMDA-induced retinal neuronal death. The beneficial effects may occur via the mitochondria pathway. PACAP derivatives like CHC may serve as a promising candidate for neuroprotection in glaucoma.

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

Czech Academy of Sciences Publication Activity Database

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

2014-01-01

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

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....

Effect of forskolin and prostaglandin E1 on stimulus secretion coupling in cultured bovine adrenal chromaffin cells.

Science.gov (United States)

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

1988-02-01

Treatment of adrenal chromaffin cells with forskolin (0.1-10 microM) stimulated cyclic AMP levels, reduced the maximal stimulation of release of noradrenaline by nicotine, and increased release in response to elevated external potassium and the calcium ionophore A23187. The presence of the phosphodiesterase inhibitor Ro 20-17-24 with forskolin potentiated both the stimulation of cyclic AMP and the inhibition of nicotine-induced noradrenaline release. Dibutyryl cyclic AMP, and the elevation of cyclic AMP with prostaglandin E1, also attenuated nicotine-stimulated release. However, when the stimulation of intracellular cyclic AMP production by prostaglandin E1 was potentiated by low levels of forskolin, there was not a concomitant potentiation of effect on noradrenaline release. Dideoxyforskolin, an analogue of forskolin which does not stimulate adenylate cyclase, inhibited both potassium- and nicotine-stimulated release, probably by a mechanism unrelated to the action of forskolin in these experiments. Using Fura-2 to estimate free intracellular calcium levels, both forskolin and dideoxyforskolin (at 10 microM) reduced the calcium transient in response to nicotine. These results support a model in which elevation of cyclic AMP inhibits the activation of nicotinic receptors, but augments stimulus secretion coupling downstream of calcium entry. The data, however, do not indicate a simple relationship between total intracellular cyclic AMP levels and the attenuation of nicotinic stimulation of release.

Energy metabolism in human melanoma cells under hypoxic and acidic conditions in vitro.

Science.gov (United States)

Skøyum, R; Eide, K; Berg, K; Rofstad, E K

1997-01-01

The response to treatment and the malignant progression of tumours are influenced by the ability of the tumour cells to withstand severe energy deprivation during prolonged exposure to hypoxia at normal or low extracellular pH (pHe). The objective of the present work was to demonstrate intertumour heterogeneity under conditions of microenvironment-induced energy deprivation and to investigate whether the heterogeneity can be attributed to differences in the capacity of the tumour cells to generate energy in an oxygen-deficient microenvironment. Cultures of four human melanoma cell lines (BEX-c, COX-c, SAX-c, WIX-c) were exposed to hypoxia in vitro at pHe 7.4, 7.0 or 6.6 for times up to 31 h by using the steel-chamber method. High-performance liquid chromatography was used to assess adenylate energy charge as a function of exposure time. Cellular rates of glucose uptake and lactate release were determined by using standard enzymatic test kits. The adenylate energy charge decreased with time under hypoxia in all cell lines. The decrease was most pronounced shortly after the treatment had been initiated and then tapered off. BEX-c and SAX-c showed a significantly higher adenylate energy charge under hypoxic conditions than did COX-c and WIX-c whether the pHe was 7.4, 7.0 or 6.6, showing that tumours can differ in the ability to avoid energy deprivation during microenvironmental stress. There was no correlation between the adenylate energy charge and the rates of glucose uptake and lactate release. Intertumour heterogeneity in the ability to withstand energy deprivation in an oxygen-deficient microenvironment cannot therefore be attributed mainly to differences in the capacity of the tumour cells to generate energy by anaerobic metabolism. The data presented here suggest that the heterogeneity is rather caused by differences in the capacity of the tumour cells to reduce the rate of energy consumption when exposed to hypoxia.

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

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...

Glycated albumin suppresses glucose-induced insulin secretion by impairing glucose metabolism in rat pancreatic β-cells

Directory of Open Access Journals (Sweden)

Muto Takashi

2011-04-01

Full Text Available Abstract Background Glycated albumin (GA is an Amadori product used as a marker of hyperglycemia. In this study, we investigated the effect of GA on insulin secretion from pancreatic β cells. Methods Islets were collected from male Wistar rats by collagenase digestion. Insulin secretion in the presence of non-glycated human albumin (HA and GA was measured under three different glucose concentrations, 3 mM (G3, 7 mM (G7, and 15 mM (G15, with various stimulators. Insulin secretion was measured with antagonists of inducible nitric oxide synthetase (iNOS, and the expression of iNOS-mRNA was investigated by real-time PCR. Results Insulin secretion in the presence of HA and GA was 20.9 ± 3.9 and 21.6 ± 5.5 μU/3 islets/h for G3 (P = 0.920, and 154 ± 9.3 and 126.1 ± 7.3 μU/3 islets/h (P = 0.046, for G15, respectively. High extracellular potassium and 10 mM tolbutamide abrogated the inhibition of insulin secretion by GA. Glyceraldehyde, dihydroxyacetone, methylpyruvate, GLP-1, and forskolin, an activator of adenylate cyclase, did not abrogate the inhibition. Real-time PCR showed that GA did not induce iNOS-mRNA expression. Furthermore, an inhibitor of nitric oxide synthetase, aminoguanidine, and NG-nitro-L-arginine methyl ester did not abrogate the inhibition of insulin secretion. Conclusion GA suppresses glucose-induced insulin secretion from rat pancreatic β-cells through impairment of intracellular glucose metabolism.

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.

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)

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

Science.gov (United States)

Li, Peng; Waldman, Scott A

2010-08-01

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

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

Science.gov (United States)

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

2018-04-01

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

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

International Nuclear Information System (INIS)

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

1988-01-01

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

Identification of cDNA encoding an additional α subunit of a human GTP-binding protein: Expression of three αi subtypes in human tissues and cell lines

International Nuclear Information System (INIS)

Kim, S.; Ang, S.L.; Bloch, D.B.; Bloch, K.D.; Kawahara, Y.; Tolman, C.; Lee, R.; Seidman, J.G.; Neer, E.J.

1988-01-01

The guanine nucleotide-binding proteins (G proteins), which mediate hormonal regulation of many membrane functions, are composed of α, β, and γ subunits. The authors have cloned and characterized cDNA from a human T-cell library encoding a form of α i that is different from the human α i subtypes previously reported. α i is the α subunit of a class of G proteins that inhibits adenylate cyclase and regulates other enzymes and ion channels. This cDNA encodes a polypeptide of 354 amino acids and is assigned to encode the α i-3 subtype of G proteins on the basis of its similarity to other α i -like cDNAs and the presence of a predicted site for ADP ribosylation by pertussis toxin. They have determined the expression of mRNA for this and two other subtypes of human α i (α i-1 and α i-2 ) in a variety of human fetal tissues and in human cell lines. All three α i subtypes were present in the tissues tested. However, analysis of individual cell types reveals specificity of α i-1 expression. mRNA for α i-1 is absent in T cells, B cells, and monocytes but is present in other cell lines. The finding of differential expression of α i-1 genes may permit characterization of distinct physiological roles for this α i subunit. mRNA for α i-2 and α i-3 was found in all the primary and transformed cell lines tested. Thus, some cells contain all three α i subtypes. This observation raises the question of how cells prevent cross talk among receptors that are coupled to effectors through such similar α proteins

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

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.

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

The transcription factor Swi4 is target for PKA regulation of cell size at the G1 to S transition in Saccharomyces cerevisiae.

Science.gov (United States)

Amigoni, Loredana; Colombo, Sonia; Belotti, Fiorella; Alberghina, Lilia; Martegani, Enzo

2015-08-03

To investigate the specific target of PKA in the regulation of cell cycle progression and cell size we developed a new approach using the yeast strain GG104 bearing a deletion in adenylate cyclase gene and permeable to cAMP ( cyr1Δ, pde2Δ, msn2Δ, msn4Δ). In this strain the PKA activity is absent and can be activated by addition of cAMP in the medium, without any other change of the growth conditions. In the present work we show that the activation of PKA by exogenous cAMP in the GG104 strain exponentially growing in glucose medium caused a marked increase of cell size and perturbation of cell cycle with a transient arrest of cells in G1, followed by an accumulation of cells in G2/M phase with a minimal change in the growth rate. Deletion of CLN1 gene, but not of CLN2, abolished the transient G1 phase arrest. Consistently we found that PKA activation caused a transcriptional repression of CLN1 gene. Transcription of CLN1 is controlled by SBF and MBF dual-regulated promoter. We found that also the deletion of SWI4 gene abolished the transient G1 arrest suggesting that Swi4 is a target responsible for PKA modulation of G1/S phase transition. We generated a SWI4 allele mutated in the consensus site for PKA (Swi4(S159A)) and we found that expression of Swi4(S159A) protein in the GG104-Swi4Δ strain did not restore the transient G1 arrest induced by PKA activation, suggesting that Swi4 phosphorylation by PKA regulates CLN1 gene expression and G1/S phase transition.

Free-energy carriers in human cultured muscle cells

NARCIS (Netherlands)

Bolhuis, P. A.; de Zwart, H. J.; Ponne, N. J.; de Jong, J. M.

1985-01-01

Creatine phosphate (CrP), adenosine triphosphate (ATP), creatine kinase (CK), adenylate kinase (AK), protein, and DNA were quantified in human muscle cell cultures undergoing transition from dividing myoblasts to multinucleate myotubes. CrP is negligible in cultures grown in commonly applied media

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

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.

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

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

Science.gov (United States)

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

2003-12-05

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

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.

Andrographolide protects liver cells from H2O2 induced cell death by upregulation of Nrf-2/HO-1 mediated via adenosine A2a receptor signalling.

Science.gov (United States)

Mittal, Smriti P K; Khole, Swati; Jagadish, Nidhi; Ghosh, Debjani; Gadgil, Vijay; Sinkar, Vilas; Ghaskadbi, Saroj S

2016-11-01

Andrographolide, principle constituent of Andrographis paniculata Nees is used in traditional medicine in Southeast Asia and is known to exhibit various biological activities. Its antioxidant activity is due to its ability to activate one of the antioxidant enzymes, heme oxygenase-1 (HO-1) which is regulated transcriptionally through Nrf-2. However, molecular mechanism underlying activation of Nrf-2/HO-1 has not yet been clearly understood. Protective effect of andrographolide against H2O2 induced cell death, reactive oxygen species and lipid peroxidation was observed in HepG2 cells. Ability of andrographolide to modulate G-protein coupled receptor (GPCR) mediated signalling was determined using in silico docking and gene expression was analyzed by qRT-PCR, confocal microscopy and western blot analysis. We clearly show that andrographolide via adenosine A2A receptor signalling leads to activation of p38 MAP kinase, resulting in upregulation of Nrf-2, its translocation to nucleus and activation of HO-1. Additionally, it activates adenylate cyclase resulting in cAMP formation which in turn activates protein kinase A leading to inhibition of GSK-3β by phosphorylation. Inactivated GSK-3β leads to retention of Nrf-2 in the nucleus leading to sustained expression of HO-1 by binding to its antioxidant response element (ARE). Thus, andrographolide probably by binding to adenosine A2a receptor activates Nrf-2 transcription and also inhibits its exclusion from the nucleus by inactivating GSK-3β, together resulting in activation of HO-1. We speculate that andrographolide can be used as a therapeutic drug to combat oxidative stress implicated in pathogenesis of various diseases such as diabetes, osteoporosis, neurodegenerative diseases etc. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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

Directory of Open Access Journals (Sweden)

Lucèia Fàtima Souza

2016-09-01

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

Radiolabelling of cholera toxin

International Nuclear Information System (INIS)

Santos, R.G.; Neves, Nicoli M.J.; Abdalla, L.F.; Brandao, R.L.; Etchehebehere, L.; Lima, M.E. de; Nicoli, J.R.

1999-01-01

Binding of cholera toxin to ganglioside receptors of enterocyte microvilli catalyzes the activation of adenylate cyclase causing a rise in cAMP which final result is a copious diarrhea. Saccharomyces boulardii, a nonpathogenic yeast has been used to prevent diarrhea. Although the antidiarrheic properties of S. boulardii are widely recognized, this yeast has been used on empirical basis, and the mechanism of this protective effect is unknown. The addition of cholera toxin to S. boulardii induces the raising of cAMP that triggers the activation of neutral trehalase. This suggests that toxin specifically binding to cells, is internalized and active the protein phosphorylation cascade. Our objective is labeling the cholera toxin to verify the presence of binding sites on yeast cell surfaces for the cholera toxin. Cholera toxin was radiolabelled with Na 125 I by a chloramine-T method modified from Cuatrecasas and Griffiths et alii. The 125 I-Cholera toxin showed a specific radioactivity at about 1000 cpm/fmol toxin. Biological activity of labeled cholera toxin measured by trehalase activation was similar to the native toxin. (author)

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

Science.gov (United States)

Souza, Lucèia Fàtima; Caputo, Lucia; Inchausti De Barros, Ingrid Bergman; Fratianni, Florinda; Nazzaro, Filomena; De Feo, Vincenzo

2016-09-03

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.

Identification of poly(ADP-ribose) polymerase-1 as the OXPHOS-generated ATP sensor of nuclei of animal cells

International Nuclear Information System (INIS)

Kun, Ernest; Kirsten, Eva; Hakam, Alaeddin; Bauer, Pal I.; Mendeleyev, Jerome

2008-01-01

Our results show that in the intact normal animal cell mitochondrial ATP is directly connected to nuclear PARP-1 by way of a specific adenylate kinase enzymatic path. This mechanism is demonstrated in two models: (a) by its inhibition with a specific inhibitor of adenylate kinase, and (b) by disruption of ATP synthesis through uncoupling of OXPHOS. In each instance the de-inhibited PARP-1 is quantitatively determined by enzyme kinetics. The nuclear binding site of PARP-1 is Topo I, and is identified as a critical 'switchpoint' indicating the nuclear element that connects OXPHOS with mRNA synthesis in real time. The mitochondrial-nuclear PARP-1 pathway is not operative in cancer cells

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

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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

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

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

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

Science.gov (United States)

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.

Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists

Science.gov (United States)

Charfi, Iness; Nagi, Karim; Mnie-Filali, Ouissame; Thibault, Dominic; Balboni, Gianfranco; Schiller, Peter W.; Trudeau, Louis-Eric

2014-01-01

Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of Emax values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase Emax values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells. PMID:24022593

Arginine vasopressin increases cellular free calcium concentration and adenosine 3',5'-monophosphate production in rat renal papillary collecting tubule cells in culture

International Nuclear Information System (INIS)

Ishikawa, S.; Okada, K.; Saito, T.

1988-01-01

The role of calcium (Ca) in the cellular action of arginine vasopressin (AVP) was examined in rat renal papillary collecting tubule cells in culture. AVP increased both the cellular free Ca concentration ([Ca2+]i) using fura-2, and cAMP production in a dose-dependent manner. AVP-induced cellular Ca mobilization was totally blocked by the antagonist to the antidiuretic action of AVP, and somewhat weakened by the antagonist to the vascular action of AVP. 1-Deamino-8-D-AVP (dDAVP). an antidiuretic analog of AVP, also increased [Ca2+] significantly. Cellular Ca mobilization was not obtained with cAMP, forskolin (a diterpene activator of adenylate cyclase), or phorbol-12-myristate-13-acetate. The early phase of [Ca2+]i depended on the intracellular Ca pool, since an AVP-induced rise in [Ca2+]i was obtained in cells pretreated with Ca-free medium containing 1 mM EGTA, verapamil, or cobalt, which blocked cellular Ca uptake. Also, AVP increased 45 Ca2+ influx during the initial 10 min, which initiated the sustained phase of cellular Ca mobilization. However, cellular cAMP production induced by AVP during the 10-min observation period was diminished in the cells pretreated with Ca-free medium, verapamil, or cobalt, but was still significantly higher than the basal level. This was also diminished by a high Ca concentration in medium. These results indicate that 1) AVP concomitantly regulates cellular free Ca as well as its second messenger cAMP production; 2) AVP-induced elevation of cellular free Ca is dependent on both the cellular Ca pool and extracellular Ca; and 3) there is an optimal level of extracellular Ca to modulate the AVP action in renal papillary collecting tubule cells

cAMP-dependent cell differentiation triggered by activated CRHR1 in hippocampal neuronal cells.

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Inda, Carolina; Bonfiglio, Juan José; Dos Santos Claro, Paula A; Senin, Sergio A; Armando, Natalia G; Deussing, Jan M; Silberstein, Susana

2017-05-16

Corticotropin-releasing hormone receptor 1 (CRHR1) activates the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). Both cAMP sources were shown to be required for the phosphorylation of ERK1/2 triggered by activated G protein coupled receptor (GPCR) CRHR1 in neuronal and neuroendocrine contexts. Here, we show that activated CRHR1 promotes growth arrest and neurite elongation in neuronal hippocampal cells (HT22-CRHR1 cells). By characterising CRHR1 signalling mechanisms involved in the neuritogenic effect, we demonstrate that neurite outgrowth in HT22-CRHR1 cells takes place by a sAC-dependent, ERK1/2-independent signalling cascade. Both tmACs and sAC are involved in corticotropin-releasing hormone (CRH)-mediated CREB phosphorylation and c-fos induction, but only sAC-generated cAMP pools are critical for the neuritogenic effect of CRH, further highlighting the engagement of two sources of cAMP downstream of the activation of a GPCR, and reinforcing the notion that restricted cAMP microdomains may regulate independent cellular processes.

MT1 melatonin receptors and their role in the oncostatic action of melatonin

Directory of Open Access Journals (Sweden)

Karolina Danielczyk

2009-09-01

Full Text Available Melatonin, the main hormone produced by the pineal gland, strongly inhibits the growth of cancer cells [i]in vitro[/i] and [i]in vivo[/i]. Some publications indicate that the addition of melatonin to culture medium slows the proliferation of some cancer cell lines. It is also suggested that melatonin used as an adjuvant benefits the effectiveness and tolerance of chemotherapy. The mechanisms of this are not fully understood, but melatonin receptors might be one of the most important elements. Two distinct types of membrane-bound melatonin receptors have been identified in humans: MT1 (Mel1a and MT2 (Mel1b receptors. These subtypes are 60�0homologous at the amino-acid level. MT1 receptors are G-protein-coupled receptors. Through the α subunit of G protein, melatonin receptors stimulate an adenylate cyclase and decrease the level of cAMP. This has a significant influence on cell proliferation and has been confirmed in many tests on different cell lines, such as S-19, B-16 murine melanoma cells, and breast cancer cells. It seems that expression of the MT1 melatonin receptors benefits the efficacy of melatonin treatment. Melatonin and its receptors may provide a promising way to establish new alternative therapeutic approaches in human cancer prevention.

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