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Sample records for regulated adenylyl cyclases

  1. 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.

  2. 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

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

    Directory of Open Access Journals (Sweden)

    Nawreen eRahman

    2013-11-01

    Full Text Available Soluble adenylyl cyclase (sAC is a source of the second messenger cyclic adenosine 3',5' monophosphate (cAMP. sAC is directly regulated by bicarbonate (HCO3- ions. In living cells, HCO3- ions are in nearly instantaneous equilibrium with carbon dioxide (CO2 and pH due to the ubiquitous presence of carbonic anhydrases. Numerous biological processes are regulated by CO2, HCO3-, and/or pH, and in a number of these, sAC has been shown to function as a physiological CO2/HCO3/pH sensor. In this review, we detail the known pH sensing functions of sAC, and we discuss two highly-studied, pH-dependent pathways in which sAC might play a role.

  4. 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.

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

    Science.gov (United States)

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

    2011-12-02

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

  6. 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

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

    Directory of Open Access Journals (Sweden)

    Jung-Chin eChang

    2014-02-01

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

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

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

    Science.gov (United States)

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

    2012-06-01

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

  18. 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

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Lucie Hejnova

    2014-01-01

    Full Text Available The adenylyl cyclase (AC signaling system plays a crucial role in the regulation of cardiac contractility. Here we analyzed the key components of myocardial AC signaling in the developing chick embryo and assessed the impact of selected β-blocking agents on this system. Application of metoprolol and carvedilol, two commonly used β-blockers, at embryonic day (ED 8 significantly downregulated (by about 40% expression levels of AC5, the dominant cardiac AC isoform, and the amount of Gsα protein at ED9. Activity of AC stimulated by forskolin was also significantly reduced under these conditions. Interestingly, when administered at ED4, these drugs did not produce such profound changes in the myocardial AC signaling system, except for markedly increased expression of Giα protein. These data indicate that β-blocking agents can strongly derange AC signaling during the first half of embryonic heart development.

  1. 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

  2. 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

  3. 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

  4. 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

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

    Czech Academy of Sciences Publication Activity Database

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

    2013-01-01

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

  6. 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...

  7. Adenylyl cyclase plays a regulatory role in development, stress resistance and secondary metabolism in Fusarium fujikuroi.

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    Jorge García-Martínez

    Full Text Available The ascomycete fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C produces secondary metabolites of biotechnological interest, such as gibberellins, bikaverin, and carotenoids. Production of these metabolites is regulated by nitrogen availability and, in a specific manner, by other environmental signals, such as light in the case of the carotenoid pathway. A complex regulatory network controlling these processes is recently emerging from the alterations of metabolite production found through the mutation of different regulatory genes. Here we show the effect of the targeted mutation of the acyA gene of F. fujikuroi, coding for adenylyl cyclase. Mutants lacking the catalytic domain of the AcyA protein showed different phenotypic alterations, including reduced growth, enhanced production of unidentified red pigments, reduced production of gibberellins and partially derepressed carotenoid biosynthesis in the dark. The phenotype differs in some aspects from that of similar mutants of the close relatives F. proliferatum and F. verticillioides: contrary to what was observed in these species, ΔacyA mutants of F. fujikuroi showed enhanced sensitivity to oxidative stress (H(2O(2, but no change in heavy metal resistance or in the ability to colonize tomato tissue, indicating a high versatility in the regulatory roles played by cAMP in this fungal group.

  8. Gene Expression Profiles of Main Olfactory Epithelium in Adenylyl Cyclase 3 Knockout Mice

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    Zhenshan Wang

    2015-11-01

    Full Text Available Adenylyl Cyclase 3 (AC3 plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE. In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3−/− and wild-type (AC3+/+ mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3−/− mice was significantly altered, compared to AC3+/+ mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3−/− and AC3+/+ mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE.

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

    Science.gov (United States)

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

    2017-08-31

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

  10. Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

    Science.gov (United States)

    Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

    2013-11-15

    Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses. © 2013 Published by Elsevier B.V.

  11. AmTAR2: Functional characterization of a honeybee tyramine receptor stimulating adenylyl cyclase activity.

    Science.gov (United States)

    Reim, Tina; Balfanz, Sabine; Baumann, Arnd; Blenau, Wolfgang; Thamm, Markus; Scheiner, Ricarda

    2017-01-01

    The biogenic monoamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they employ octopamine and tyramine for comparable physiological functions. These biogenic amines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Based on pharmacological data obtained on heterologously expressed receptors, α- and β-adrenergic-like octopamine receptors are better activated by octopamine than by tyramine. Conversely, GPCRs forming the type 1 tyramine receptor clade (synonymous to octopamine/tyramine receptors) are better activated by tyramine than by octopamine. More recently, receptors were characterized which are almost exclusively activated by tyramine, thus forming an independent type 2 tyramine receptor clade. Functionally, type 1 tyramine receptors inhibit adenylyl cyclase activity, leading to a decrease in intracellular cAMP concentration ([cAMP] i ). Type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . We here provide evidence that the honeybee tyramine receptor 2 (AmTAR2), when heterologously expressed in flpTM cells, exclusively causes an increase in [cAMP] i . The receptor displays a pronounced preference for tyramine over octopamine. Its activity can be blocked by a series of established antagonists, of which mianserin and yohimbine are most efficient. The functional characterization of two tyramine receptors from the honeybee, AmTAR1 (previously named AmTYR1) and AmTAR2, which respond to tyramine by changing cAMP levels in opposite direction, is an important step towards understanding the actions of tyramine in honeybee behavior and physiology, particularly in comparison to the effects of octopamine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. [BETA-ADRENERGIC REGULATION OF THE ADENYLYL CYCLASE SIGNALING SYSTEM IN MYOCARDIUM AND BRAIN OF RATS WITH OBESITY AND TYPES 2 DIABETES MELLITUS AND THE EFFECT OF LONG-TERM INTRANASAL INSULIN TREATMENT].

    Science.gov (United States)

    Kuznetsova, L A; Sharova, T S; Pertseva, M N; Shpakov, A O

    2015-01-01

    The stimulating effect of norepinephrine, isoproterenol and selective β-adrenoceptor (β3-AR) agonists BRL 37344 and CL 316.243 on the adenylyl cyclase signaling system (ACSS) in the brain and myocardium of young and mature rats (disease induction at 2 and 4 months, respectively) with experimental obesity and type 2 diabetes mellitus (DM2), and the influence of long-term treatment of animals with intranasal insulin (I-I) were studied. The AC stimulatory effects of β-agonist isoproterenol in animals with obesity and DM2 was shown to be practically unchanged. The respective effects of norepinephrine on the AC activity were attenuated in the brain of young and mature rats and in the myocardium if mature rats, and the I-I treatment led to their partial recovery. In the brain and myocardium of mature rats with obesity and DM2, the enhancement of the AC stimulatory effects of β3-AR agonists was observed, white in young rats the influence of the same pathological conditions was lacking. The I-I treatment decreased the AC stimulatory effects of β3-agonists to their levels in the control. Since functional disruption of the adrenergic agonist-sensitive ACSS can lead to metabolic syndrome and DM2, the recovery of this system by the I-I treatment offers one of the ways to correct these diseases and their complications in the nervous and cardiovascular systems.

  13. 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.

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

    Science.gov (United States)

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

    2003-05-30

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

  15. 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.

  16. 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.

  17. Elevated Adenylyl Cyclase 9 Expression Is a Potential Prognostic Biomarker for Patients with Colon Cancer.

    Science.gov (United States)

    Yi, Hua; Wang, Kun; Jin, Jun-Feng; Jin, He; Yang, Lihua; Zou, Yidan; Du, Biaoyan; Liu, Xiaodong

    2018-01-02

    BACKGROUND Adenylyl cyclase 9 (ADCY9) is an enzyme that modulates signal transduction by producing the second messenger, cyclic adenosine monophosphate (cAMP). The aim of the present study was to investigate the association of ADCY9 expression with clinicopathological features and disease-free survival of colon cancer patients. MATERIAL AND METHODS Immunohistochemistry staining with ADCY9 antibody was performed on a tissue microarray. Immunoreactivity scores (IRS) were recorded and applied for association analysis. ADCY9 mRNA expression and clinicopathogical information were also extracted from TCGA colon cancer dataset and analyzed using univariate and multivariate Cox proportional hazards models.  RESULTS ADCY9 IRS was significantly higher (P=0.002) in tumor tissues (6.40±1.26, n=200) than in adjacent normal samples (4.13±0.83, n=8). The IRS and mRNA expression of ADCY9 were correlated to colon cancer TNM staging. Longer disease-free survival was observed in patients with lower ADCY9 expression (P=0.001). In the multivariate models, ADCY9 expression level (hazard ratio [HR] 5.495, 95% confidence interval [CI] 1.753-17.227, P=0.003), and distant metastasis (HR 4.329, 95% CI 1.374-13.636, P=0.012) were still associated with disease-free survival. CONCLUSIONS High ADCY9 expression is a poor prognostic factor for disease-free survival in colon cancer.

  18. 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.

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

    Directory of Open Access Journals (Sweden)

    Emily Qualls-Creekmore

    2017-07-01

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

  20. Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora.

    Science.gov (United States)

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-09-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    Zhang, Ming; Wang, Hongbing

    2013-01-01

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

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

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    Dong Hongmei

    2010-12-01

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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Elena Elizabeth Bagley

    2014-06-01

    Full Text Available Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1 currents in periaqueductal gray (PAG neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1

  6. Regulation of brain adenylate cyclase by calmodulin

    International Nuclear Information System (INIS)

    Harrison, J.K.

    1988-01-01

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

  7. Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

    Science.gov (United States)

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-01-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different α-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Δgsa1, Δgsa2, and Δgsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Gα-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Δgsa1Δgsa2 and Δgsa1Δgsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Gα-subunits, two recently generated Δpre strains were crossed with all Δgsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three ΔgsaΔsac1 double mutants and one Δgsa2Δgsa3Δsac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1–GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Gα-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora. PMID:18723884

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

    Science.gov (United States)

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

    2014-04-01

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

  9. 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.

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

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    Rosemary C Challis

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2016-12-01

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

  13. Expression and Immunohistochemical Localisation of the G beta gamma activated and Calcineurin-inhibited Adenylyl Cyclase Isoforms in Rat Articular Chondrocytes

    International Nuclear Information System (INIS)

    Memon, I.; Khan, K.M.; Siddiqui, S.; Perveen, S.; Ishaq, M.

    2016-01-01

    Objective: To determine the expression and localisation of the Gβγ-activated adenylyl cyclase (AC) isoforms 2, 4, and 7 and calcineurin-inhibited AC isoform 9 in rat articular chondrocytes. Study Design: Experimental study. Place and Duration of Study: Jumma Research Laboratory and Histology Laboratory, The Aga Khan University, Karachi, from 2009 to 2011. Methodology: Fresh slices of articular cartilage were taken from various synovial joints of rats of different age groups. The expression of AC isoforms was determined by RT-PCR and immunohistochemistry was performed to localise these isoforms in articular chondrocytes. Tissue sections were processed for immunostaining with respective antibodies. The color was developed by diaminobenzidine. Results: All the studied AC isoforms were found to be differentially expressed in different zones of the rat articular cartilage. Generally, expression of all AC isoforms studied increased with age. The expression of the AC isoforms through PCR was almost consistent with the localisation of these isoforms by immunohistochemistry. Conclusion: These data add to the information about signalling cascades possibly involved in articular chondrocytes. Variable expression of AC isoforms 2, 4, 7, and 9 suggest a role for the signalling cascades regulated by the AC isoforms in articular chondrocytes. (author)

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    LinQuan Ge

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

  16. [Attenuation of inhibitory influence of hormones on adenylyl cyclase systems in the myocardium and brain of rats with obesity and type 2 diabetes mellitus and effect of intranasal insulin on it].

    Science.gov (United States)

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

    2014-01-01

    The functional state of the adenylyl cyclase signaling system (ACSS) and its regulation by hormones, the inhibitors of adenylyl cyclase (AC)--somatostatin (SST) in the brain and myocardium and 5-nonyloxytryptamine (5-NOT) in the brain of rats of different ages (5- and 7-month-old) with experimental obesity and a combination of obesity and type 2 diabetes mellitus (DM2), and the effect of long-term treatment of animals with intranasally administered insulin (II) on ACSS were studied. It was shown that the basal AC activity in rats with obesity and DM2 was increased in the myocardium, and to the lesser extent in the brain, the treatment with II reducing this parameter. The AC stimulating effects of forskolin are decreased in the myocardium, but not in the brain, of rats with obesity and DM2. The treatment with II restored the AC action of forskolin in the 7-month-old animals, but has little effect on it in the 5-month-old rats. In obesity the basal AC activity and its stimulation by forskolin varied insignificantly and weakly changed in treatment of animals with II. The AC inhibitory effects of SST and 5-NOT in the investigated pathology are essentially attenuated, the effect of SST to the greatest extent, which we believe to be associated with a reduction in the functional activity of Gi-proteins. The II treatment of animals with obesity and with a combination of obesity and DM2 restored completely or partially the AC inhibiting effects of hormones, to the greatest extent in the brain. Since impaired functioning of ACSS is one of the causes of the metabolic syndrome and DM2, their elimination by treatments with II can be an effective approach to treat these diseases and their CNS and cardiovascular system complications.

  17. Adenylyl cyclase-5 in the dorsal striatum function as a molecular switch for the generation of behavioral preferences for cue-directed food choices.

    Science.gov (United States)

    Kim, Hannah; Kim, Tae-Kyung; Kim, Ji-Eun; Park, Jin-Young; Lee, Yunjin; Kang, Minkyung; Kim, Kyoung-Shim; Han, Pyung-Lim

    2014-11-07

    Behavioral choices in habits and innate behaviors occur automatically in the absence of conscious selection. These behaviors are not easily modified by learning. Similar types of behaviors also occur in various mental illnesses including drug addiction, obsessive-compulsive disorder, schizophrenia, and autism. However, underlying mechanisms are not clearly understood. In the present study, we investigated the molecular mechanisms regulating unconditioned preferred behaviors in food-choices. Mice lacking adenylyl cyclase-5 (AC5 KO mice), which is preferentially expressed in the dorsal striatum, consumed food pellets nearly one after another in cages. AC5 KO mice showed aversive behaviors to bitter tasting quinine, but they compulsively chose quinine-containing AC5 KO-pellets over fresh pellets. The unusual food-choice behaviors in AC5 KO mice were due to the gain of behavioral preferences for food pellets containing an olfactory cue, which wild-type mice normally ignored. Such food-choice behaviors in AC5 KO mice disappeared when whiskers were trimmed. Conversely, whisker trimming in wildtype mice induced behavioral preferences for AC5 KO food pellets, indicating that preferred food-choices were not learned through prior experience. Both AC5 KO mice and wildtype mice with trimmed whiskers had increased glutamatergic input from the barrel cortex into the dorsal striatum, resulting in an increase in the mGluR1-dependent signaling cascade. The siRNA-mediated inhibition of mGluR1 in the dorsal striatum in AC5 KO mice and wildtype mice with trimmed whiskers abolished preferred choices for AC5 KO food pellets, whereas siRNA-mediated inhibition of mGluR3 glutamate receptors in the dorsal striatum in wildtype mice induced behavioral preferences for AC5 KO food pellets, thus mimicking AC5 KO phenotypes. Our results show that the gain and loss of behavioral preferences for a specific cue-directed option were regulated by specific cellular factors in the dorsal striatum, such

  18. 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.

  19. [THE CHANGES OF NOCICEPTIVE THRESHOLD AND ACTIVITY OF THE ADENYLYL CYCLASE SYSTEM IN THE SKELETAL MUSCLES OF RATS WITH ACUTE AND MILD TYPE 1 DIABETES MELLITUS ].

    Science.gov (United States)

    Shipilov, V N; Trost, A M; Chistyakova, O V; Derkach, K V; Shpakov, A O

    2016-02-01

    Diabetic peripheral neuropathy (DPN) is one of the most common complications of the type 1 diabetes mellitus (DM1). The aim of the work was to study the dynamics of a painful DPN and functional state of the hormone-sensitive ACSS in the skeletal muscles of rats with the models of acute and mild DM1, as well as the study of impact on them of insulin therapy with different ways of hormone delivery - intranasal and peripheral. In both models of DM1, the level of nociceptive threshold in rats decreased and the stimulatory effects of guanine nucleotides (GppNHp) and adrenergic agonists (isoproterenol, BRL-37344) on adenylyl cyclase (AC) activity were attenuated. The AC stimulating effect of relaxin decreased in animals with acute DM1, but in mild DM1, the decrease was insignificant. Peripheral administration of insulin in rats with acute DM1 increased the nociceptive threshold and partially restored the AC effect of ß 3-agonist BRL-37344. Intranasal administration of insulin in rats with DM1 also increased the nociceptive threshold and partially restored the basal and BRL-37344-stimulated AC activity in the skeletal muscles of diabetic animals. Thus, in the skeletal muscles of rats with acute and mild DM1 the nociceptive sensitivity and the functions of ACSS were disturbed, and they were partially restored by the treatment with peripheral (acute DM1) or intranasal (mild DM1) insulin.

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

    Science.gov (United States)

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

    2017-03-22

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

  1. Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice

    Directory of Open Access Journals (Sweden)

    Margarita O. Shleeva

    2017-08-01

    Full Text Available Earlier we demonstrated that the adenylyl cyclase (AC encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis (Mtb, the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro, the AC-overexpressing pMindRv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo, AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMindRv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo.

  2. 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.

  3. 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

  4. Essential Control of the Function of the Striatopallidal Neuron by Pre-coupled Complexes of Adenosine A2A-Dopamine D2 Receptor Heterotetramers and Adenylyl Cyclase

    Directory of Open Access Journals (Sweden)

    Sergi Ferré

    2018-04-01

    Full Text Available The central adenosine system and adenosine receptors play a fundamental role in the modulation of dopaminergic neurotransmission. This is mostly achieved by the strategic co-localization of different adenosine and dopamine receptor subtypes in the two populations of striatal efferent neurons, striatonigral and striatopallidal, that give rise to the direct and indirect striatal efferent pathways, respectively. With optogenetic techniques it has been possible to dissect a differential role of the direct and indirect pathways in mediating “Go” responses upon exposure to reward-related stimuli and “NoGo” responses upon exposure to non-rewarded or aversive-related stimuli, respectively, which depends on their different connecting output structures and their differential expression of dopamine and adenosine receptor subtypes. The striatopallidal neuron selectively expresses dopamine D2 receptors (D2R and adenosine A2A receptors (A2AR, and numerous experiments using multiple genetic and pharmacological in vitro, in situ and in vivo approaches, demonstrate they can form A2AR-D2R heteromers. It was initially assumed that different pharmacological interactions between dopamine and adenosine receptor ligands indicated the existence of different subpopulations of A2AR and D2R in the striatopallidal neuron. However, as elaborated in the present essay, most evidence now indicates that all interactions can be explained with a predominant population of striatal A2AR-D2R heteromers forming complexes with adenylyl cyclase subtype 5 (AC5. The A2AR-D2R heteromer has a tetrameric structure, with two homodimers, which allows not only multiple allosteric interactions between different orthosteric ligands, agonists, and antagonists, but also the canonical Gs-Gi antagonistic interaction at the level of AC5. We present a model of the function of the A2AR-D2R heterotetramer-AC5 complex, which acts as an integrative device of adenosine and dopamine signals that

  5. Inhibition of the adenylyl cyclase toxin, edema factor, from Bacillus anthracis by a series of 18 mono- and bis-(M)ANT-substituted nucleoside 5'-triphosphates.

    Science.gov (United States)

    Taha, Hesham; Dove, Stefan; Geduhn, Jens; König, Burkhard; Shen, Yuequan; Tang, Wei-Jen; Seifert, Roland

    2012-01-01

    Bacillus anthracis causes anthrax disease and exerts its deleterious effects by the release of three exotoxins, i.e. lethal factor, protective antigen and edema factor (EF), a highly active calmodulin-dependent adenylyl cyclase (AC). Conventional antibiotic treatment is ineffective against either toxaemia or antibiotic-resistant strains. Thus, more effective drugs for anthrax treatment are needed. Our previous studies showed that EF is differentially inhibited by various purine and pyrimidine nucleotides modified with N-methylanthraniloyl (MANT)- or anthraniloyl (ANT) groups at the 2'(3')-O-ribosyl position, with the unique preference for the base cytosine (Taha et al., Mol Pharmacol 75:693 (2009)). MANT-CTP was the most potent EF inhibitor (K (i), 100 nM) among 16 compounds studied. Here, we examined the interaction of EF with a series of 18 2',3'-O-mono- and bis-(M)ANT-substituted nucleotides, recently shown to be very potent inhibitors of the AC toxin from Bordetella pertussis, CyaA (Geduhn et al., J Pharmacol Exp Ther 336:104 (2011)). We analysed purified EF and EF mutants in radiometric AC assays and in fluorescence spectroscopy studies and conducted molecular modelling studies. Bis-MANT nucleotides inhibited EF competitively. Propyl-ANT-ATP was the most potent EF inhibitor (K (i), 80 nM). In contrast to the observations made for CyaA, introduction of a second (M)ANT-group decreased rather than increased inhibitor potency at EF. Activation of EF by calmodulin resulted in effective fluorescence resonance energy transfer (FRET) from tryptophan and tyrosine residues located in the vicinity of the catalytic site to bis-MANT-ATP, but FRET to bis-MANT-CTP was only small. Mutations N583Q, K353A and K353R differentially altered the inhibitory potencies of bis-MANT-ATP and bis-MANT-CTP. The nucleotide binding site of EF accommodates bulky bis-(M)ANT-substituted purine and pyrimidine nucleotides, but the fit is suboptimal compared to CyaA. These data provide a basis

  6. 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

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

    Directory of Open Access Journals (Sweden)

    Sarah J. Casey

    2014-03-01

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

  8. Adenylate cyclase regulation in intact cultured myocardial cells

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  9. 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

  10. 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...

  11. 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

  12. 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.

  13. 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.

  14. 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

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

    Directory of Open Access Journals (Sweden)

    Matthew M. Hurley

    2016-08-01

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

  16. 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.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    International Nuclear Information System (INIS)

    Beebe, L.E.

    1989-01-01

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

  19. 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

  20. 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

  1. 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.

  2. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

    Directory of Open Access Journals (Sweden)

    A Theron

    Full Text Available Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

  3. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform.

    Science.gov (United States)

    Theron, A; Roth, R L; Hoppe, H; Parkinson, C; van der Westhuyzen, C W; Stoychev, S; Wiid, I; Pietersen, R D; Baker, B; Kenyon, C P

    2017-01-01

    Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay.

  4. 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.

  5. 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

  6. 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.

  7. 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.

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

    Science.gov (United States)

    Han, P; Lucero, M T

    2005-01-01

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

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

    International Nuclear Information System (INIS)

    Johnson, G.P.

    1987-01-01

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

  10. Diguanylate cyclase null mutant reveals that C-Di-GMP pathway regulates the motility and adherence of the extremophile bacterium Acidithiobacillus caldus.

    Directory of Open Access Journals (Sweden)

    Matías Castro

    Full Text Available An understanding of biofilm formation is relevant to the design of biological strategies to improve the efficiency of the bioleaching process and to prevent environmental damages caused by acid mine/rock drainage. For this reason, our laboratory is focused on the characterization of the molecular mechanisms involved in biofilm formation in different biomining bacteria. In many bacteria, the intracellular levels of c-di-GMP molecules regulate the transition from the motile planktonic state to sessile community-based behaviors, such as biofilm development, through different kinds of effectors. Thus, we recently started a study of the c-di-GMP pathway in several biomining bacteria including Acidithiobacillus caldus. C-di-GMP molecules are synthesized by diguanylate cyclases (DGCs and degraded by phosphodiesterases (PDEs. We previously reported the existence of intermediates involved in c-di-GMP pathway from different Acidithiobacillus species. Here, we report our work related to At. caldus ATCC 51756. We identified several putative-ORFs encoding DGC and PDE and effector proteins. By using total RNA extracted from At. caldus cells and RT-PCR, we demonstrated that these genes are expressed. We also demonstrated the presence of c-di-GMP by mass spectrometry and showed that genes for several of the DGC enzymes were functional by heterologous genetic complementation in Salmonella enterica serovar Typhimurium mutants. Moreover, we developed a DGC defective mutant strain (Δc1319 that strongly indicated that the c-di-GMP pathway regulates the swarming motility and adherence to sulfur surfaces by At. caldus. Together, our results revealed that At. caldus possesses a functional c-di-GMP pathway which could be significant for ores colonization during the bioleaching process.

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

    Directory of Open Access Journals (Sweden)

    Jimena P Cabilla

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

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

    Science.gov (United States)

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

    2011-01-01

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

  13. 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

  14. 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.

  15. 17 beta-estradiol modifies nitric oxide-sensitive guanylyl cyclase expression and down-regulates its activity in rat anterior pituitary gland.

    Science.gov (United States)

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

    2006-09-01

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

  16. Role of adenylyl cyclase 6 in the development of lithium-induced nephrogenic diabetes insipidus

    DEFF Research Database (Denmark)

    Poulsen, Søren Brandt; Kristensen, Tina Bøgelund; Brooks, Heddwen L

    2017-01-01

    knockout (AC6(loxloxCre)) mice had approximately 50% lower urine osmolality and doubled water intake under baseline conditions compared with controls. Dietary Li(+) administration increased water intake and reduced urine osmolality in control, AC6(-/-), and AC6(loxloxCre) mice. Consistent with AC6......(-/-) mice, medullary AQP2 and pS256-AQP2 abundances were lower in AC6(loxloxCre) mice compared with controls under standard conditions, and levels were further reduced after Li(+) administration. AC6(loxloxCre) and control mice had a similar increase in the numbers of proliferating cell nuclear antigen......-positive cells in response to Li(+). However, AC6(loxloxCre) mice had a higher number of H(+)-ATPase B1 subunit-positive cells under standard conditions and after Li(+) administration. Collectively, AC6 has a minor role in Li-NDI development but may be important for determining the intercalated cell...

  17. The Emerging Role of Soluble Adenylyl Cyclase in Primary Biliary Cholangitis

    NARCIS (Netherlands)

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

    2017-01-01

    Primary biliary cholangitis (PBC; previously referred to as primary biliary cirrhosis) is a chronic fibrosing cholangiopathy with the signature of an autoimmune disease and features of intrahepatic cholestasis. Immunosuppressing treatments are largely unsuccessful. Responsiveness to ursodeoxycholic

  18. Altered gene regulation and synaptic morphology in Drosophila learning and memory mutants

    Science.gov (United States)

    Guan, Zhuo; Buhl, Lauren K.; Quinn, William G.; Littleton, J. Troy

    2011-01-01

    Genetic studies in Drosophila have revealed two separable long-term memory pathways defined as anesthesia-resistant memory (ARM) and long-lasting long-term memory (LLTM). ARM is disrupted in radish (rsh) mutants, whereas LLTM requires CREB-dependent protein synthesis. Although the downstream effectors of ARM and LLTM are distinct, pathways leading to these forms of memory may share the cAMP cascade critical for associative learning. Dunce, which encodes a cAMP-specific phosphodiesterase, and rutabaga, which encodes an adenylyl cyclase, both disrupt short-term memory. Amnesiac encodes a pituitary adenylyl cyclase-activating peptide homolog and is required for middle-term memory. Here, we demonstrate that the Radish protein localizes to the cytoplasm and nucleus and is a PKA phosphorylation target in vitro. To characterize how these plasticity pathways may manifest at the synaptic level, we assayed synaptic connectivity and performed an expression analysis to detect altered transcriptional networks in rutabaga, dunce, amnesiac, and radish mutants. All four mutants disrupt specific aspects of synaptic connectivity at larval neuromuscular junctions (NMJs). Genome-wide DNA microarray analysis revealed ∼375 transcripts that are altered in these mutants, suggesting defects in multiple neuronal signaling pathways. In particular, the transcriptional target Lapsyn, which encodes a leucine-rich repeat cell adhesion protein, localizes to synapses and regulates synaptic growth. This analysis provides insights into the Radish-dependent ARM pathway and novel transcriptional targets that may contribute to memory processing in Drosophila. PMID:21422168

  19. 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.

  20. 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...

  1. Altered Regulation of the Diguanylate Cyclase YaiC Reduces Production of Type 1 Fimbriae in a Pst Mutant of Uropathogenic Escherichia coli CFT073.

    Science.gov (United States)

    Crépin, Sébastien; Porcheron, Gaëlle; Houle, Sébastien; Harel, Josée; Dozois, Charles M

    2017-12-15

    The pst gene cluster encodes the phosphate-specific transport (Pst) system. Inactivation of the Pst system constitutively activates the two-component regulatory system PhoBR and attenuates the virulence of pathogenic bacteria. In uropathogenic Escherichia coli strain CFT073, attenuation by inactivation of pst is predominantly attributed to the decreased expression of type 1 fimbriae. However, the molecular mechanisms connecting the Pst system and type 1 fimbriae are unknown. To address this, a transposon library was constructed in the pst mutant, and clones were tested for a regain in type 1 fimbrial production. Among them, the diguanylate cyclase encoded by yaiC ( adrA in Salmonella ) was identified to connect the Pst system and type 1 fimbrial expression. In the pst mutant, the decreased expression of type 1 fimbriae is connected by the induction of yaiC This is predominantly due to altered expression of the FimBE-like recombinase genes ipuA and ipbA , affecting at the same time the inversion of the fim promoter switch ( fimS ). In the pst mutant, inactivation of yaiC restored fim -dependent adhesion to bladder cells and virulence. Interestingly, the expression of yaiC was activated by PhoB, since transcription of yaiC was linked to the PhoB-dependent phoA-psiF operon. As YaiC is involved in cyclic di-GMP (c-di-GMP) biosynthesis, an increased accumulation of c-di-GMP was observed in the pst mutant. Hence, the results suggest that one mechanism by which deletion of the Pst system reduces the expression of type 1 fimbriae is through PhoBR-mediated activation of yaiC , which in turn increases the accumulation of c-di-GMP, represses the fim operon, and, consequently, attenuates virulence in the mouse urinary tract infection model. IMPORTANCE Urinary tract infections (UTIs) are common bacterial infections in humans. They are mainly caused by uropathogenic Escherichia coli (UPEC). We previously showed that interference with phosphate homeostasis decreases the

  2. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform

    CSIR Research Space (South Africa)

    Theron, Anjo

    2017-10-01

    Full Text Available mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does...

  3. 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.

  4. 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...

  5. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    Science.gov (United States)

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. © 2015 Lama and Ryan; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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

    Science.gov (United States)

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

    1988-03-01

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

  7. 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...

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

    Science.gov (United States)

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

    2005-07-01

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

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Justin Schleede

    2015-10-01

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

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

    Science.gov (United States)

    Schleede, Justin; Blair, Seth S

    2015-10-01

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

  12. 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

  13. 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

  14. Corticotropin-releasing factor critical for zebrafish camouflage behavior is regulated by light and sensitive to ethanol.

    Science.gov (United States)

    Wagle, Mahendra; Mathur, Priya; Guo, Su

    2011-01-05

    The zebrafish camouflage response is an innate "hard-wired" behavior that offers an excellent opportunity to explore neural circuit assembly and function. Moreover, the camouflage response is sensitive to ethanol, making it a tractable system for understanding how ethanol influences neural circuit development and function. Here we report the identification of corticotropin-releasing factor (CRF) as a critical component of the camouflage response pathway. We further show that ethanol, having no direct effect on the visual sensory system or the melanocytes, acts downstream of retinal ganglion cells and requires the CRF-proopiomelanocortin pathway to exert its effect on camouflage. Treatment with ethanol, as well as alteration of light exposure that changes sensory input into the camouflage circuit, robustly modifies CRF expression in subsets of neurons. Activity of both adenylyl cyclase 5 and extracellular signal-regulated kinase (ERK) is required for such ethanol-induced or light-induced plasticity of crf expression. These results reveal an essential role of a peptidergic pathway in camouflage that is regulated by light and influenced by ethanol at concentrations relevant to abuse and anxiolysis, in a cAMP-dependent and ERK-dependent manner. We conclude that this ethanol-modulated camouflage response represents a novel and relevant system for molecular genetic dissection of a neural circuit that is regulated by light and sensitive to ethanol.

  15. Up-regulation of adenylylcyclases I and II induced by long-term adaptation of rats to morphine fades away 20 days after morphine withdrawal

    Czech Academy of Sciences Publication Activity Database

    Ujčíková, Hana; Dlouhá, Kateřina; Roubalová, Lenka; Vošahlíková, Miroslava; Kagan, Dmytro; Svoboda, Petr

    2011-01-01

    Roč. 1810, č. 12 (2011), s. 1220-1229 ISSN 0304-4165 R&D Projects: GA MŠk(CZ) LC554; GA MŠk(CZ) LC06063; GA ČR(CZ) GD305/08/H037 Institutional research plan: CEZ:AV0Z50110509 Keywords : morphine * long-term adaptation * adenylyl cyclase isoforms I–IX * forebrain cortex * isolated plasma membranes Subject RIV: FH - Neurology Impact factor: 5.000, year: 2011

  16. Delivery of Large Heterologous Polypeptides across the Cytoplasmic Membrane of Antigen-Presenting Cells by the Bordetella RTX Hemolysin Moiety Lacking the Adenylyl Cyclase Domain

    Czech Academy of Sciences Publication Activity Database

    Holubová, Jana; Kamanová, Jana; Jelínek, J.; Tomala, Jakub; Mašín, Jiří; Kosová, Martina; Staněk, Ondřej; Bumba, Ladislav; Michálek, J.; Kovář, Marek; Šebo, Peter

    2012-01-01

    Roč. 80, č. 3 (2012), s. 1181-1192 ISSN 0019-9567 R&D Projects: GA AV ČR IAA500200914; GA ČR(CZ) GAP207/11/0717; GA ČR GAP301/11/0325; GA MŠk 1M0506; GA MŠk 2B06161 Institutional research plan: CEZ:AV0Z50200510 Keywords : MHC CLASS-I * ESCHERICHIA-COLI * PRESENTATION PATHWAY Subject RIV: EE - Microbiology, Virology Impact factor: 4.074, year: 2012

  17. Cyclic adenosine monophosphate metabolism in synaptic growth, strength, and precision: neural and behavioral phenotype-specific counterbalancing effects between dnc phosphodiesterase and rut adenylyl cyclase mutations.

    Science.gov (United States)

    Ueda, Atsushi; Wu, Chun-Fang

    2012-03-01

    Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cyclic adenosine monophosphate (cAMP) synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. We ask how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. Our study at larval neuromuscular junctions (NMJs) demonstrates that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29-30°C) decreased synaptic transmission in rut, but did not alter dnc and wild-type (WT). Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, we reviewed previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. We discuss factors that could contribute to the effectiveness of counterbalancing interactions between dnc and rut mutations for phenotypic rescue.

  18. Odorant Receptor 51E2 Agonist β-ionone Regulates RPE Cell Migration and Proliferation

    Directory of Open Access Journals (Sweden)

    Nikolina Jovancevic

    2017-11-01

    Full Text Available The odorant receptor 51E2 (OR51E2, which is well-characterized in prostate cancer cells and epidermal pigment cells, was identified for the first time as the most highly expressed OR in human fetal and adult retinal pigment epithelial (RPE cells. Immunofluorescence staining and Western blot analysis revealed OR51E2 localization throughout the cytosol and in the plasma membrane. Additionally, immunohistochemical staining of diverse layers of the eye showed that the expression of OR51E2 is restricted to the pigment cells of the RPE and choroid. The results of Ca2+-imaging experiments demonstrate that activation of OR51E2 triggers a Ca2+ dependent signal pathway in RPE cells. Downstream signaling of OR51E2 involves the activation of adenylyl cyclase, ERK1/2 and AKT. The activity of these protein kinases likely accounts for the demonstrated increase in the migration and proliferation of RPE cells upon stimulation with the OR51E2 ligand β-ionone. These findings suggest that OR51E2 is involved in the regulation of RPE cell growth. Thus, OR51E2 represents a potential target for the treatment of proliferative disorders.

  19. Regulation of melanogenesis: the role of cAMP and MITF

    Directory of Open Access Journals (Sweden)

    Michał Otręba

    2012-01-01

    Full Text Available The article presents the melanogenesis pathway and the role of cyclic adenosine monophosphate (cAMP and microphthalmia transcription factor (MITF in regulation of this process. Products of melanogenesis are eu- and/or pheomelanins synthesized in a multistage process of tyrosine oxidation and polymerization. The conversions require the presence of tyrosinase (TYR, key enzyme, tyrosine hydroxylase isoform I (THI and tyrosinase related proteins (TRP1 and TRP2. Many types of signal molecules and transcription factors participate in regulation of melanin synthesis, but the most important are cAMP and MITF. cAMP is the second messenger in the intracellular signal cascade, which is synthesized from adenosine triphosphate (ATP by adenylyl cyclase, activated among others by the melanocortin receptor and the αS subunit of G protein. The signal molecule cAMP regulates MITF, TYR, THI, GTP-cyclohydroxylase I (GTP-CHI transcription and phenylalanine hydroxylase (PAH phosphorylation at Ser16 by protein kinase A (PKA. Mutations of genes encoding proteins belonging to the cAMP signal cascade may lead to McCune-Albright and Carney syndromes. MITF is one of the most important nuclear transcription factors regulating melanogenesis. Currently 10 isoforms of human MITF are known, but in melanocytes only MITF-M, MITF-Mdel, MITF-A and MITF-H occur. MITF transcription factor regulates melanogenesis by activation of tyrosinase, TRP1 and TRP2 transcription. It also affects expression of other factors regulating melanosome maturation, biogenesis and transport. Moreover, it regulates melanocyte proliferation and protection against apoptosis. Mutations of the MITF gene may lead to hereditary diseases: Waardenburg type II and Tietz syndromes.

  20. 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

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

    Directory of Open Access Journals (Sweden)

    Yasmeen Faiz Kazi

    2012-06-01

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

  2. DMT efficiently inhibits hepatic gluconeogenesis by regulating the Gαq signaling pathway.

    Science.gov (United States)

    Zhou, Ting-Ting; Ma, Fei; Shi, Xiao-Fan; Xu, Xin; Du, Te; Guo, Xiao-Dan; Wang, Gai-Hong; Yu, Liang; Rukachaisirikul, Vatcharin; Hu, Li-Hong; Chen, Jing; Shen, Xu

    2017-08-01

    Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with complicated pathogenesis and targeting gluconeogenesis inhibition is a promising strategy for anti-diabetic drug discovery. G protein-coupled receptors (GPCRs) are classified as distinct families by heterotrimeric G proteins, primarily including Gαs, Gαi and Gαq. Gαs-coupled GPCRs function potently in the regulation of hepatic gluconeogenesis by activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and Gαi-coupled GPCRs exhibit inhibitory effect on adenylyl cyclase and reduce intracellular cAMP level. However, little is known about the regulation of Gαq-coupled GPCRs in hepatic gluconeogenesis. Here, small-molecule 2-(2,4-dimethoxy-3-methylphenyl)-7-(thiophen-2-yl)-9-(trifluoromethyl)-2,3-dihydropyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4( 1H )-one (DMT) was determined to suppress hepatic glucose production and reduce mRNA levels of gluconeogenic genes. Treatment of DMT in db/db mice decreased fasting blood glucose and hemoglobin A1C (HbA1c) levels, while improved glucose tolerance and pyruvate tolerance. Mechanism study demonstrated that DMT-inhibited gluconeogenesis by regulating the Gαq/phospholipase C (PLC)/inositol-1,4,5-triphosphate receptor (IP3R)-mediated calcium (Ca 2+ )/calmodulin (CaM)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT)/forkhead box protein O1 (FOXO1) signaling pathway. To our knowledge, DMT might be the first reported small molecule able to suppress hepatic gluconeogenesis by regulating Gαq signaling, and our current work has also highlighted the potential of DMT in the treatment of T2DM. © 2017 Society for Endocrinology.

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

    Science.gov (United States)

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

    1981-08-01

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

  4. Activation of Adenosine Receptor A2A Increases HSC Proliferation and Inhibits Death and Senescence by Down-regulation of p53 and Rb

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    Md. Kaimul eAhsan

    2014-04-01

    Full Text Available Background & Aims: During fibrosis hepatic stellate cells (HSC undergo activation, proliferation and senescence but the regulation of these important processes is poorly understood. The adenosine A2A receptor (A2A is known to be present on HSC, and its activation results in liver fibrosis. In this study, we tested if A2A has a role in the regulation of HSC proliferation, apoptosis, senescence, and the relevant molecular mechanism.Methods: The ability of adenosine to regulate p53 and Rb protein levels, proliferation, apoptosis and senescence was tested in the human HSC cell line LX-2 and rat primary HSC.Results: Adenosine receptor activation down-regulates p53 and Rb protein levels, increases BrdU incorporation and increases cell survival in LX-2 cells and in primary rat HSC. These effects of NECA were reproduced by an adenosine A2A receptor specific agonist (CGS21680 and blocked by a specific antagonist (ZM241385. By day twenty-one of culture primary rat HSC entered senescence and expressed -gal which was significantly inhibited by NECA. Furthermore, NECA induced down regulation of p53 and Rb and Rac1, and decreased phosphorylation of p44-42 MAP Kinase in LX-2 cells and primary rat HSC. These effects were reproduced by the cAMP analog 8-Bromo-cAMP, and the adenylyl cyclase activator forskolin, and were blocked by PKA inhibitors.Conclusions: These results demonstrate that A2A receptor regulates a number of HSC fate decisions and induces greater HSC proliferation, reduces apoptosis and senescence by decreasing p53 and Rb through cAMP-PKA/Rac1/p38 MAPK pathway. This provides a mechanism for adenosine induced HSC regulation and liver fibrosis.

  5. Adenylyl cylases 1 and 8 mediate select striatal-dependent behaviors and sensitivity to ethanol stimulation in the adolescent period following acute neonatal ethanol exposure.

    Science.gov (United States)

    Susick, Laura L; Lowing, Jennifer L; Bosse, Kelly E; Hildebrandt, Clara C; Chrumka, Alexandria C; Conti, Alana C

    2014-08-01

    Neonatal alcohol exposure in rodents causes dramatic neurodegenerative effects throughout the developing nervous system, particularly in the striatum, acutely after exposure. These acute neurodegenerative effects are augmented in mice lacking adenylyl cyclases 1 and 8 (AC1/8) as neonatal mice with a genetic deletion of both AC isoforms (DKO) have increased vulnerability to ethanol-induced striatal neurotoxicity compared to wild type (WT) controls. While neonatal ethanol exposure is known to negatively impact cognitive behaviors, such as executive functioning and working memory in adolescent and adult animals, the threshold of ethanol exposure required to impinge upon developmental behaviors in mice has not been extensively examined. Therefore, the purpose of this study was to determine the behavioral effects of neonatal ethanol exposure using various striatal-dependent developmental benchmarks and to assess the impact of AC1/8 deletion on this developmental progression. WT and DKO mice were treated with 2.5 g/kg ethanol or saline on postnatal day (P)6 and later subjected to the wire suspension, negative geotaxis, postural reflex, grid hang, tail suspension and accelerating rotarod tests at various time points. At P30, mice were evaluated for their hypnotic responses to 4.0 g/kg ethanol by using the loss of righting reflex assay and ethanol-induced stimulation of locomotor activity after 2.0 g/kg ethanol. Ethanol exposure significantly impaired DKO performance in the negative geotaxis test while genetic deletion of AC1/8 alone increased grid hang time and decreased immobility time in the tail suspension test with a concomitant increase in hindlimb clasping behavior. Locomotor stimulation was significantly increased in animals that received ethanol as neonates, peaking significantly in ethanol-treated DKO mice compared to ethanol-treated WT controls, while sedation duration following high-dose ethanol challenge was unaffected. These data indicate that the

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

    Directory of Open Access Journals (Sweden)

    Teresa eDuda

    2014-03-01

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

  7. Diphtheria toxin can simultaneously bind to its receptor and adenylyl-(3',5')-uridine 3'-monophosphate

    International Nuclear Information System (INIS)

    Barbieri, J.T.; Collins, C.M.; Collier, R.J.

    1986-01-01

    Diphtheria toxin (DT) that was bound to receptors on BS-C-1 cells was able to bind approximately 1 molar equiv of adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). In contrast, receptor-bound CRM197, a mutant form of toxin with greatly diminished affinity for dinucleotides, did not bind ApUp. Affinity of the dinucleotide for receptor-bound toxin differed from that for free toxin by less than an order of magnitude. These results indicate that the receptor site and the ApUp site on the toxin do not significantly overlap. BS-C-1 cells were incubated with or without 125 I-DT or CRM 197. They were then incubated with [ 32 P]ApUp, and assayed

  8. 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

  9. 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

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

    Directory of Open Access Journals (Sweden)

    Dev Churamani

    2007-08-01

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

  11. 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.

  12. Haloperidol Regulates the State of Phosphorylation of Ribosomal Protein S6 via Activation of PKA and Phosphorylation of DARPP-32

    Science.gov (United States)

    Valjent, Emmanuel; Bertran-Gonzalez, Jesus; Bowling, Heather; Lopez, Sébastien; Santini, Emanuela; Matamales, Miriam; Bonito-Oliva, Alessandra; Hervé, Denis; Hoeffer, Charles; Klann, Eric; Girault, Jean-Antoine; Fisone, Gilberto

    2011-01-01

    Administration of typical antipsychotic drugs, such as haloperidol, promotes cAMP-dependent signaling in the medium spiny neurons (MSNs) of the striatum. In this study, we have examined the effect of haloperidol on the state of phosphorylation of the ribosomal protein S6 (rpS6), a component of the small 40S ribosomal subunit. We found that haloperidol increases the phosphorylation of rpS6 at the dual site Ser235/236, which is involved in the regulation of mRNA translation. This effect was exerted in the MSNs of the indirect pathway, which express specifically dopamine D2 receptors (D2Rs) and adenosine A2 receptors (A2ARs). The effect of haloperidol was decreased by blockade of A2ARs or by genetic attenuation of the Gαolf protein, which couples A2ARs to activation of adenylyl cyclase. Moreover, stimulation of cAMP-dependent protein kinase A (PKA) increased Ser235/236 phosphorylation in cultured striatal neurons. The ability of haloperidol to promote rpS6 phosphorylation was abolished in knock-in mice deficient for PKA activation of the protein phosphatase-1 inhibitor, dopamine- and cAMP-regulated phosphoprotein of 32 kDa. In contrast, pharmacological or genetic inactivation of p70 rpS6 kinase 1, or extracellular signal-regulated kinases did not affect haloperidol-induced rpS6 phosphorylation. These results identify PKA as a major rpS6 kinase in neuronal cells and suggest that regulation of protein synthesis through rpS6 may be a potential target of antipsychotic drugs. PMID:21814187

  13. 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

  14. Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology

    Directory of Open Access Journals (Sweden)

    Callihan Phillip

    2008-12-01

    Full Text Available Abstract Background Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development. Results Our results demonstrate that Lysophosphatidic Acid (LPA and Sphingosine-1-phosphate (S1P receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK. Conclusion Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

  15. 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.

  16. 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

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

    Science.gov (United States)

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

    2012-07-03

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

  18. 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

  19. 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

  20. 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

  1. Regulation of estradiol and progesterone production by CRH-R1 and -R2 is through divergent signaling pathways in cultured human placental trophoblasts.

    Science.gov (United States)

    Gao, Lu; Tao, Yi; Hu, Tianxiao; Liu, Weina; Xu, Chen; Liu, Jie; You, Xingji; Gu, Hang; Ni, Xin

    2012-10-01

    CRH and its related peptides urocortins (UCN) have been identified in placenta and implicated to play pivotal roles in the regulation of pregnancy and parturition in humans. The objectives of present study were to investigate the effects of endogenous CRH and its related peptides in the regulation of steroid production in placenta. Placental trophoblasts were isolated from term placenta tissues and cultured for 72 h. Estradiol (E(2)) and progesterone (P(4)) contents in culture media were determined by radioimmunoassay. Treatment of cultured trophoblasts with CRH or UCNI antibody showed decreased E(2), whereas increased P(4) production. Treatment of cells with CRH receptor type 1 antagonist antalarmin or CRH receptor type 2 (CRH-R2) antagonist astressin-2b also decreased E(2) but increased P(4) production. Knockdown of CRH receptor type 1 or CRH-R2 cells showed a decrease in E(2) production and an increase in P(4) production. In CRH-R2 knockdown cells, CRH stimulated GTP-bound Gαs protein and phosphorylated phospholipase C-β3. Adenylyl cyclase and protein kinase A inhibitors blocked CRH-induced increased E(2) production but not decreased P(4) production. PLC inhibitor U73122 and protein kinase C inhibitor chelerythrine blocked the effects of CRH on E(2) and P(4) production in CRH-R2 knockdown cells. UCNIII, the specific CRH-R2 agonist, stimulated GTP-bound Gαi protein and phosphorylated phospholipase C-β3 expression. Both U73122 and chelerythrine blocked UCNIII-induced increased E(2) production and decreased P(4) production. We suggest that CRH and its related peptides might be involved in changes in the progesterone to estrogen ratio during human pregnancy.

  2. The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules.

    Science.gov (United States)

    Simon, Katharina; Hennen, Stephanie; Merten, Nicole; Blättermann, Stefanie; Gillard, Michel; Kostenis, Evi; Gomeza, Jesus

    2016-01-08

    Recent studies have recognized G protein-coupled receptors as important regulators of oligodendrocyte development. GPR17, in particular, is an orphan G protein-coupled receptor that has been identified as oligodendroglial maturation inhibitor because its stimulation arrests primary mouse oligodendrocytes at a less differentiated stage. However, the intracellular signaling effectors transducing its activation remain poorly understood. Here, we use Oli-neu cells, an immortalized cell line derived from primary murine oligodendrocytes, and primary rat oligodendrocyte cultures as model systems to identify molecular targets that link cell surface GPR17 to oligodendrocyte maturation blockade. We demonstrate that stimulation of GPR17 by the small molecule agonist MDL29,951 (2-carboxy-4,6-dichloro-1H-indole-3-propionic acid) decreases myelin basic protein expression levels mainly by triggering the Gαi/o signaling pathway, which in turn leads to reduced activity of the downstream cascade adenylyl cyclase-cAMP-PKA-cAMP response element-binding protein (CREB). In addition, we show that GPR17 activation also diminishes myelin basic protein abundance by lessening stimulation of the exchange protein directly activated by cAMP (EPAC), thus uncovering a previously unrecognized role for EPAC to regulate oligodendrocyte differentiation. Together, our data establish PKA and EPAC as key downstream effectors of GPR17 that inhibit oligodendrocyte maturation. We envisage that treatments augmenting PKA and/or EPAC activity represent a beneficial approach for therapeutic enhancement of remyelination in those demyelinating diseases where GPR17 is highly expressed, such as multiple sclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Towards a multiscale description of microvascular flow regulation: O2-dependent release of ATP from human erythrocytes and the distribution of ATP in capillary networks

    Directory of Open Access Journals (Sweden)

    Daniel eGoldman

    2012-07-01

    Full Text Available Integration of the numerous mechanisms that have been suggested to contribute to optimization of O2 supply to meet O2 need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O2 tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP. This cAMP then activates PKA and subsequently CFTR culminating in ATP release via pannexin 1. A critical control point in this pathway is the level of cAMP which is regulated by pathway-specific phosphodiesterases. Using time constants (~100ms that are consistent with measured erythrocyte ATP release, we have constructed a dynamic model of this pathway. The model predicts levels of ATP release consistent with measurements obtained over a wide range of hemoglobin O2 saturations (sO2. The model further predicts how insulin, at concentrations found in prediabetes, enhances the activity of PDE3 and reduces intracellular cAMP levels leading to decreased low O2-induced ATP release from erythrocytes. The second model, which couples O2 and ATP transport in capillary networks, shows how intravascular ATP and the resulting conducted vasodilation are affected by local sO2, convection and ATP degradation. This model also predicts network-level effects of decreased ATP release resulting from elevated insulin levels. Taken together, these models lay the groundwork for investigating the systems biology of the regulation of microvascular perfusion distribution by

  4. Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

    Science.gov (United States)

    Li, Ting; Liu, Nannan

    2017-12-01

    This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

  5. Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling

    KAUST Repository

    Gehring, Christoph A.

    2017-10-04

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

  6. 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.

  7. 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

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

    Directory of Open Access Journals (Sweden)

    Chinnusamy Viswanathan

    2010-04-01

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

  9. 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.

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

    International Nuclear Information System (INIS)

    Katz, M.S.

    1988-01-01

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

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

    International Nuclear Information System (INIS)

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

    1976-01-01

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

  12. 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.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  14. 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.

  15. Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP mediated PKA-dependent Ca2+ cycling with surface membrane channels

    Science.gov (United States)

    Vinogradova, Tatiana M.; Lakatta, Edward G.

    2009-01-01

    Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca2+ and, specifically Ca2+-release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca2+ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cells spontaneous firing. Local Ca2+ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca2+ releases activate an inward Na+-Ca2+ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca2+ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca2+ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation. PMID:19573534

  16. 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.

  17. 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

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

    Science.gov (United States)

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

    1975-08-22

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

  19. 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...

  20. 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.

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  2. 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.

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

    Science.gov (United States)

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

    2014-01-17

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

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Science.gov (United States)

    Lin, Jieru E; Li, Peng; Pitari, Giovanni M; Schulz, Stephanie; Waldman, Scott A

    2009-05-01

    Colorectal cancer is one of the leading causes of tumor-related morbidity and mortality worldwide. While mechanisms underlying this disease have been elucidated over the past two decades, these molecular insights have failed to translate into efficacious therapy. The oncogenomic view of cancer suggests that terminal transformation reflects the sequential corruption of signal transduction circuits regulating key homeostatic mechanisms, whose multiplicity underlies the therapeutic resistance of most tumors to interventions targeting individual pathways. Conversely, the paucity of mechanistic insights into proximal pathophysiological processes that initiate and amplify oncogenic circuits preceding accumulation of mutations and transformation impedes development of effective prevention and therapy. In that context, guanylyl cyclase C (GCC), the intestinal receptor for the paracrine hormones guanylin and uroguanylin, whose early loss characterizes colorectal transformation, has emerged as a component of lineage-specific homeostatic programs organizing spatiotemporal patterning along the crypt-surface axis. Dysregulation of GCC signaling, reflecting hormone loss, promotes tumorigenesis through reprogramming of replicative and bioenergetic circuits and genomic instability. Compensatory upregulation of GCC in response to hormone loss provides a unique translational opportunity for prevention and treatment of colorectal tumors by hormone-replacement therapy.

  6. 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.

  7. Cyclic Nucleotide-Gated Channels, Calmodulin, Adenylyl Cyclase, and Calcium/Calmodulin-Dependent Protein Kinase II Are Required for Late, but Not Early, Long-Term Memory Formation in the Honeybee

    Science.gov (United States)

    Matsumoto, Yukihisa; Sandoz, Jean-Christophe; Devaud, Jean-Marc; Lormant, Flore; Mizunami, Makoto; Giurfa, Martin

    2014-01-01

    Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee "Apis mellifera," olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM)…

  8. 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.

  9. Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yan Dong

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

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

    Directory of Open Access Journals (Sweden)

    Xiaonan Han

    2011-01-01

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

  12. 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...

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

    Directory of Open Access Journals (Sweden)

    Tian-Jun Cao

    2017-04-01

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

  14. 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.

  15. 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.

  16. Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana

    Science.gov (United States)

    Wang, Zhonghua; Yeats, Trevor; Han, Hong; Jetter, Reinhard

    2010-01-01

    The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C30H50O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761–779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores. PMID:20610397

  17. Polarized Th1 and Th2 cells are less responsive to negative feedback by receptors coupled to the AC/cAMP system compared to freshly isolated T cells

    NARCIS (Netherlands)

    Heijink, Irene H; Vellenga, Edo; Borger, Peter; Postma, Dirkje S; Monchy, Jan G R de; Kauffman, Henk F

    1 The adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP) system is known to negatively regulate transcriptional activity of T cells, thereby possibly modulating T-cell-mediated responses at the sites of inflammation. Effects of cAMP have been widely studied in freshly isolated T cells and

  18. Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches

    NARCIS (Netherlands)

    Postma, M.; Roelofs, J.; Goedhart, J.; Loovers, H.M.; Visser, A.J.W.G.; Haastert, van P.J.M.

    2004-01-01

    The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate

  19. Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches.

    NARCIS (Netherlands)

    Postma, M.; Roelofs, J.; Goedhart, J.; Loovers, H.M.; Visser, A.J.; van Haastert, P.J.

    2004-01-01

    The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. 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

  5. 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...

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

    KAUST Repository

    Turek, Ilona

    2016-03-05

    The functional homologues of vertebrate natriuretic peptides (NPs), the plant natriuretic peptides (PNPs), are a novel class of peptidic hormones that signal via guanosine 3′,5′-cyclic monophosphate (cGMP) and systemically affect plant salt and water balance and responses to biotrophic plant pathogens. Although there is increasing understanding of the complex roles of PNPs in plant responses at the systems level, little is known about the underlying signaling mechanisms. Here we report isolation and identification of a novel Leucine-Rich Repeat (LRR) protein that directly interacts with A. thaliana PNP, AtPNP-A. In vitro binding studies revealed that the Arabidopsis AtPNP-A binds specifically to the LRR protein, termed AtPNP-R1, and the active region of AtPNP-A is sufficient for the interaction to occur. Importantly, the cytosolic part of the AtPNP-R1, much like in some vertebrate NP receptors, harbors a catalytic center diagnostic for guanylyl cyclases and the recombinant AtPNP-R1 is capable of catalyzing the conversion of guanosine triphosphate to cGMP. In addition, we show that AtPNP-A causes rapid increases of cGMP levels in wild type (WT) leaf tissue while this response is significantly reduced in the atpnp-r1 mutants. AtPNP-A also causes cGMP-dependent net water uptake into WT protoplasts, and hence volume increases, whereas responses of the protoplasts from the receptor mutant are impaired. Taken together, our results suggest that the identified LRR protein is an AtPNP-A receptor essential for the PNP-dependent regulation of ion and water homeostasis in plants and that PNP- and vertebrate NP-receptors and their signaling mechanisms share surprising similarities. © 2016 Springer Science+Business Media Dordrecht

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

    Directory of Open Access Journals (Sweden)

    Satoko eHattori

    2012-10-01

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

  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.

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

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

  10. 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.

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

    KAUST Repository

    Hussein, Rana

    2012-01-01

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

  12. 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

  13. 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

  14. 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.

  15. 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 ...

  16. 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.

  17. 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.

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

    Science.gov (United States)

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

    2018-06-01

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

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

    Directory of Open Access Journals (Sweden)

    Jennifer C Irvine

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

  20. Acute up-regulation of the rat brain somatostatin receptor-effector system by leptin is related to activation of insulin signaling and may counteract central leptin actions.

    Science.gov (United States)

    Perianes-Cachero, A; Burgos-Ramos, E; Puebla-Jiménez, L; Canelles, S; Frago, L M; Hervás-Aguilar, A; de Frutos, S; Toledo-Lobo, M V; Mela, V; Viveros, M P; Argente, J; Chowen, J A; Arilla-Ferreiro, E; Barrios, V

    2013-11-12

    Leptin and somatostatin (SRIF) have opposite effects on food seeking and ingestive behaviors, functions partially regulated by the frontoparietal cortex and hippocampus. Although it is known that the acute suppression of food intake mediated by leptin decreases with time, the counter-regulatory mechanisms remain unclear. Our aims were to analyze the effect of acute central leptin infusion on the SRIF receptor-effector system in these areas and the implication of related intracellular signaling mechanisms in this response. We studied 20 adult male Wister rats including controls and those treated intracerebroventricularly with a single dose of 5 μg of leptin and sacrificed 1 or 6h later. Density of SRIF receptors was unchanged at 1h, whereas leptin increased the density of SRIF receptors at 6h, which was correlated with an elevated capacity of SRIF to inhibit forskolin-stimulated adenylyl cyclase activity in both areas. The functional capacity of SRIF receptors was unaltered as cell membrane levels of αi1 and αi2 subunits of G inhibitory proteins were unaffected in both brain areas. The increased density of SRIF receptors was due to enhanced SRIF receptor subtype 2 (sst2) protein levels that correlated with higher mRNA levels for this receptor. These changes in sst2 mRNA levels were concomitant with increased activation of the insulin signaling, c-Jun and cyclic AMP response element-binding protein (CREB); however, activation of signal transducer and activator of transcription 3 was reduced in the cortex and unchanged in the hippocampus and suppressor of cytokine signaling 3 remained unchanged in these areas. In addition, the leptin antagonist L39A/D40A/F41A blocked the leptin-induced changes in SRIF receptors, leptin signaling and CREB activation. In conclusion, increased activation of insulin signaling after leptin infusion is related to acute up-regulation of the SRIF receptor-effector system that may antagonize short-term leptin actions in the rat brain

  1. Regulation of rat hepatocyte function by P2Y receptors: focus on control of glycogen phosphorylase and cyclic AMP by 2-methylthioadenosine 5'-diphosphate.

    Science.gov (United States)

    Dixon, C Jane; Hall, John F; Webb, Tania E; Boarder, Michael R

    2004-10-01

    Hepatocyte function is regulated by several P2Y receptor subtypes. Here we report that 2-methylthioadenosine 5'-diphosphate (2-MeSADP), an agonist at P2Y(1), P2Y(12), and P2Y(13) receptors, potently (threshold 30 nM) stimulates glycogen phosphorylase in freshly isolated rat hepatocytes. Antagonism by N(6)-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS 2179) confirms that this response is mediated by P2Y(1) receptors. In addition, in these cells, both 2-MeSADP and UTP inhibited glucagon-stimulated cyclic AMP accumulation. This inhibitory effect of 2-MeSADP was not reversed by the P2Y(1) antagonists, adenosine-3'-phosphate-5'-phosphate (A3P5P) or MRS 2179, both in the range 1 to 300 microM, indicating that it was not mediated by P2Y(1) receptors. This contrasts with the increase in cytosolic free Ca(2+) concentration ([Ca(2+)](c)) induced by 2-MeSADP, which has shown to be inhibited by A3P5P. Pertussis toxin abolished the inhibitory effect of both UTP and 2-MeSADP. After culture of cells for 48 h, the ability of 2-MeSADP to inhibit cyclic AMP accumulation was greatly diminished. Reverse transcriptase-polymerase chain reaction analysis revealed that during this culture period, there was a decline in the ability to detect transcripts for P2Y(12) and P2Y(13) receptors, both of which are activated by 2-MeSADP and negatively coupled to adenylyl cyclase. However, in freshly isolated cells, the P2Y(12) and P2Y(13) receptor antagonist, 2-propylthio-beta,gamma-dichloromethylene-d-ATP (AR-C67085) (10 nM to 300 microM) did not alter the ability of 2-MeSADP to inhibit glucagon-stimulated cyclic AMP accumulation. We conclude that 2-MeSADP regulates rat hepatocyte glycogen phosphorylase by acting on P2Y(1) receptors coupled to raised [Ca(2+)](c), and by inhibiting cyclic AMP levels by an unknown G(i)-coupled receptor subtype, distinct from P2Y(1), P2Y(12), or P2Y(13) receptors.

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

    International Nuclear Information System (INIS)

    Chatterjee, A.; Bhattacharya, A.K.

    1988-01-01

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

  3. 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.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  5. 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.

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

    OpenAIRE

    Arango, Y; Heise, K P

    1998-01-01

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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Ma’ayan Israeli

    2016-08-01

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

  16. 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.

  17. 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.

  18. 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.

  19. Regulation

    International Nuclear Information System (INIS)

    Ballereau, P.

    1999-01-01

    The different regulations relative to nuclear energy since the first of January 1999 are given here. Two points deserve to be noticed: the decree of the third august 1999 authorizing the national Agency for the radioactive waste management to install and exploit on the commune of Bures (Meuse) an underground laboratory destined to study the deep geological formations where could be stored the radioactive waste. The second point is about the uranium residues and the waste notion. The judgment of the administrative tribunal of Limoges ( 9. july 1998) forbidding the exploitation of a storage installation of depleted uranium considered as final waste and qualifying it as an industrial waste storage facility has been annulled bu the Court of Appeal. It stipulated that, according to the law number 75663 of the 15. july 1965, no criteria below can be applied to depleted uranium: production residue (possibility of an ulterior enrichment), abandonment of a personal property or simple intention to do it ( future use aimed in the authorization request made in the Prefecture). This judgment has devoted the primacy of the waste notion on this one of final waste. (N.C.)

  20. [Regulation of terpene metabolism]. [Mentha piperita, Mentha spicata

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.

    1989-01-01

    Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

  1. 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.

  2. [Regulation of terpene metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.

    1989-11-09

    Terpenoid oils, resins, and waxes from plants are important renewable resources. The objective of this project is to understand the regulation of terpenoid metabolism using the monoterpenes (C[sub 10]) as a model. The pathways of monoterpene biosynthesis and catabolism have been established, and the relevant enzymes characterized. Developmental studies relating enzyme levels to terpene accumulation within the oil gland sites of synthesis, and work with bioregulators, indicate that monoterpene production is controlled by terpene cyclases, the enzymes catalyzing the first step of the monoterpene pathway. As the leaf oil glands mature, cyclase levels decline and monoterpene biosynthesis ceases. Yield then decreases as the monoterpenes undergo catabolism by a process involving conversion to a glycoside and transport from the leaf glands to the root. At this site, the terpenoid is oxidatively degraded to acetate that is recycled into other lipid metabolites. During the transition from terpene biosynthesis to catabolism, the oil glands undergo dramatic ultrastructural modification. Degradation of the producing cells results in mixing of previously compartmentized monoterpenes with the catabolic enzymes, ultimately leading to yield decline. This regulatory model is being applied to the formation of other terpenoid classes (C[sub 15] C[sub 20], C[sub 30], C[sub 40]) within the oil glands. Preliminary investigations on the formation of sesquiterpenes (C[sub 15]) suggest that the corresponding cyclases may play a lesser role in determining yield of these products, but that compartmentation effects are important. From these studies, a comprehensive scheme for the regulation of terpene metabolism is being constructed. Results from this project wail have important consequences for the yield and composition of terpenoid natural products that can be made available for industrial exploitation.

  3. 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.

  4. [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.

  5. 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.

  6. 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.

  7. 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

  8. 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...

  9. 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

  10. 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

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

    International Nuclear Information System (INIS)

    Padh, Harish; Venkitsubramanian, T.A.

    1979-01-01

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

  12. Increases in guanylin and uroguanylin in a mouse model of osmotic diarrhea are guanylate cyclase C-independent.

    Science.gov (United States)

    Steinbrecher, K A; Mann, E A; Giannella, R A; Cohen, M B

    2001-11-01

    Guanylin and uroguanylin are peptide hormones that are homologous to the diarrhea-causing Escherichia coli enterotoxins. These secretagogues are released from the intestinal epithelia into the intestinal lumen and systemic circulation and bind to the receptor guanylate cyclase C (GC-C). We hypothesized that a hypertonic diet would result in osmotic diarrhea and cause a compensatory down-regulation of guanylin/uroguanylin. Gut-to-carcass weights were used to measure fluid accumulation in the intestine. Northern and/or Western analysis was used to determine the levels of guanylin, uroguanylin, and GC-C in mice with osmotic diarrhea. Wild-type mice fed a polyethylene glycol or lactose-based diet developed weight loss, diarrhea, and an increased gut-to-carcass ratio. Unexpectedly, 2 days on either diet resulted in increased guanylin/uroguanylin RNA and prohormone throughout the intestine, elevated uroguanylin RNA, and prohormone levels in the kidney and increased levels of circulating prouroguanylin. GC-C-deficient mice given the lactose diet reacted with higher gut-to-carcass ratios. Although they did not develop diarrhea, GC-C-sufficient and -deficient mice on the lactose diet responded with elevated levels of guanylin and uroguanylin RNA and protein. A polyethylene glycol drinking water solution resulted in diarrhea, higher gut-to-carcass ratios, and induction of guanylin and uroguanylin in both GC-C heterozygous and null animals. We conclude that this model of osmotic diarrhea results in a GC-C-independent increase in intestinal fluid accumulation, in levels of these peptide ligands in the epithelia of the intestine, and in prouroguanylin in the kidney and blood.

  13. 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.

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

    Science.gov (United States)

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

    2015-05-01

    Fibrates are a class of drugs widely used to treat dyslipidaemias. They regulate lipid metabolism and act as PPARα agonists. Clinical trials demonstrate that besides changes in lipid profiles, fibrates decrease the incidence of cardiovascular events, with gemfibrozil exhibiting the most pronounced benefit. This study aims to characterize the effect of gemfibrozil on the activity and function of soluble guanylyl cyclase (sGC), the key mediator of NO signalling. High-throughput screening of a drug library identified gemfibrozil as a direct sGC activator. Activation of sGC is unique to gemfibrozil and is not shared by other fibrates. Gemfibrozil activated purified sGC, induced endothelium-independent relaxation of aortic rings and inhibited platelet aggregation. Gemfibrozil-dependent activation was absent when the sGC haem domain was deleted, but was significantly enhanced when sGC haem was lacking or oxidized. Oxidation of sGC haem enhanced the vasoactive and anti-platelet effects of gemfibrozil. Gemfibrozil competed with the haem-independent sGC activators ataciguat and cinaciguat. Computational modelling predicted that gemfibrozil occupies the space of the haem group and interacts with residues crucial for haem stabilization. This is consistent with structure-activity data which revealed an absolute requirement for gemfibrozil's carboxyl group. These data suggest that in addition to altered lipid and lipoprotein state, the cardiovascular preventive benefits of gemfibrozil may derive from direct activation and protection of sGC function. A sGC-directed action may explain the more pronounced cardiovascular benefit of gemfibrozil observed over other fibrates and some of the described side effects of gemfibrozil. © 2014 The British Pharmacological Society.

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

    Directory of Open Access Journals (Sweden)

    Misquitta Stephanie A

    2004-02-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Chatterjee, A; Bhattacharya, A K

    1988-06-01

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

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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)

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

    Science.gov (United States)

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

    2015-06-01

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

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Didier Salmon

    2018-04-01

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

  5. Lipid Signaling via Pkh1/2 Regulates Fungal CO2 Sensing through the Kinase Sch9.

    Science.gov (United States)

    Pohlers, Susann; Martin, Ronny; Krüger, Thomas; Hellwig, Daniela; Hänel, Frank; Kniemeyer, Olaf; Saluz, Hans Peter; Van Dijck, Patrick; Ernst, Joachim F; Brakhage, Axel; Mühlschlegel, Fritz A; Kurzai, Oliver

    2017-01-31

    pathogenic yeast by direct activation of adenylyl cyclase. The second pathway, although playing a fundamental role in fungal metabolism, is much less understood. Here the transcription factor Cst6/Rca1 controls carbon homeostasis by regulating carbonic anhydrase expression. Upstream signaling in this pathway remains elusive. We identify Sch9 as the kinase controlling Cst6/Rca1 activity in yeast and demonstrate that this pathway is conserved in pathogenic yeast species, which highlights identified key players as potential pharmacological targets. Furthermore, we provide a direct link between adaptation to changing CO 2 conditions and lipid/Pkh1/2 signaling in yeast, thus establishing a new signaling cascade central to metabolic adaptation. Copyright © 2017 Pohlers et al.

  6. 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

  7. 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

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

    Directory of Open Access Journals (Sweden)

    Tobias Haase

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

  9. 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.

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

    Science.gov (United States)

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

    2010-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Tomoya eNakamachi

    2012-11-01

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

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

    Science.gov (United States)

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

    2013-09-12

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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. [Regulation of terpene metabolism]. Annual progress report, March 15, 1988--March 14, 1989

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.

    1989-12-31

    Progress in understanding of the metabolism of monoterpenes by peppermint and spearmint is recorded including the actions of two key enzymes, geranyl pyrophosphate:limonene cyclase and a UDP-glucose dependent glucosyl transferase; concerning the ultrastructure of oil gland senescence; enzyme subcellular localization; regulation of metabolism; and tissue culture systems.

  20. 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

  1. 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

  2. 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...

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

    KAUST Repository

    Al-Younis, Inas

    2015-11-27

    Adenylate Cyclases (ACs) catalyze the formation of the second messenger cyclic adenosine 3′, 5′-monophosphate (cAMP) from adenosine 5’-triphosphate (ATP). Although cAMP is increasingly recognized as an important signaling molecule in higher plants, ACs have remained somewhat elusive. Here we used a search motif derived from experimentally tested guanylyl cyclases (GCs), substituted the residues essential for substrate specificity and identified the Arabidopsis thaliana K+-uptake permease 7 (AtKUP7) as one of several candidate ACs. Firstly, we show that a recombinant N-terminal, cytosolic domain of AtKUP71-100 is able to complement the AC-deficient mutant cyaA in Escherichia coli and thus restoring the fermentation of lactose, and secondly, we demonstrate with both enzyme immunoassays and mass spectrometry that a recombinant AtKUP71-100 generates cAMP in vitro.

  4. 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

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

    Directory of Open Access Journals (Sweden)

    Anthony R West

    2011-06-01

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

  6. 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.

  7. 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

  8. 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...

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

    Science.gov (United States)

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

    2006-10-12

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

  10. 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 -

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

    Czech Academy of Sciences Publication Activity Database

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

    2008-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT{sub 1C} receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing ({sup 3}H)serotonin, ({sup 3}H)lysergic acid diethylamide or ({sup 3}H)dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor.

  13. 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

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

    International Nuclear Information System (INIS)

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT 1C receptor, which modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing [ 3 H]serotonin, [ 3 H]lysergic acid diethylamide or [ 3 H]dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor

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

    Directory of Open Access Journals (Sweden)

    Sarangan Ravichandran

    2017-06-01

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

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

    Science.gov (United States)

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

    2018-03-23

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

  17. Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

    Science.gov (United States)

    Rawashdeh, Oliver; Jilg, Antje; Maronde, Erik; Fahrenkrug, Jan; Stehle, Jörg H

    2016-09-01

    Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6 kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus

  18. 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

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

    Science.gov (United States)

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

    2014-01-01

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

  20. Bioinformatics analysis of the oxidosqualene cyclase gene and the amino acid sequence in mangrove plants

    Science.gov (United States)

    Basyuni, M.; Wati, R.

    2017-01-01

    This study described the bioinformatics methods to analyze seven oxidosqualene cyclase (OSC) genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, similarity, subcellular localization and phylogenetic. The physical and chemical properties of seven mangrove OSC showed variation among the genes. The percentage of the secondary structure of seven mangrove OSC genes followed the order of a helix > random coil > extended chain structure. The values of chloroplast or signal peptide were too low, indicated that no chloroplast transit peptide or signal peptide of secretion pathway in mangrove OSC genes. The target peptide value of mitochondria varied from 0.163 to 0.430, indicated it was possible to exist. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove OSC genes. To clarify the relationship among the mangrove OSC gene, a phylogenetic tree was constructed. The phylogenetic tree shows that there are three clusters, Kandelia KcMS join with Bruguiera BgLUS, Rhizophora RsM1 was close to Bruguiera BgbAS, and Rhizophora RcCAS join with Kandelia KcCAS. The present study, therefore, supported the previous results that plant OSC genes form distinct clusters in the tree.

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

    Directory of Open Access Journals (Sweden)

    Tatjana Arsenijevic

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

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

    Science.gov (United States)

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

    2017-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Charlotte Detremmerie

    2017-07-01

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

  4. 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

  5. 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.

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

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    Erin J Heckler

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

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

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    Mounira Tlili

    2015-01-01

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

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

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    Hadi Danaee

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

  9. 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.

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

    Science.gov (United States)

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

    2017-11-16

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

  11. Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase

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    Papacchioli Velia

    2006-06-01

    Full Text Available Abstract Background Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity. Results We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e, by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold. Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed. Conclusion The data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified.

  12. Differential control of Yersinia pestis biofilm formation in vitro and in the flea vector by two c-di-GMP diguanylate cyclases.

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    Yi-Cheng Sun

    2011-04-01

    Full Text Available Yersinia pestis forms a biofilm in the foregut of its flea vector that promotes transmission by flea bite. As in many bacteria, biofilm formation in Y. pestis is controlled by intracellular levels of the bacterial second messenger c-di-GMP. Two Y. pestis diguanylate cyclase (DGC enzymes, encoded by hmsT and y3730, and one phosphodiesterase (PDE, encoded by hmsP, have been shown to control biofilm production in vitro via their opposing c-di-GMP synthesis and degradation activities, respectively. In this study, we provide further evidence that hmsT, hmsP, and y3730 are the only three genes involved in c-di-GMP metabolism in Y. pestis and evaluated the two DGCs for their comparative roles in biofilm formation in vitro and in the flea vector. As with HmsT, the DGC activity of Y3730 depended on a catalytic GGDEF domain, but the relative contribution of the two enzymes to the biofilm phenotype was influenced strongly by the environmental niche. Deletion of y3730 had a very minor effect on in vitro biofilm formation, but resulted in greatly reduced biofilm formation in the flea. In contrast, the predominant effect of hmsT was on in vitro biofilm formation. DGC activity was also required for the Hms-independent autoaggregation phenotype of Y. pestis, but was not required for virulence in a mouse model of bubonic plague. Our results confirm that only one PDE (HmsP and two DGCs (HmsT and Y3730 control c-di-GMP levels in Y. pestis, indicate that hmsT and y3730 are regulated post-transcriptionally to differentially control biofilm formation in vitro and in the flea vector, and identify a second c-di-GMP-regulated phenotype in Y. pestis.

  13. 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

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

    Science.gov (United States)

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

    2018-04-01

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

  15. Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules.

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    Anna Zdyb

    Full Text Available Jasmonic acid (JA, its derivatives and its precursor cis-12-oxo phytodienoic acid (OPDA form a group of phytohormones, the jasmonates, representing signal molecules involved in plant stress responses, in the defense against pathogens as well as in development. Elevated levels of JA have been shown to play a role in arbuscular mycorrhiza and in the induction of nitrogen-fixing root nodules. In this study, the gene families of two committed enzymes of the JA biosynthetic pathway, allene oxide synthase (AOS and allene oxide cyclase (AOC, were characterized in the determinate nodule-forming model legume Lotus japonicus JA levels were to be analysed in the course of nodulation. Since in all L. japonicus organs examined, JA levels increased upon mechanical disturbance and wounding, an aeroponic culture system was established to allow for a quick harvest, followed by the analysis of JA levels in whole root and shoot systems. Nodulated plants were compared with non-nodulated plants grown on nitrate or ammonium as N source, respectively, over a five week-period. JA levels turned out to be more or less stable independently of the growth conditions. However, L. japonicus nodules formed on aeroponically grown plants often showed patches of cells with reduced bacteroid density, presumably a stress symptom. Immunolocalization using a heterologous antibody showed that the vascular systems of these nodules also seemed to contain less AOC protein than those of nodules of plants grown in perlite/vermiculite. Hence, aeroponically grown L. japonicus plants are likely to be habituated to stress which could have affected JA levels.

  16. 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

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

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    Catalina Sanz

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

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

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    Victor L Jensen

    2010-11-01

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

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  20. 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.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-10

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

  3. 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

  4. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3′-5′)-cyclic-GMP in virulence

    Science.gov (United States)

    Kulesekara, Hemantha; Lee, Vincent; Brencic, Anja; Liberati, Nicole; Urbach, Jonathan; Miyata, Sachiko; Lee, Daniel G.; Neely, Alice N.; Hyodo, Mamoru; Hayakawa, Yoshihiro; Ausubel, Frederick M.; Lory, Stephen

    2006-01-01

    The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease in patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles in the regulation of virulence-related factors in pathogenic bacteria. A set of P. aeruginosa genes, encoding proteins that contain putative domains characteristic of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that are responsible for the maintenance of cellular levels of the second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) was identified in the annotated genomes of P. aeruginosa strains PAO1 and PA14. Although the majority of these genes are components of the P. aeruginosa core genome, several are located on presumptive horizontally acquired genomic islands. A comprehensive analysis of P. aeruginosa genes encoding the enzymes of c-di-GMP metabolism (DGC- and PDE-encoding genes) was carried out to analyze the function of c-di-GMP in two disease-related phenomena, cytotoxicity and biofilm formation. Analysis of the phenotypes of DGC and PDE mutants and overexpressing clones revealed that certain virulence-associated traits are controlled by multiple DGCs and PDEs through alterations in c-di-GMP levels. A set of mutants in selected DGC- and PDE-encoding genes exhibited attenuated virulence in a mouse infection model. Given that insertions in different DGC and PDE genes result in distinct phenotypes, it seems likely that the formation or degradation of c-di-GMP by these enzymes is in highly localized and intimately linked to particular targets of c-di-GMP action. PMID:16477007

  5. 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

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

    KAUST Repository

    Wong, Aloysius Tze

    2013-07-08

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

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

    Directory of Open Access Journals (Sweden)

    Miriam M. Cortese-Krott

    2018-04-01

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

  8. Comparative analysis of diguanylate cyclase and phosphodiesterase genes in Klebsiella pneumoniae.

    Science.gov (United States)

    Cruz, Diana P; Huertas, Mónica G; Lozano, Marcela; Zárate, Lina; Zambrano, María Mercedes

    2012-07-09

    Klebsiella pneumoniae can be found in environmental habitats as well as in hospital settings where it is commonly associated with nosocomial infections. One of the factors that contribute to virulence is its capacity to form biofilms on diverse biotic and abiotic surfaces. The second messenger Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a ubiquitous signal in bacteria that controls biofilm formation as well as several other cellular processes. The cellular levels of this messenger are controlled by c-di-GMP synthesis and degradation catalyzed by diguanylate cyclase (DGC) and phophodiesterase (PDE) enzymes, respectively. Many bacteria contain multiple copies of these proteins with diverse organizational structure that highlight the complex regulatory mechanisms of this signaling network. This work was undertaken to identify DGCs and PDEs and analyze the domain structure of these proteins in K. pneumoniae. A search for conserved GGDEF and EAL domains in three sequenced K. pneumoniae genomes showed that there were multiple copies of GGDEF and EAL containing proteins. Both single domain and hybrid GGDEF proteins were identified: 21 in K. pneumoniae Kp342, 18 in K. pneumoniae MGH 78578 and 17 in K. pneumoniae NTUH-K2044. The majority had only the GGDEF domain, most with the GGEEF motif, and hybrid proteins containing both GGDEF and EAL domains were also found. The I site for allosteric control was identified only in single GGDEF domain proteins and not in hybrid proteins. EAL-only proteins, containing either intact or degenerate domains, were also identified: 15 in Kp342, 15 in MGH 78578 and 10 in NTUH-K2044. Several input sensory domains and transmembrane segments were identified, which together indicate complex regulatory circuits that in many cases can be membrane associated. The comparative analysis of proteins containing GGDEF/EAL domains in K. pneumoniae showed that most copies were shared among the three strains and that some were unique to a particular strain

  9. Comparative analysis of diguanylate cyclase and phosphodiesterase genes in Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    Cruz Diana P

    2012-07-01

    Full Text Available Abstract Background Klebsiella pneumoniae can be found in environmental habitats as well as in hospital settings where it is commonly associated with nosocomial infections. One of the factors that contribute to virulence is its capacity to form biofilms on diverse biotic and abiotic surfaces. The second messenger Bis-(3’-5’-cyclic dimeric GMP (c-di-GMP is a ubiquitous signal in bacteria that controls biofilm formation as well as several other cellular processes. The cellular levels of this messenger are controlled by c-di-GMP synthesis and degradation catalyzed by diguanylate cyclase (DGC and phophodiesterase (PDE enzymes, respectively. Many bacteria contain multiple copies of these proteins with diverse organizational structure that highlight the complex regulatory mechanisms of this signaling network. This work was undertaken to identify DGCs and PDEs and analyze the domain structure of these proteins in K. pneumoniae. Results A search for conserved GGDEF and EAL domains in three sequenced K. pneumoniae genomes showed that there were multiple copies of GGDEF and EAL containing proteins. Both single domain and hybrid GGDEF proteins were identified: 21 in K. pneumoniae Kp342, 18 in K. pneumoniae MGH 78578 and 17 in K. pneumoniae NTUH-K2044. The majority had only the GGDEF domain, most with the GGEEF motif, and hybrid proteins containing both GGDEF and EAL domains were also found. The I site for allosteric control was identified only in single GGDEF domain proteins and not in hybrid proteins. EAL-only proteins, containing either intact or degenerate domains, were also identified: 15 in Kp342, 15 in MGH 78578 and 10 in NTUH-K2044. Several input sensory domains and transmembrane segments were identified, which together indicate complex regulatory circuits that in many cases can be membrane associated. Conclusions The comparative analysis of proteins containing GGDEF/EAL domains in K. pneumoniae showed that most copies were shared among the

  10. Gomisin N Inhibits Melanogenesis through Regulating the PI3K/Akt and MAPK/ERK Signaling Pathways in Melanocytes

    Directory of Open Access Journals (Sweden)

    Jae Kyoung Chae

    2017-02-01

    Full Text Available Gomisin N, one of the lignan compounds found in Schisandra chinensis has been shown to possess anti-oxidative, anti-tumorigenic, and anti-inflammatory activities in various studies. Here we report, for the first time, the anti-melenogenic efficacy of Gomisin N in mammalian cells as well as in zebrafish embryos. Gomisin N significantly reduced the melanin content without cellular toxicity. Although it was not capable of modulating the catalytic activity of mushroom tyrosinase in vitro, Gomisin N downregulated the expression levels of key proteins that function in melanogenesis. Gomisin N downregulated melanocortin 1 receptor (MC1R, adenylyl cyclase 2, microphthalmia-associated transcription factor (MITF, tyrosinase, tyrosinase-related protein-1 (TRP-1, and tyrosinase-related protein-2 (TRP-2. In addition, Gomisin N-treated Melan-A cells exhibited increased p-Akt and p-ERK levels, which implies that the activation of the PI3K/Akt and MAPK/ERK pathways may function to inhibit melanogenesis. We also validated that Gomisin N reduced melanin production by repressing the expression of MITF, tyrosinase, TRP-1, and TRP-2 in mouse and human cells as well as in developing zebrafish embryos. Collectively, we conclude that Gomisin N inhibits melanin synthesis by repressing the expression of MITF and melanogenic enzymes, probably through modulating the PI3K/Akt and MAPK/ERK pathways.

  11. 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

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  13. 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

  14. 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...

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Identification of a novel Arabidopsis thaliana nitric oxide-binding molecule with guanylate cyclase activity in vitro

    KAUST Repository

    Mulaudzi, Takalani

    2011-09-01

    While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5′-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O 2 and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. 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.

  2. Synechocystis sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll a for activity.

    Science.gov (United States)

    Xiong, Wei; Shen, Gaozhong; Bryant, Donald A

    2017-03-01

    The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA 6803 ) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA 6803 gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA 6803 was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA 6803 was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA 6803 is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.

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

    Directory of Open Access Journals (Sweden)

    Natalia López-del Hoyo

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

  4. α/sub i/-3 cDNA encodes the α subunit of G/sub k/, the stimulatory G protein of receptor-regulated K+ channels

    International Nuclear Information System (INIS)

    Codina, J.; Olate, J.; Abramowitz, J.; Mattera, R.; Cook, R.G.; Birnbaumer, L.

    1988-01-01

    cDNA cloning has identified the presence in the human genome of three genes encoding α subunits of pertussis toxin substrates, generically called G/sub i/. They are named α/sub i/-1, α/sub i/-2 and α/sub i/-3. However, none of these genes has been functionally identified with any of the α subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A 2 , G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K + channels. The authors now report the nucleotide sequence and the complete predicted amino acid sequence of human liver α/sub i/-3 and the partial amino acid sequence of proteolytic fragments of the α subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of α/sub i/-3, thus identifying it as α/sub k/. The probable identity of α/sub i/-1 with α/sub p/ and possible roles for α/sub i/-2, as well as additional roles for α/sub i/-1 and α/sub i/-3 (α/sub k/) are discussed

  5. 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.

  6. Proteomic signatures implicate cAMP in light and temperature responses in Arabidopsis thaliana

    KAUST Repository

    Thomas, Ludivine; Marondedze, Claudius; Ederli, Luisa; Pasqualini, Stefania; Gehring, Christoph A

    2013-01-01

    The second messenger 3'-5'-cyclic adenosine monophosphate (cAMP) and adenylyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, are increasingly recognized as important signaling molecules in a number of physiological responses

  7. Dispatch. Dictyostelium chemotaxis: fascism through the back door?

    Science.gov (United States)

    Insall, Robert

    2003-04-29

    Aggregating Dictyostelium cells secrete cyclic AMP to attract their neighbours by chemotaxis. It has now been shown that adenylyl cyclase is enriched in the rear of cells, and this localisation is required for normal aggregation.

  8. 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...

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  10. 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

  11. 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...

  12. ORF Alignment: NC_002755 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002755 gi|15841371 >1wc3A 2 197 10 178 6e-16 ... pdb|1YBU|D Chain D, Mycobacterium Tuberculosis...C, ... Mycobacterium Tuberculosis Adenylyl Cyclase Rv1900c Chd, ... In Complex With A Substrat...e Analog. pdb|1YBU|B Chain B, ... Mycobacterium Tuberculosis Adenylyl Cycl...ase Rv1900c Chd, ... In Complex With A Substrate Analog. pdb|1YBU|A Chain A, ... Mycobacterium Tuberculosis

  13. ORF Alignment: NC_000962 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_000962 gi|15609037 >1wc3A 2 197 10 178 6e-16 ... pdb|1YBU|D Chain D, Mycobacterium Tuberculosis...C, ... Mycobacterium Tuberculosis Adenylyl Cyclase Rv1900c Chd, ... In Complex With A Substrat...e Analog. pdb|1YBU|B Chain B, ... Mycobacterium Tuberculosis Adenylyl Cycl...ase Rv1900c Chd, ... In Complex With A Substrate Analog. pdb|1YBU|A Chain A, ... Mycobacterium Tuberculosis

  14. ORF Alignment: NC_002945 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available NC_002945 gi|31793093 >1wc3A 2 197 10 178 6e-16 ... pdb|1YBU|D Chain D, Mycobacterium Tuberculosis...C, ... Mycobacterium Tuberculosis Adenylyl Cyclase Rv1900c Chd, ... In Complex With A Substrat...e Analog. pdb|1YBU|B Chain B, ... Mycobacterium Tuberculosis Adenylyl Cycl...ase Rv1900c Chd, ... In Complex With A Substrate Analog. pdb|1YBU|A Chain A, ... Mycobacterium Tuberculosis

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

    KAUST Repository

    Kwezi, Lusisizwe; Ruzvidzo, Oziniel; Wheeler, Janet I.; Govender, Kershini; Iacuone, Sylvana; Thompson, Philip E.; Gehring, Christoph A; Irving, Helen R.

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tóth Gábor

    2002-02-01

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

  17. {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.)

  18. Cloning of the Lycopene β-cyclase Gene in Nicotiana tabacum and Its Overexpression Confers Salt and Drought Tolerance

    Directory of Open Access Journals (Sweden)

    Yanmei Shi

    2015-12-01

    Full Text Available Carotenoids are important pigments in plants that play crucial roles in plant growth and in plant responses to environmental stress. Lycopene β cyclase (β-LCY functions at the branch point of the carotenoid biosynthesis pathway, catalyzing the cyclization of lycopene. Here, a β-LCY gene from Nicotiana tabacum, designated as Ntβ-LCY1, was cloned and functionally characterized. Robust expression of Ntβ-LCY1 was found in leaves, and Ntβ-LCY1 expression was obviously induced by salt, drought, and exogenous abscisic acid treatments. Strong accumulation of carotenoids and expression of carotenoid biosynthesis genes resulted from Ntβ-LCY1 overexpression. Additionally, compared to wild-type plants, transgenic plants with overexpression showed enhanced tolerance to salt and drought stress with higher abscisic acid levels and lower levels of malondialdehyde and reactive oxygen species. Conversely, transgenic RNA interference plants had a clear albino phenotype in leaves, and some plants did not survive beyond the early developmental stages. The suppression of Ntβ-LCY1 expression led to lower expression levels of genes in the carotenoid biosynthesis pathway and to reduced accumulation of carotenoids, chlorophyll, and abscisic acid. These results indicate that Ntβ-LCY1 is not only a likely cyclization enzyme involved in carotenoid accumulation but also confers salt and drought stress tolerance in Nicotiana tabacum.

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

    Science.gov (United States)

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

    2016-05-20

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

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

    Directory of Open Access Journals (Sweden)

    Stephanie Hielscher-Michael

    2016-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Tamás Körtési

    2018-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    Padmamalini Baskaran

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

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

    KAUST Repository

    Kwezi, Lusisizwe

    2011-04-19

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

  6. 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

  7. 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

  8. Sumatriptan increases the proliferation of peripheral blood mononuclear cells from HIV-infected individuals and healthy blood donors in vitro

    DEFF Research Database (Denmark)

    Afzelius, P; Nielsen, Jens Ole

    2000-01-01

    responsible for regulation of the intracellular levels of cAMP. In a preliminary study sumatriptan increased the proliferative responses of PBMC to a polyclonal activator in vitro in 9 of 10 HIV-seropositive individuals (p=0.007), and in 7 of 9 healthy blood donors (p=0.05). This was probably due...... of the intracellular second messenger adenosine 3',5'-cyclic monophosphate (cAMP) has been shown to cause impaired proliferative capacity of peripheral blood mononuclear cells (PBMC) from HIV-infected individuals in vitro. Sumatriptan, a 5HT1d receptor agonist, inhibits the activity of adenylyl cyclases, the enzymes...

  9. Overexpression of allene oxide cyclase improves the biosynthesis of artemisinin in Artemisia annua L.

    Directory of Open Access Journals (Sweden)

    Xu Lu

    Full Text Available Jasmonates (JAs are important signaling molecules in plants and play crucial roles in stress responses, secondary metabolites' regulation, plant growth and development. In this study, the promoter of AaAOC, which was the key gene of jasmonate biosynthetic pathway, had been cloned. GUS staining showed that AaAOC was expressed ubiquitiously in A. annua. AaAOC gene was overexpressed under control of 35S promoter. RT-Q-PCR showed that the expression levels of AaAOC were increased from 1.6- to 5.2-fold in AaAOC-overexpression transgenic A. annua. The results of GC-MS showed that the content of endogenous jasmonic acid (JA was 2- to 4.7-fold of the control level in AaAOC-overexpression plants. HPLC showed that the contents of artemisinin, dihydroartemisinic acid and artemisinic acid were increased significantly in AaAOC-overexpression plants. RT-Q-PCR showed that the expression levels of FPS (farnesyl diphosphate synthase, CYP71AV1 (cytochrome P450 dependent hydroxylase and DBR2 (double bond reductase 2 were increased significantly in AaAOC-overexpression plants. All data demonstrated that increased endogenous JA could significantly promote the biosynthesis of artemisinin in AaAOC-overexpression transgenic A. annua.

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

    Directory of Open Access Journals (Sweden)

    Sandra Geschka

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

  11. Lentiviral expression of retinal guanylate cyclase-1 (RetGC1 restores vision in an avian model of childhood blindness.

    Directory of Open Access Journals (Sweden)

    Melissa L Williams

    2006-06-01

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

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

    Science.gov (United States)

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

    2016-06-01

    Dysregulated nitric oxide (NO) signaling contributes to the pathogenesis of hypertension, a prevalent and often sex-specific risk factor for cardiovascular disease. We previously reported that mice deficient in the α1-subunit of the NO receptor soluble guanylate cyclase (sGCα1 (-/-) mice) display sex- and strain-specific hypertension: male but not female sGCα1 (-/-) mice are hypertensive on an 129S6 (S6) but not a C57BL6/J (B6) background. We aimed to uncover the genetic and molecular basis of the observed sex- and strain-specific blood pressure phenotype. Via linkage analysis, we identified a suggestive quantitative trait locus associated with elevated blood pressure in male sGCα1 (-/-)S6 mice. This locus encompasses Cyp4a12a, encoding the predominant murine synthase of the vasoconstrictor 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE). Renal expression of Cyp4a12a in mice was associated with genetic background, sex, and testosterone levels. In addition, 20-HETE levels were higher in renal preglomerular microvessels of male sGCα1 (-/-)S6 than of male sGCα1 (-/-)B6 mice. Furthermore, treating male sGCα1 (-/-)S6 mice with the 20-HETE antagonist 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE) lowered blood pressure. Finally, 20-HEDE rescued the genetic background- and testosterone-dependent impairment of acetylcholine-induced relaxation in renal interlobar arteries associated with sGCα1 deficiency. Elevated Cyp4a12a expression and 20-HETE levels render mice susceptible to hypertension and vascular dysfunction in a setting of sGCα1 deficiency. Our data identify Cyp4a12a as a candidate sex-specific blood pressure-modifying gene in the context of deficient NO-sGC signaling. Copyright © 2016 the American Physiological Society.

  13. A conserved hydrogen-bond network in the catalytic centre of animal glutaminyl cyclases is critical for catalysis.

    Science.gov (United States)

    Huang, Kai-Fa; Wang, Yu-Ruei; Chang, En-Cheng; Chou, Tsung-Lin; Wang, Andrew H-J

    2008-04-01

    QCs (glutaminyl cyclases; glutaminyl-peptide cyclotransferases, EC 2.3.2.5) catalyse N-terminal pyroglutamate formation in numerous bioactive peptides and proteins. The enzymes were reported to be involved in several pathological conditions such as amyloidotic disease, osteoporosis, rheumatoid arthritis and melanoma. The crystal structure of human QC revealed an unusual H-bond (hydrogen-bond) network in the active site, formed by several highly conserved residues (Ser(160), Glu(201), Asp(248), Asp(305) and His(319)), within which Glu(201) and Asp(248) were found to bind to substrate. In the present study we combined steady-state enzyme kinetic and X-ray structural analyses of 11 single-mutation human QCs to investigate the roles of the H-bond network in catalysis. Our results showed that disrupting one or both of the central H-bonds, i.e., Glu(201)...Asp(305) and Asp(248)...Asp(305), reduced the steady-state catalysis dramatically. The roles of these two COOH...COOH bonds on catalysis could be partly replaced by COOH...water bonds, but not by COOH...CONH(2) bonds, reminiscent of the low-barrier Asp...Asp H-bond in the active site of pepsin-like aspartic peptidases. Mutations on Asp(305), a residue located at the centre of the H-bond network, raised the K(m) value of the enzyme by 4.4-19-fold, but decreased the k(cat) value by 79-2842-fold, indicating that Asp(305) primarily plays a catalytic role. In addition, results from mutational studies on Ser(160) and His(319) suggest that these two residues might help to stabilize the conformations of Asp(248) and Asp(305) respectively. These data allow us to propose an essential proton transfer between Glu(201), Asp(305) and Asp(248) during the catalysis by animal QCs.

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

    Directory of Open Access Journals (Sweden)

    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

  15. 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.

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

    Science.gov (United States)

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

    2011-11-01

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

  17. 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

  18. 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

  19. Stimulatory effects of adenosine on prolactin secretion in the pituitary gland of the rat

    Directory of Open Access Journals (Sweden)

    D.L.W. Picanço-Diniz

    2002-07-01

    Full Text Available We investigated the effects of adenosine on prolactin (PRL secretion from rat anterior pituitaries incubated in vitro. The administration of 5-N-methylcarboxamidoadenosine (MECA, an analog agonist that preferentially activates A2 receptors, induced a dose-dependent (1 nM to 1 µM increase in the levels of PRL released, an effect abolished by 1,3-dipropyl-7-methylxanthine, an antagonist of A2 adenosine receptors. In addition, the basal levels of PRL secretion were decreased by the blockade of cyclooxygenase or lipoxygenase pathways, with indomethacin and nordihydroguaiaretic acid (NDGA, respectively. The stimulatory effects of MECA on PRL secretion persisted even after the addition of indomethacin, but not of NDGA, to the medium. MECA was unable to stimulate PRL secretion in the presence of dopamine, the strongest inhibitor of PRL release that works by inducing a decrease in adenylyl cyclase activity. Furthermore, the addition of adenosine (10 nM mimicked the effects of MECA on PRL secretion, an effect that persisted regardless of the presence of LiCl (5 mM. The basal secretion of PRL was significatively reduced by LiCl, and restored by the concomitant addition of both LiCl and myo-inositol. These results indicate that PRL secretion is under a multifactorial regulatory mechanism, with the participation of different enzymes, including adenylyl cyclase, inositol-1-phosphatase, cyclooxygenase, and lipoxygenase. However, the increase in PRL secretion observed in the lactotroph in response to A2 adenosine receptor activation probably was mediated by mechanisms involving regulation of adenylyl cyclase, independent of membrane phosphoinositide synthesis or cyclooxygenase activity and partially dependent on lipoxygenase arachidonic acid-derived substances.

  20. 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

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

    OpenAIRE

    Ly, Thuy Thi Bich

    2011-01-01

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

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

    Science.gov (United States)

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

    2017-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Melissa H Costell

    2012-07-01

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

  4. Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis.

    Science.gov (United States)

    Shailubhai, Kunwar; Palejwala, Vaseem; Arjunan, Krishna Priya; Saykhedkar, Sayali; Nefsky, Bradley; Foss, John A; Comiskey, Stephen; Jacob, Gary S; Plevy, Scott E

    2015-11-06

    To evaluate the effect of orally administered plecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models. The cyclic guanosine monophosphate (cGMP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cell-based assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid (5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium (DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic (TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout (TCRα(-/-)) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity. Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C (GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cGMP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs (0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity (P < 0.05) and disease activity index (P < 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα(-/-) mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. This is the first-ever study reporting

  5. 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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-09-23

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

  7. 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.

  8. 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.

  9. 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

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

    Science.gov (United States)

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

    2016-04-01

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

  11. 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

  12. 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.

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

    KAUST Repository

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

    2010-01-01

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

  14. 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)

  15. Protein kinase-dependent oxidative regulation of the cardiac Na+-K+ pump: evidence from in vivo and in vitro modulation of cell signalling.

    Science.gov (United States)

    Galougahi, Keyvan Karimi; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A S; Hamilton, Elisha J; Rasmussen, Helge H; Figtree, Gemma A

    2013-06-15

    The widely reported stimulation of the cardiac Na(+)-K(+) pump by protein kinase A (PKA) should oppose other effects of PKA to increase contractility of the normal heart. It should also reduce harmful raised myocyte Na(+) levels in heart failure, yet blockade of the β1 adrenergic receptor (AR), coupled to PKA signalling, is beneficial. We treated rabbits with the β1 AR antagonist metoprolol to modulate PKA activity and studied cardiac myocytes ex vivo. Metoprolol increased electrogenic pump current (Ip) in voltage clamped myocytes and reduced glutathionylation of the β1 pump subunit, an oxidative modification causally related to pump inhibition. Activation of adenylyl cyclase with forskolin to enhance cAMP synthesis or inclusion of the catalytic subunit of PKA in patch pipette solutions abolished the increase in Ip in voltage clamped myocytes induced by treatment with metoprolol, supporting cAMP/PKA-mediated pump inhibition. Metoprolol reduced myocardial PKA and protein kinase C (PKC) activities, reduced coimmunoprecipitation of cytosolic p47(phox) and membranous p22(phox) NADPH oxidase subunits and reduced myocardial O2(•-)-sensitive dihydroethidium fluorescence. Treatment also enhanced coimmunoprecipitation of the β1 pump subunit with glutaredoxin 1 that catalyses de-glutathionylation. Since angiotensin II induces PKC-dependent activation of NADPH oxidase, we examined the effects of angiotensin-converting enzyme inhibition with captopril. This treatment had no effect on PKA activity but reduced the activity of PKC, reduced β1 subunit glutathionylation and increased Ip. The PKA-induced Na(+)-K(+) pump inhibition we report should act with other mechanisms that enhance contractility of the normal heart but accentuate the harmful effects of raised cytosolic Na(+) in the failing heart. This scheme is consistent with the efficacy of β1 AR blockade in the treatment of heart failure.

  16. Protein kinase-dependent oxidative regulation of the cardiac Na+–K+ pump: evidence from in vivo and in vitro modulation of cell signalling

    Science.gov (United States)

    Galougahi, Keyvan Karimi; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A S; Hamilton, Elisha J; Rasmussen, Helge H; Figtree, Gemma A

    2013-01-01

    The widely reported stimulation of the cardiac Na+–K+ pump by protein kinase A (PKA) should oppose other effects of PKA to increase contractility of the normal heart. It should also reduce harmful raised myocyte Na+ levels in heart failure, yet blockade of the β1 adrenergic receptor (AR), coupled to PKA signalling, is beneficial. We treated rabbits with the β1 AR antagonist metoprolol to modulate PKA activity and studied cardiac myocytes ex vivo. Metoprolol increased electrogenic pump current (Ip) in voltage clamped myocytes and reduced glutathionylation of the β1 pump subunit, an oxidative modification causally related to pump inhibition. Activation of adenylyl cyclase with forskolin to enhance cAMP synthesis or inclusion of the catalytic subunit of PKA in patch pipette solutions abolished the increase in Ip in voltage clamped myocytes induced by treatment with metoprolol, supporting cAMP/PKA-mediated pump inhibition. Metoprolol reduced myocardial PKA and protein kinase C (PKC) activities, reduced coimmunoprecipitation of cytosolic p47phox and membranous p22phox NADPH oxidase subunits and reduced myocardial O2•−-sensitive dihydroethidium fluorescence. Treatment also enhanced coimmunoprecipitation of the β1 pump subunit with glutaredoxin 1 that catalyses de-glutathionylation. Since angiotensin II induces PKC-dependent activation of NADPH oxidase, we examined the effects of angiotensin-converting enzyme inhibition with captopril. This treatment had no effect on PKA activity but reduced the activity of PKC, reduced β1 subunit glutathionylation and increased Ip. The PKA-induced Na+–K+ pump inhibition we report should act with other mechanisms that enhance contractility of the normal heart but accentuate the harmful effects of raised cytosolic Na+ in the failing heart. This scheme is consistent with the efficacy of β1 AR blockade in the treatment of heart failure. PMID:23587884

  17. Cholesterol regulates contractility and inotropic response to β2-adrenoceptor agonist in the mouse atria: Involvement of Gi-protein-Akt-NO-pathway.

    Science.gov (United States)

    Odnoshivkina, Yulia G; Sytchev, Vaycheslav I; Petrov, Alexey M

    2017-06-01

    Majority of cardiac β2-adrenoceptors is located in cholesterol-rich microdomains. Here, we have investigated the underlying mechanisms by which a slight to moderate cholesterol depletion with methyl-β-cyclodextrin (MβCD, 1 and 5mM) interferes with contractility and inotropic effect of β2-adrenergic agonist (fenoterol, 50μM) in the mouse atria. Treatment with MβCD itself increased amplitude of Ca 2+ transient but did not change the contraction amplitude due to a clamping action of elevated NO. Cholesterol depletion significantly attenuated the positive inotropic response to fenoterol which is accompanied by increase in NO generation and decrease in Ca 2+ transient. Influence of 1mM MβCD on the fenoterol-driven changes in both contractility and NO level was strongly attenuated by inhibition of G i -protein (pertussis toxin), Akt (Akt 1/2 kinase inhibitor) or NO-synthase (L-NAME). After exposure to 5mM MβCD, pertussis toxin or Akt inhibitor could recover the β2-agonist effects on contractility, NO production and Ca 2+ transient, while L-NAME only reduced NO level. An adenylyl cyclase activator (forskolin, 50nM) had no influence on the MβCD-induced changes in the β2-agonist effects. Obtained results suggest that slight cholesterol depletion upregulates G i -protein/Akt/NO-synthase signaling that attenuates the positive inotropic response to β2-adrenergic stimulation without altering the Ca 2+ transient. Whilst moderate cholesterol depletion additionally could suppress the enhancement of the Ca 2+ transient amplitude caused by the β2-adrenergic agonist administration in G i -protein/Akt-dependent but NO-independent manner. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. 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

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

    Science.gov (United States)

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

    2011-04-08

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

  20. 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

  1. 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.

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

    Science.gov (United States)

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

    2010-07-01

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

  3. [Regulation of terpene metabolism]. Annual progress report, March 15, 1989--March 14, 1990

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.

    1989-11-09

    Terpenoid oils, resins, and waxes from plants are important renewable resources. The objective of this project is to understand the regulation of terpenoid metabolism using the monoterpenes (C{sub 10}) as a model. The pathways of monoterpene biosynthesis and catabolism have been established, and the relevant enzymes characterized. Developmental studies relating enzyme levels to terpene accumulation within the oil gland sites of synthesis, and work with bioregulators, indicate that monoterpene production is controlled by terpene cyclases, the enzymes catalyzing the first step of the monoterpene pathway. As the leaf oil glands mature, cyclase levels decline and monoterpene biosynthesis ceases. Yield then decreases as the monoterpenes undergo catabolism by a process involving conversion to a glycoside and transport from the leaf glands to the root. At this site, the terpenoid is oxidatively degraded to acetate that is recycled into other lipid metabolites. During the transition from terpene biosynthesis to catabolism, the oil glands undergo dramatic ultrastructural modification. Degradation of the producing cells results in mixing of previously compartmentized monoterpenes with the catabolic enzymes, ultimately leading to yield decline. This regulatory model is being applied to the formation of other terpenoid classes (C{sub 15} C{sub 20}, C{sub 30}, C{sub 40}) within the oil glands. Preliminary investigations on the formation of sesquiterpenes (C{sub 15}) suggest that the corresponding cyclases may play a lesser role in determining yield of these products, but that compartmentation effects are important. From these studies, a comprehensive scheme for the regulation of terpene metabolism is being constructed. Results from this project wail have important consequences for the yield and composition of terpenoid natural products that can be made available for industrial exploitation.

  4. Regulating the Regulator

    Energy Technology Data Exchange (ETDEWEB)

    1992-08-26

    The article reports on a challenge to the UK electricity regulator to defend his record by the Coalition for Fair Electricity Regulation (COFFER). The challenge centres on whether the obligation for the regional electric companies (REC) to purchase power from the cheapest source is being enforced. This is related to the wider issue of whether the REC's support of combined-cycle gas turbine (CCGT) is economic. COFFER considers that uneconomic gas-fired power plants are being allowed to displace economic coal-fired stations. Aspects discussed include the background to the dispute and the costs of CCGT and coal fired power generation. 1 fig., 1 tab.

  5. Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling.

    Science.gov (United States)

    Inda, Carolina; Dos Santos Claro, Paula A; Bonfiglio, Juan J; Senin, Sergio A; Maccarrone, Giuseppina; Turck, Christoph W; Silberstein, Susana

    2016-07-18

    Corticotropin-releasing hormone receptor 1 (CRHR1) activates G protein-dependent and internalization-dependent signaling mechanisms. Here, we report that the cyclic AMP (cAMP) response of CRHR1 in physiologically relevant scenarios engages separate cAMP sources, involving the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). cAMP produced by tmACs and sAC is required for the acute phase of extracellular signal regulated kinase 1/2 activation triggered by CRH-stimulated CRHR1, but only sAC activity is essential for the sustained internalization-dependent phase. Thus, different cAMP sources are involved in different signaling mechanisms. Examination of the cAMP response revealed that CRH-activated CRHR1 generates cAMP after endocytosis. Characterizing CRHR1 signaling uncovered a specific link between CRH-activated CRHR1, sAC, and endosome-based signaling. We provide evidence of sAC being involved in an endocytosis-dependent cAMP response, strengthening the emerging model of GPCR signaling in which the cAMP response does not occur exclusively at the plasma membrane and introducing the notion of sAC as an alternative source of cAMP. © 2016 Inda et al.

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

    Science.gov (United States)

    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.

  7. Market, Regulation, Market, Regulation

    DEFF Research Database (Denmark)

    Frankel, Christian; Galland, Jean-Pierre

    2015-01-01

    barriers to trade in Europe, realized the free movement of products by organizing progressively several orders of markets and regulation. Based on historical and institutional documents, on technical publications, and on interviews, this article relates how the European Commission and the Member States had......This paper focuses on the European Regulatory system which was settled both for opening the Single Market for products and ensuring the consumers' safety. It claims that the New Approach and Standardization, and the Global Approach to conformity assessment, which suppressed the last technical...... alternatively recourse to markets and to regulations, at the three main levels of the New Approach Directives implementation. The article focuses also more specifically on the Medical Devices sector, not only because this New Approach sector has long been controversial in Europe, and has recently been concerned...

  8. Guanylin and uroguanylin mRNA expression is increased following Roux-en-Y gastric bypass, but guanylins do not play a significant role in body weight regulation and glycemic control

    DEFF Research Database (Denmark)

    Fernandez-Cachon, María L; Pedersen, Søren L; Rigbolt, Kristoffer T

    2018-01-01

    AIM: To determine whether intestinal expression of guanylate cyclase activator 2A (GUCA2A) and guanylate cyclase activator 2B (GUCA2B) genes is regulated in obese humans following Roux-en-Y gastric bypass (RYGB), and to evaluate the corresponding guanylin (GN) and uroguanylin (UGN) peptides...... for potentially contributing to the beneficial metabolic effects of RYGB. METHODS: Enteroendocrine cells were harvested peri- and post-RYGB, and GUCA2A/GUCA2B mRNA expression was compared. GN, UGN and their prohormones (proGN, proUGN) were administered subcutaneously in normal-weight mice to evaluate effects...... on food intake. GN and UGN, as well as their prohormones, were evaluated for effects on glucose-stimulated insulin secretion (GSIS) in rat pancreatic islets and perfused rat pancreas. RESULTS: GUCA2A and GUCA2B mRNA expression was significantly upregulated in enteroendocrine cells after RYGB. Peripheral...

  9. Intracellular pH in sperm physiology.

    Science.gov (United States)

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Carotenoids in staple cereals: Metabolism, regulation, and genetic manipulation

    Directory of Open Access Journals (Sweden)

    shengnan zhai

    2016-08-01

    Full Text Available Carotenoids play a critical role in animal and human health. Animals and humans are unable to synthesize carotenoids de novo, and therefore rely upon diet as sources of these compounds. However, major staple cereals often contain only small amounts of carotenoids in their grain. Consequently, there is considerable interest in genetic manipulation of carotenoid content in cereal grain. In this review, we focus on carotenoid metabolism and regulation in non-green plant tissues, as well as genetic manipulation in staple cereals such as rice, maize, and wheat. Significant progress has been made in three aspects: (1 seven carotenogenes play vital roles in carotenoid regulation in non-green plant tissues, including DXS (1-deoxyxylulose-5-phosphate synthase influencing isoprenoid precursor supply, PSY (phytoene synthase, LCYB (β-cyclase and LCYE (ε-cyclase controlling biosynthesis, HYDB (1-hydroxy-2-methyl-2-(E-butenyl 4-diphosphate reductase and CCDs (carotenoid cleavage dioxygenases responsible for degradation, and OR (orange conditioning sequestration sink; (2 pro-vitamin A-biofortified crops, such as rice and maize, were developed by either metabolic engineering or marker-assisted breeding; (3 QTLs for carotenoid content on chromosomes 3B, 7A, and 7B were consistently identified, eight carotenogenes including 23 loci were detected, and ten gene-specific markers for carotenoid accumulation were developed and applied in wheat improvement. A comprehensive and deeper understanding of the regulatory mechanisms of carotenoid metabolism in crops will be benefitical in improving our precision in improving carotenoid contents. Genomic selection and gene editing are emerging as transformative technologies for vitamin A biofortification.

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

    Science.gov (United States)

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

    1999-09-01

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

  12. 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.

  13. 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.

  14. 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

  15. Endothelium-dependent relaxation of rat aorta to a histamine H3 agonist is reduced by inhibitors of nitric oxide synthase, guanylate cyclase and Na+,K+-ATPase

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    D. M. Djuric

    1996-01-01

    Full Text Available The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, β-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na+,K+-ATPase was evaluated in a histamine H3 receptor agonist-induced ((Rα-methylhistamine, (Rα-MeHA endothelium-dependent rat aorta relaxation assay. (Rα-MeHA (0.1 nM – 0.01 mM relaxed endothelium-dependent rat aorta, with a pD2 value of 8.22 ± 0.06, compared with a pD2 value of 7.98 ± 0.02 caused by histamine (50% and 70% relaxation, respectively. The effect of (Rα-MeHA (0.1 nM – 0.01 mM was competitively antagonized by thioperamide (1, 10 and 30 nM (pA2 = 9.21 ± 0.40; slope = 1.03 ± 0.35 but it was unaffected by pyrilamine (100 nM, cimetidine (1 μM, atropine (10 μM, propranolol (1 μM, indomethacin (10 μM or nordthydroguaiaretic acid (0.1 mM. Inhibitors of nitric oxide synthase, L-NG-monomethylarginine (L-NMMA, 10 μM and NG-nitro-L-arginine methylester (L-NOARG, 10 μM inhibited the relaxation effect of (Rα-MeHA, by approximately 52% and 70%, respectively. This inhibitory effect of L-NMMA was partially reversed by L-arginine (10 μM. Methylene blue (10 μM and ouabain (10 μM inhibited relaxation (Rα-MeHA-induced by approximately 50% and 90%, respectively. The products of cyclooxygenase and lipoxygenase are not involved in (Rα-MeHA-induced endothelium-dependent rat aorta relaxation nor are the muscarinic cholinergic and β-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na+,K+-ATPase in (Rα-MeHA-induced endothelium-dependent rat aorta relaxation.

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

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    Janna eBlechman

    2013-05-01

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

  17. Identification of potential glutaminyl cyclase inhibitors from lead-like libraries by in silico and in vitro fragment-based screening.

    Science.gov (United States)

    Szaszkó, Mária; Hajdú, István; Flachner, Beáta; Dobi, Krisztina; Magyar, Csaba; Simon, István; Lőrincz, Zsolt; Kapui, Zoltán; Pázmány, Tamás; Cseh, Sándor; Dormán, György

    2017-02-01

    A glutaminyl cyclase (QC) fragment library was in silico selected by disconnection of the structure of known QC inhibitors and by lead-like 2D virtual screening of the same set. The resulting fragment library (204 compounds) was acquired from commercial suppliers and pre-screened by differential scanning fluorimetry followed by functional in vitro assays. In this way, 10 fragment hits were identified ([Formula: see text]5 % hit rate, best inhibitory activity: 16 [Formula: see text]). The in vitro hits were then docked to the active site of QC, and the best scoring compounds were analyzed for binding interactions. Two fragments bound to different regions in a complementary manner, and thus, linking those fragments offered a rational strategy to generate novel QC inhibitors. Based on the structure of the virtual linked fragment, a 77-membered QC target focused library was selected from vendor databases and docked to the active site of QC. A PubChem search confirmed that the best scoring analogues are novel, potential QC inhibitors.

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

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    Valentina Spinelli

    2016-01-01

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

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

    KAUST Repository

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

    2010-01-01

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

  20. 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.

  1. Necrotic enteritis locus 1 diguanylate cyclase and phosphodiesterase (cyclic-di-GMP) gene mutation attenuates virulence in an avian necrotic enteritis isolate of Clostridium perfringens.

    Science.gov (United States)

    Parreira, Valeria R; Ojha, Shivani; Lepp, Dion; Mehdizadeh Gohari, Iman; Zhou, Hongzhuan; Susta, Leonardo; Gong, Jianhua; Prescott, John F

    2017-09-01

    Necrotic enteritis (NE) caused by netB-positive strains of Clostridium perfringens is an important disease of intensively-reared broiler chickens. It is widely controlled by antibiotic use, but this practice that has come under increasing scrutiny and alternative approaches are required. As part of the search for alternative approaches over the last decade, advances have been made in understanding its pathogenesis but much remains to be understood and applied to the control of NE. The objective of this work was to assess the effect on virulence of mutation of the cyclic-di-GMP signaling genes present on the large pathogenicity locus (NELoc-1) in the tcp-encoding conjugative virulence plasmid, pNetB. For this purpose, the diguanylate cyclase (dgc) and phosphodiesterase (pde) genes were individually insertionally inactivated and the two mutants were subsequently complemented with their respective genes. Southern blotting showed that a single gene insertion was present. Mutation of either gene resulted in almost total attenuation of the mutants to cause NE in experimentally-infected broiler chickens, which was fully restored in each case by complementation of the respective mutated gene. Production of NetB-associated cytotoxicity for Leghorn male hepatoma (LMH) cells was unaffected in mutants. We conclude that the cyclic-di-GMP signaling system is important in controlling virulence in a NE C. perfringens strain and might be a target for control of the disease. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load. PMID:26230691

  3. 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.

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

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    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.

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

    KAUST Repository

    Meier, Stuart

    2010-01-26

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

  6. 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

  7. Metabolic regulation of carotenoid-enriched Golden rice line

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    Dipak Gayen

    2016-10-01

    Full Text Available Vitamin A deficiency (VAD is the leading cause of blindness among children and is associated with high risk of maternal mortality. In order to enhance the bioavailability of vitamin A, high carotenoid transgenic golden rice has been developed by manipulating enzymes, such as phytoene synthase (psy and phytoene desaturase (crtI. In this study, proteome and metabolite analyses were carried out to comprehend metabolic regulation and adaptation of transgenic golden rice after the manipulation of endosperm specific carotenoid pathways. The main alteration was observed in carbohydrate metabolism pathways of the transgenic seeds. The 2D based proteomic studies demonstrated that carbohydrate metabolism-related enzymes, such as pullulanase, UDP-glucose pyrophosphorylase and glucose-1-phosphate adenylyl transferase, were primarily up-regulated in transgenic rice seeds. In addition, the enzyme PPDK was also elevated in transgenic seeds thus enhancing pyruvate biosynthesis, which is the precursor in the carotenoids biosynthetic pathway. GC-MS based metabolite profiling demonstrated an increase in the levels of glyceric acid, fructo-furanose, and galactose, while decrease in galactonic acid and gentiobiose in the transgenic rice compared to WT. It is noteworthy to mention that the carotenoid content, especially β-carotene level in transgenic rice (4.3 µg/g was significantly enhanced. The present study highlights the metabolic adaptation process of a transgenic golden rice line (homozygous T4 progeny of SKBR-244 after enhancing carotenoid biosynthesis. The presented information would be helpful in the development of crops enriched in carotenoids by expressing metabolic flux of pyruvate biosynthesis.

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

    Science.gov (United States)

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

    2018-07-05

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

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

    Science.gov (United States)

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

    2017-09-01

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

  10. Isolation and characterization of an oxidosqualene cyclase gene encoding a β-amyrin synthase involved in Polygala tenuifolia Willd. saponin biosynthesis.

    Science.gov (United States)

    Jin, Mei Lan; Lee, Dae Young; Um, Yurry; Lee, Jeong Hoon; Park, Chun Geun; Jetter, Reinhard; Kim, Ok Tae

    2014-03-01

    Expression of PtBS (Polygala tenuifolia β-amyrin synthase) led to the production of β-amyrin as sole product. Polygala tenuifolia Willdenow is a rich source of triterpene saponins, onjisaponins and polygalasaponins, used as herbal medicine to treat phlegms and for detumescence in traditional Asian healing. The Polygala saponins share the oleanane backbone structure and are, therefore, likely synthesized via β-amyrin as a common precursor. We hypothesized that, in analogy to diverse other plant species, this central intermediate should be formed by a β-amyrin synthase catalyzing the complex cyclization of oxidosqualene. This member of the oxidosqualene cyclase (OSC) family of enzymes is thus defining an important branch point between primary and secondary metabolisms, and playing a crucial role in the control of oleanane-type triterpene saponin biosynthesis. From P. tenuifolia roots, we isolated an OSC cDNA containing a reading frame of 2,289 bp nucleotides. The predicted protein of 763 amino acids (molecular weight 87.353 kDa) showed particularly high amino acid sequence identities to known β-amyrin synthases (85-87 %) and was, therefore, named PtBS. Expression of PtBS in the triterpenoid synthase-deficient yeast mutant GIL77 led to the production of β-amyrin as sole product. qRT-PCR analysis of various P. tenuifolia organs showed that PtBS transcript levels were highest in the roots, consistent with onjisaponin accumulation patterns. Therefore, we conclude that PtBS is the β-amyrin synthase enzyme catalyzing the first committed step in the biosynthesis of onjisaponins and polygalasaponins in P. tenuifolia.

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

    Directory of Open Access Journals (Sweden)

    Gabor Maasz

    2017-02-01

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

  12. Cloning and characterization of oxidosqualene cyclases from Kalanchoe daigremontiana: enzymes catalyzing up to 10 rearrangement steps yielding friedelin and other triterpenoids.

    Science.gov (United States)

    Wang, Zhonghua; Yeats, Trevor; Han, Hong; Jetter, Reinhard

    2010-09-24

    The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C(30)H(50)O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761-779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores.

  13. 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.

  14. Radiation regulation

    International Nuclear Information System (INIS)

    Braithwaite, J.; Grabosky, P.

    1985-01-01

    The five main areas of radiation regulation considered are radiation exposure in the mining of uranium and other minerals, exposure in the use of uranium in nuclear reactors, risks in the transport of radioactive materials and hazards associated with the disposal of used materials. In Australia these problems are regulated by mines departments, the Australian Atomic Energy Commission and radiation control branches in state health departments. Each of these instutional areas of regulation is examined

  15. A novel CDX2 isoform regulates alternative splicing.

    Directory of Open Access Journals (Sweden)

    Matthew E Witek

    Full Text Available Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain. CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-β1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2 and pre-mRNA processing (CDX2/AS.

  16. 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

  17. Regulative environmental policy. Regulative Umweltpolitik

    Energy Technology Data Exchange (ETDEWEB)

    Goerlitz, A; Voigt, R [Universitaet der Bundeswehr Muenchen, Neubiberg (Germany, F.R.). Fakultaet fuer Sozialwissenschaften; eds.

    1991-01-01

    Regulative policy means those governmental attempts to steer the course of things which can fall back on a certain repertoire of instruments for actions in order to warrant the causal and temporal connection between the making available and the employment of means. The fact that environmental protection needs regulative policy is substantiated by the thesis that the market has failed; consequently only government can manage the public goods 'environment' in a suitable way, and it is a matter of fact that environmental protection at present is operated preferably via regulative policy. The problems of regulative enviromental policy are manifold. Its implementation often miscarries because of limited administrative resources on the one hand - making sufficient control impossible for instance -, and because of poor quality regulative instruments on the other hand. One way out would be to increase the efficiency of regulative policy by sophisticating judicial techniques. Other ways out point to the executing level and aim at improving implementation strategies or are concerned with post-regulative law. The latter refers to a new legal quality which demonstrates itself already in corporatistical crisis regulation or in induction programs such as pollution limits. A final way out favours deregulation strategies which includes the introduction of environmental levies or the allocation of environmental licences. An interdisciplinary discourse is to find out what would happen if these ways were taken. Pointers to solutions from varying scientific disciplines resulting from this discourse are to be found in this volume. (orig./HSCH).

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

    Science.gov (United States)

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

    2011-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Ina M Ott

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

  20. The Autonomic Nervous System Regulates the Heart Rate through cAMP-PKA Dependent and Independent Coupled-Clock Pacemaker Cell Mechanisms.

    Science.gov (United States)

    Behar, Joachim; Ganesan, Ambhighainath; Zhang, Jin; Yaniv, Yael

    2016-01-01

    Sinoatrial nodal cells (SANCs) generate spontaneous action potentials (APs) that control the cardiac rate. The brain modulates SANC automaticity, via the autonomic nervous system, by stimulating membrane receptors that activate (adrenergic) or inactivate (cholinergic) adenylyl cyclase (AC). However, these opposing afferents are not simply additive. We showed that activation of adrenergic signaling increases AC-cAMP/PKA signaling, which mediates the increase in the SANC AP firing rate (i.e., positive chronotropic modulation). However, there is a limited understanding of the underlying internal pacemaker mechanisms involved in the crosstalk between cholinergic receptors and the decrease in the SANC AP firing rate (i.e., negative chronotropic modulation). We hypothesize that changes in AC-cAMP/PKA activity are crucial for mediating either decrease or increase in the AP firing rate and that the change in rate is due to both internal and membrane mechanisms. In cultured adult rabbit pacemaker cells infected with an adenovirus expressing the FRET sensor AKAR3, PKA activity and AP firing rate were tightly linked in response to either adrenergic receptor stimulation (by isoproterenol, ISO) or cholinergic stimulation (by carbachol, CCh). To identify the main molecular targets that mediate between PKA signaling and pacemaker function, we developed a mechanistic computational model. The model includes a description of autonomic-nervous receptors, post- translation signaling cascades, membrane molecules, and internal pacemaker mechanisms. Yielding results similar to those of the experiments, the model simulations faithfully reproduce the changes in AP firing rate in response to CCh or ISO or a combination of both (i.e., accentuated antagonism). Eliminating AC-cAMP-PKA signaling abolished the core effect of autonomic receptor stimulation on the AP firing rate. Specifically, disabling the phospholamban modulation of the SERCA activity resulted in a significantly reduced effect

  1. Vasoactive intestinal polypeptide excites medial pontine reticular formation neurons in the brainstem rapid eye movement sleep-induction zone

    DEFF Research Database (Denmark)

    Kohlmeier, Kristi Anne; Reiner, P B

    1999-01-01

    Although it has long been known that microinjection of the cholinergic agonist carbachol into the medial pontine reticular formation (mPRF) induces a state that resembles rapid eye movement (REM) sleep, it is likely that other transmitters contribute to mPRF regulation of behavioral states. A key...... candidate is the peptide vasoactive intestinal polypeptide (VIP), which innervates the mPRF and induces REM sleep when injected into this region of the brainstem. To begin understanding the cellular mechanisms underlying this phenomenon, we examined the effects of VIP on mPRF cells using whole-cell patch...... conclude that VIP excites mPRF neurons by activation of a sodium current. This effect is mediated at least in part by G-protein stimulation of adenylyl cyclase, cAMP, and protein kinase A. These data suggest that VIP may play a physiological role in REM induction by its actions on mPRF neurons....

  2. Glycogen synthase kinase-3 regulation of urinary concentrating ability.

    Science.gov (United States)

    Rao, Reena

    2012-09-01

    Glycogen synthase kinase-3 (GSK3) is an enzyme that is gaining prominence as a critical signaling molecule in the epithelial cells of renal tubules. This review will focus on recent findings exploring the role of GSK3 in renal collecting ducts, especially its role in urine concentration involving vasopressin signaling. Recent studies using inhibition or tissue-specific gene deletion of GSK3 revealed the mechanism by which GSK3 regulates aquaporin 2 water channels via adenylate cyclase or the prostaglandin-E2 pathway. In other studies, postnatal treatment with lithium, an inhibitor of GSK3, increased cell proliferation and led to microcyst formation in rat kidneys. These studies suggest that loss of GSK3 activity could interfere with renal water transport at two levels. In the short term, it could disrupt vasopressin signaling in collecting duct cells and in the long term it could alter the structure of the collecting ducts, making them less responsive to the hydro-osmotic effects of vasopressin. Ongoing studies reveal the crucial role played by GSK3 in the regulation of vasopressin action in the renal collecting ducts and suggest a possible use of GSK3 inhibitors in disease conditions associated with disrupted vasopressin signaling.

  3. NORM regulations

    Energy Technology Data Exchange (ETDEWEB)

    Gray, P. [ed.

    1997-02-01

    The author reviews the question of regulation for naturally occuring radioactive material (NORM), and the factors that have made this a more prominent concern today. Past practices have been very relaxed, and have often involved very poor records, the involvment of contractors, and the disposition of contaminated equipment back into commercial service. The rationale behind the establishment of regulations is to provide worker protection, to exempt low risk materials, to aid in scrap recycling, to provide direction for remediation and to examine disposal options. The author reviews existing regulations at federal and state levels, impending legislation, and touches on the issue of site remediation and potential liabilities affecting the release of sites contaminated by NORM.

  4. Fisheries regulation

    DEFF Research Database (Denmark)

    Jensen, Frank; Frost, Hans Staby; Abildtrup, Jens

    2017-01-01

    Economists normally claim that a stock externality arises within fisheries because each individual fisherman does not take the effect on stock size into account when making harvest decisions. Due to the stock externality, it is commonly argued that fisheries regulation is necessary, but regulatory...... decisions are complicated by a tremendous amount of uncertainty and asymmetric information. This paper provides an overview of selected parts of the literature on the regulation of fisheries under uncertainty and asymmetric information, and possible areas for future research are identified. Specifically...

  5. French regulations

    International Nuclear Information System (INIS)

    Ballereau, P.

    1998-01-01

    In this issue are given the new French regulations relative to radiation protection of temporary personnel, the licensing to release gaseous and liquid wastes and the licensing granted to thirty two laboratories using beta and gamma decay radioisotopes. (N.C.)

  6. The small molecule triclabendazole decreases the intracellular level of cyclic AMP and increases resistance to stress in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Yong Joo Lee

    Full Text Available The Ras-adenylyl cyclase-protein kinase A nutrient-sensing pathway controls metabolism, proliferation and resistance to stress in Saccharomyces cerevisiae. The genetic disruption of this pathway increases resistance to a variety of stresses. We show here that the pharmacological inhibition of this pathway by the drug triclabendazole increases resistance to oxidants, heat stress and extends the chronological life. Evidence is presented that triclabendazole decreases the intracellular level of cyclic AMP by inhibiting adenylyl cyclase and triggers the parallel rapid translocation of the stress-resistance transcription factor Msn2 from the cytosol into the nucleus, as deduced from experiments employing a strain in which MSN2 is replaced with MSN2-GFP (GFP, green fluorescent protein. Msn2 and Msn4 are responsible for activating the transcription of numerous genes that encode proteins that protect cells from stress. The results are consistent with triclabendazole either inhibiting the association of Ras with adenylyl cyclase or directly inhibiting adenylyl cyclase, which in turn triggers Msn2/4 to enter the nucleus and activate stress-responsible element gene expression.

  7. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; DeVree, Brian T; Zou, Yaozhong

    2011-01-01

    -occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs...

  8. Allergen challenge primes for IL-5 mRNA production and abrogates beta-adrenergic function in peripheral blood T lymphocytes from asthmatics

    NARCIS (Netherlands)

    Borger, P; Jonker, GJ; Vellenga, E; Postma, DS; De Monchy, JGR; Kauffman, HF

    Background In previous studies, we have found a dysfunctional adenylyl cyclase (AC) system in patients with asthma after allergen provocation, which resulted in a 40-50% decreased generation of intracellular cAMP. In addition, in activated T helper lymphocyte clones, it has been demonstrated that

  9. TMFunction data: 17 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available ens Human Rhee MH, Nevo I, Bayewitch ML, Zagoory O, Vogel Z J Neurochem. 2000 Dec;75(6):2485-91. AC activity 6.4+_1.7 Ki (nM) Ki valu...es for CP55,940. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase

  10. TMFunction data: 21 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available ens Human Rhee MH, Nevo I, Bayewitch ML, Zagoory O, Vogel Z J Neurochem. 2000 Dec;75(6):2485-91. AC activity 3.6+_0.5 Ki (nM) Ki valu...es for WIN55,212-2. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase

  11. TMFunction data: 7 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available 1.0+_ 0.3 EC50 (nM) Inhibition of AC by W158F; EC50 values for HU-210. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase

  12. TMFunction data: 8 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available 1.2+_0.2 EC50 (nM) Inhibition of AC by W158F; EC50 values for HU-210. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase

  13. TMFunction data: 18 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available ens Human Rhee MH, Nevo I, Bayewitch ML, Zagoory O, Vogel Z J Neurochem. 2000 Dec;75(6):2485-91. AC activity 6.9+_0.9 Ki (nM) Ki valu...es for CP55,940. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase

  14. TMFunction data: 25 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available 1.3+_0.6 EC50 (nM) Inhibition of AC by W172F; EC50 values for HU-210. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase

  15. TMFunction data: 24 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available 1.4+_0.3 EC50 (nM) Inhibition of AC by W172F; EC50 values for HU-210. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase

  16. TMFunction data: 23 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available 2.0+_0.6 EC50 (nM) Inhibition of AC by W172F; EC50 values for HU-210. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase

  17. TMFunction data: 22 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available ens Human Rhee MH, Nevo I, Bayewitch ML, Zagoory O, Vogel Z J Neurochem. 2000 Dec;75(6):2485-91. AC activity 3.6+_1.2 Ki (nM) Ki valu...es for WIN55,212-2. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase

  18. TMFunction data: 14 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available 0.14+_0.02 Ki (nM) Ki values for HU-210. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase ...ens Human Rhee MH, Nevo I, Bayewitch ML, Zagoory O, Vogel Z J Neurochem. 2000 Dec;75(6):2485-91. AC activity

  19. Olfactory Receptors in Non-Chemosensory Organs: The Nervous System in Health and Disease

    NARCIS (Netherlands)

    Ferrer, Isidro; Garcia-Esparcia, Paula; Carmona, Margarita; Carro, Eva; Aronica, Eleonora; Kovacs, Gabor G.; Grison, Alice; Gustincich, Stefano

    2016-01-01

    Olfactory receptors (ORs) and down-stream functional signaling molecules adenylyl cyclase 3 (AC3), olfactory G protein α subunit (Gαolf), OR transporters receptor transporter proteins 1 and 2 (RTP1 and RTP2), receptor expression enhancing protein 1 (REEP1), and UDP-glucuronosyltransferases (UGTs)

  20. A Concise Synthesis of Forskolin

    Czech Academy of Sciences Publication Activity Database

    Hylse, O.; Maier, L.; Kučera, R.; Perečko, Tomáš; Svobodová, Aneta; Kubala, Lukáš; Paruch, K.; Svenda, J.

    2017-01-01

    Roč. 56, č. 41 (2017), s. 12586-12589 ISSN 1433-7851 Institutional support: RVO:68081707 Keywords : adenylyl-cyclase isoforms * key intermediate * enantioselective route Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 11.994, year: 2016

  1. TMFunction data: 11 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available 0.16+_ 0.07 Ki (nM) Ki values for HU-210. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase ...ns Human Rhee MH, Nevo I, Bayewitch ML, Zagoory O, Vogel Z J Neurochem. 2000 Dec;75(6):2485-91. AC activity

  2. TMFunction data: 13 [TMFunction[Archive

    Lifescience Database Archive (English)

    Full Text Available 0.19+_0.04 Ki (nM) Ki values for HU-210. ... CNR2_HUMAN (P34972) Helix ... Cannabinoids; CB2 receptor; G protein; Site directed mutagensis; adenylyl cyclase ...ens Human Rhee MH, Nevo I, Bayewitch ML, Zagoory O, Vogel Z J Neurochem. 2000 Dec;75(6):2485-91. AC activity

  3. Beta-Adrenergic signaling in rat heart is similarly affected by continuous and intermittent normobaric hypoxia

    Czech Academy of Sciences Publication Activity Database

    Hahnová, K.; Kašparová, D.; Žurmanová, J.; Neckář, Jan; Kolář, František; Novotný, J.

    2016-01-01

    Roč. 35, č. 2 (2016), s. 165-173 ISSN 0231-5882 R&D Projects: GA ČR(CZ) GAP303/12/1162 Institutional support: RVO:67985823 Keywords : rat myocardium * chronic hypoxia * beta-adrenergic receptors * adenylyl cyclase Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 1.170, year: 2016

  4. Regulating Internalities

    OpenAIRE

    Sunstein, Cass Robert; Allcott, Hunt

    2015-01-01

    This paper offers a framework for regulating internalities. Using a simple economic model, we provide four principles for designing and evaluating behaviorally-motivated policy. We then outline rules for determining which contexts reliably reflect true preferences and discuss empirical strategies for measuring internalities. As a case study, we focus on energy efficiency policy, including Corporate Average Fuel Economy (CAFE) standards and appliance and lighting energy efficiency standards.

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

    Directory of Open Access Journals (Sweden)

    Alain Mazé

    2014-06-01

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

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

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    Nancy Magnus

    2017-12-01

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

  7. A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.

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    Robert W Moon

    2009-09-01

    Full Text Available The ookinete is a motile stage in the malaria life cycle which forms in the mosquito blood meal from the zygote. Ookinetes use an acto-myosin motor to glide towards and penetrate the midgut wall to establish infection in the vector. The regulation of gliding motility is poorly understood. Through genetic interaction studies we here describe a signalling module that identifies guanosine 3', 5'-cyclic monophosphate (cGMP as an important second messenger regulating ookinete differentiation and motility. In ookinetes lacking the cyclic nucleotide degrading phosphodiesterase delta (PDEdelta, unregulated signalling through cGMP results in rounding up of the normally banana-shaped cells. This phenotype is suppressed in a double mutant additionally lacking guanylyl cyclase beta (GCbeta, showing that in ookinetes GCbeta is an important source for cGMP, and that PDEdelta is the relevant cGMP degrading enzyme. Inhibition of the cGMP-dependent protein kinase, PKG, blocks gliding, whereas enhanced signalling through cGMP restores normal gliding speed in a mutant lacking calcium dependent protein kinase 3, suggesting at least a partial overlap between calcium and cGMP dependent pathways. These data demonstrate an important function for signalling through cGMP, and most likely PKG, in dynamically regulating ookinete gliding during the transmission of malaria to the mosquito.

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

    Science.gov (United States)

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

    2015-12-01

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

  9. Atrial natriuretic peptide regulates Ca channel in early developmental cardiomyocytes.

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    Lin Miao

    Full Text Available BACKGROUND: Cardiomyocytes derived from murine embryonic stem (ES cells possess various membrane currents and signaling cascades link to that of embryonic hearts. The role of atrial natriuretic peptide (ANP in regulation of membrane potentials and Ca(2+ currents has not been investigated in developmental cardiomyocytes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of ANP in regulating L-type Ca(2+ channel current (I(CaL in different developmental stages of cardiomyocytes derived from ES cells. ANP decreased the frequency of action potentials (APs in early developmental stage (EDS cardiomyocytes, embryonic bodies (EB as well as whole embryo hearts. ANP exerted an inhibitory effect on basal I(CaL in about 70% EDS cardiomyocytes tested but only in about 30% late developmental stage (LDS cells. However, after stimulation of I(CaL by isoproterenol (ISO in LDS cells, ANP inhibited the response in about 70% cells. The depression of I(CaL induced by ANP was not affected by either Nomega, Nitro-L-Arginine methyl ester (L-NAME, a nitric oxide synthetase (NOS inhibitor, or KT5823, a cGMP-dependent protein kinase (PKG selective inhibitor, in either EDS and LDS cells; whereas depression of I(CaL by ANP was entirely abolished by erythro-9-(2-Hydroxy-3-nonyl adenine (EHNA, a selective inhibitor of type 2 phosphodiesterase(PDE2 in most cells tested. CONCLUSION/SIGNIFICANCES: Taken together, these results indicate that ANP induced depression of action potentials and I(CaL is due to activation of particulate guanylyl cyclase (GC, cGMP production and cGMP-activation of PDE2 mediated depression of adenosine 3', 5'-cyclic monophophate (cAMP-cAMP-dependent protein kinase (PKA in early cardiomyogenesis.

  10. 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.

  11. 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

  12. 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.

  13. Differential regulation of c-di-GMP metabolic enzymes by environmental signals modulates biofilm formation in Yersinia pestis

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    Gai-Xian eRen

    2016-06-01

    Full Text Available Cyclic diguanylate (c-di-GMP is essential for Yersinia pestis biofilm formation, which is important for flea-borne blockage-dependent plague transmission. Two diguanylate cyclases (DGCs, HmsT and HmsD and one phosphodiesterase (PDE, HmsP are responsible for the synthesis and degradation of c-di-GMP in Y. pestis. Here, we systematically analyzed the effect of various environmental signals on regulation of the biofilm phenotype, the c-di-GMP levels, and expression of HmsT, HmsD and HmsP in Y. pestis. Biofilm formation was higher in the presence of nonlethal high concentration of CaCl2, MgCl2, CuSO4, sucrose, sodium dodecyl sulfonate, or dithiothreitol, and was lower in the presence of FeCl2 or NaCl. In addition, we found that HmsD plays a major role in biofilm formation in acidic or redox environments. These environmental signals differentially regulated expression of HmsT, HmsP and HmsD, resulting in changes in the intracellular levels of c-di-GMP in Y. pestis. Our results suggest that bacteria can sense various environmental signals, and differentially regulates their DGCs and PDEs to coordinately regulate and adapt metabolism of c-di-GMP and biofilm formation to changing environments.

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

    Science.gov (United States)

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

    2001-11-30

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

  15. The Diguanylate Cyclase HsbD Intersects with the HptB Regulatory Cascade to Control Pseudomonas aeruginosa Biofilm and Motility.

    Directory of Open Access Journals (Sweden)

    Martina Valentini

    2016-10-01

    Full Text Available The molecular basis of second messenger signaling relies on an array of proteins that synthesize, degrade or bind the molecule to produce coherent functional outputs. Cyclic di-GMP (c-di-GMP has emerged as a eubacterial nucleotide second messenger regulating a plethora of key behaviors, like the transition from planktonic cells to biofilm communities. The striking multiplicity of c-di-GMP control modules and regulated cellular functions raised the question of signaling specificity. Are c-di-GMP signaling routes exclusively dependent on a central hub or can they be locally administrated? In this study, we show an example of how c-di-GMP signaling gains output specificity in Pseudomonas aeruginosa. We observed the occurrence in P. aeruginosa of a c-di-GMP synthase gene, hsbD, in the proximity of the hptB and flagellar genes cluster. We show that the HptB pathway controls biofilm formation and motility by involving both HsbD and the anti-anti-sigma factor HsbA. The rewiring of c-di-GMP signaling into the HptB cascade relies on the original interaction between HsbD and HsbA and on the control of HsbD dynamic localization at the cell poles.

  16. Comparative Analysis of Tocopherol Biosynthesis Genes and Its Transcriptional Regulation in Soybean Seeds.

    Science.gov (United States)

    T, Vinutha; Bansal, Navita; Kumari, Khushboo; Prashat G, Rama; Sreevathsa, Rohini; Krishnan, Veda; Kumari, Sweta; Dahuja, Anil; Lal, S K; Sachdev, Archana; Praveen, Shelly

    2017-12-20

    Tocopherols composed of four isoforms (α, β, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

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

    Science.gov (United States)

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

    2003-05-01

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

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

    Science.gov (United States)

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

    2018-03-01

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

  19. 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.

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

    KAUST Repository

    Qia, Zhi

    2010-11-18

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

  1. 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.

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

    Science.gov (United States)

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

    2003-11-01

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

  3. Signaling governed by G proteins and cAMP is crucial for growth, secondary metabolism and sexual development in Fusarium fujikuroi.

    Directory of Open Access Journals (Sweden)

    Lena Studt

    Full Text Available The plant-pathogenic fungus Fusarium fujikuroi is a notorious rice pathogen causing hyper-elongation of infected plants due to the production of gibberellic acids (GAs. In addition to GAs, F. fujikuroi produces a wide range of other secondary metabolites, such as fusarins, fusaric acid or the red polyketides bikaverins and fusarubins. The recent availability of the fungal genome sequence for this species has revealed the potential of many more putative secondary metabolite gene clusters whose products remain to be identified. However, the complex regulation of secondary metabolism is far from being understood. Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA, to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs. We demonstrated that fusarubin biosynthesis is negatively regulated by at least two Gα subunits, FfG1 and FfG3, which both function as stimulators of the adenylyl cyclase FfAC. Surprisingly, the primary downstream target of the adenylyl cyclase, the PKA, is not involved in the regulation of fusarubins, suggesting that additional, yet unidentified, cAMP-binding protein(s exist. In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner. In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media. Finally, sexual crosses of ffg1 mutants showed the importance of a functional FfG1 protein for development of perithecia in the mating strain that carries the MAT1-1 idiomorph.

  4. Competition between bank regulators

    OpenAIRE

    Schindler, Dirk; Eggert, Wolfgang

    2004-01-01

    This paper examines competition between bank regulators in open economies. We use a model where credit demand of firms is endogenous and show any tendency for downward competition in regulation policy is limited by the effect of regulation on profits of nonfinancial firms. Moreover, perfect mobility on loans and deposit markets fully eliminates the incentives of regulators to set bank regulation at ine±cient low levels.

  5. Regulating through leverage: Civil regulation in China

    NARCIS (Netherlands)

    Fürst, K.

    2016-01-01

    The overarching goal of this study is to examine the efforts of Chinese NGOs to prevent and/or control industrial pollution risks and then use the findings of this research to study the nature of civil regulation in, and beyond, China’s authoritarian setting. It first argues that 'regulation through

  6. Comparative genomics reveals candidate carotenoid pathway regulators of ripening watermelon fruit

    Science.gov (United States)

    2013-01-01

    Background Many fruits, including watermelon, are proficient in carotenoid accumulation during ripening. While most genes encoding steps in the carotenoid biosynthetic pathway have been cloned, few transcriptional regulators of these genes have been defined to date. Here we describe the identification of a set of putative carotenoid-related transcription factors resulting from fresh watermelon carotenoid and transcriptome analysis during fruit development and ripening. Our goal is to both clarify the expression profiles of carotenoid pathway genes and to identify candidate regulators and molecular targets for crop improvement. Results Total carotenoids progressively increased during fruit ripening up to ~55 μg g-1 fw in red-ripe fruits. Trans-lycopene was the carotenoid that contributed most to this increase. Many of the genes related to carotenoid metabolism displayed changing expression levels during fruit ripening generating a metabolic flux toward carotenoid synthesis. Constitutive low expression of lycopene cyclase genes resulted in lycopene accumulation. RNA-seq expression profiling of watermelon fruit development yielded a set of transcription factors whose expression was correlated with ripening and carotenoid accumulation. Nineteen putative transcription factor genes from watermelon and homologous to tomato carotenoid-associated genes were identified. Among these, six were differentially expressed in the flesh of both species during fruit development and ripening. Conclusions Taken together the data suggest that, while the regulation of a common set of metabolic genes likely influences carotenoid synthesis and accumulation in watermelon and tomato fruits during development and ripening, specific and limiting regulators may differ between climacteric and non-climacteric fruits, possibly related to their differential susceptibility to and use of ethylene during ripening. PMID:24219562

  7. Identification of a molecular pH sensor in coral.

    Science.gov (United States)

    Barott, Katie L; Barron, Megan E; Tresguerres, Martin

    2017-11-15

    Maintaining stable intracellular pH (pHi) is essential for homeostasis, and requires the ability to both sense pH changes that may result from internal and external sources, and to regulate downstream compensatory pH pathways. Here we identified the cAMP-producing enzyme soluble adenylyl cyclase (sAC) as the first molecular pH sensor in corals. sAC protein was detected throughout coral tissues, including those involved in symbiosis and calcification. Application of a sAC-specific inhibitor caused significant and reversible pHi acidosis in isolated coral cells under both dark and light conditions, indicating sAC is essential for sensing and regulating pHi perturbations caused by respiration and photosynthesis. Furthermore, pHi regulation during external acidification was also dependent on sAC activity. Thus, sAC is a sensor and regulator of pH disturbances from both metabolic and external origin in corals. Since sAC is present in all coral cell types, and the cAMP pathway can regulate virtually every aspect of cell physiology through post-translational modifications of proteins, sAC is likely to trigger multiple homeostatic mechanisms in response to pH disturbances. This is also the first evidence that sAC modulates pHi in any non-mammalian animal. Since corals are basal metazoans, our results indicate this function is evolutionarily conserved across animals. © 2017 The Author(s).

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

    Science.gov (United States)

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

    2015-03-01

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

  9. Atomic Energy Control Regulations

    International Nuclear Information System (INIS)

    1992-01-01

    This is the consolidated text of the Atomic Energy Control Regulations of 17 March 1960, with amendments to 27 August 1992. The Regulations cover the licensing of nuclear facilities, radiation sources, including uranium mining, radiation protection questions, etc. (NEA)

  10. Environmental regulation and competitiveness

    NARCIS (Netherlands)

    Mulatu, A.; Florax, R.J.G.M.; Withagen, C.A.A.M.

    2001-01-01

    The potential relationship between domestic environmental regulation and international competitiveness has evoked various speculations. The common neoclassical train of thought is that strict environmental regulation is detrimental to the competitiveness of industry, and that it induces phenomena

  11. Ocean Dumping Control Regulations

    International Nuclear Information System (INIS)

    1978-01-01

    These Regulations were made further to the Ocean Dumping Control Act which provides for restrictions in dumping operations. The Regulations contain model applications for permits to dump or load a series of materials. (NEA)

  12. Regulation of Genetic Tests

    Science.gov (United States)

    ... for Genomics Research Intellectual Property Issues in Genetics Archive Online Bioethics Resources Privacy in Genomics Regulation of ... are not regulated, meaning that they go to market without any independent analysis to verify the claims ...

  13. Trout Stream Special Regulations

    Data.gov (United States)

    Minnesota Department of Natural Resources — This layer shows Minnesota trout streams that have a special regulation as described in the 2006 Minnesota Fishing Regulations. Road crossings were determined using...

  14. N-docosahexaenoylethanolamine regulates Hedgehog signaling and promotes growth of cortical axons

    Directory of Open Access Journals (Sweden)

    Giorgi Kharebava

    2015-12-01

    Full Text Available Axonogenesis, a process for the establishment of neuron connectivity, is central to brain function. The role of metabolites derived from docosahexaenoic acid (DHA, 22:6n-3 that is specifically enriched in the brain, has not been addressed in axon development. In this study, we tested if synaptamide (N-docosahexaenoylethanolamine, an endogenous metabolite of DHA, affects axon growth in cultured cortical neurons. We found that synaptamide increased the average axon length, inhibited GLI family zinc finger 1 (GLI1 transcription and sonic hedgehog (Shh target gene expression while inducing cAMP elevation. Similar effects were produced by cyclopamine, a regulator of the Shh pathway. Conversely, Shh antagonized elevation of cAMP and blocked synaptamide-mediated increase in axon length. Activation of Shh pathway by a smoothened (SMO agonist (SAG or overexpression of SMO did not inhibit axon growth mediated by synaptamide or cyclopamine. Instead, adenylate cyclase inhibitor SQ22536 abolished synaptamide-mediated axon growth indicating requirement of cAMP elevation for this process. Our findings establish that synaptamide promotes axon growth while Shh antagonizes synaptamide-mediated cAMP elevation and axon growth by a SMO-independent, non-canonical pathway.

  15. Developmentally regulated sesquiterpene production confers resistance to Colletotrichum gloeosporioides in ripe pepper fruits.

    Directory of Open Access Journals (Sweden)

    Sangkyu Park

    Full Text Available Sesquiterpenoid capsidiol, exhibiting antifungal activity against pathogenic fungus, is accumulated in infected ripe pepper fruits. In this study, we found a negative relation between the capsidiol level and lesion size in fruits infected with Colletotrichum gloeosporioides, depending on the stage of ripening. To understand the developmental regulation of capsidiol biosynthesis, fungal-induced gene expressions in the isoprenoid biosynthetic pathways were examined in unripe and ripe pepper fruits. The sterol biosynthetic pathway was almost shut down in healthy ripe fruits, showing very low expression of hydroxymethyl glutaryl CoA reductase (HMGR and squalene synthase (SS genes. In contrast, genes in the carotenoid pathway were highly expressed in ripe fruits. In the sesquiterpene pathway, 5-epi-aristolochene synthase (EAS, belonging to a sesquiterpene cyclase (STC family, was significantly induced in the ripe fruits upon fungal infection. Immunoblot and enzyme activity analyses showed that the STCs were induced both in the infected unripe and ripe fruits, while capsidiol was synthesized discriminatively in the ripe fruits, implying diverse enzymatic specificity of multiple STCs. Thereby, to divert sterol biosynthesis into sesquiterpene production, infected fruits were pretreated with an SS inhibitor, zaragozic acid (ZA, resulting in increased levels of capsidiol by more than 2-fold in the ripe fruits, with concurrent reduction of phytosterols. Taken together, the present results suggest that the enhanced expression and activity of EAS in the ripe fruits play an important role in capsidiol production, contributing to the incompatibility between the anthracnose fungus and the ripe pepper fruits.

  16. Regulation of Biofilm Formation by Hfq is Influenced by Presence of Plasmid pCD1 in Yersinia Pestis Biovar Microtus

    Directory of Open Access Journals (Sweden)

    Huiying Yang

    2017-10-01

    Full Text Available Yersinia pestis synthesizes the attached biofilms in the flea gut to promotethe flea-borne transmission of this deadly pathogen. Bellows et al. reported that the posttranscriptional regulator Hfq inhibites biofilm formation in apCD1− derivative of Y. pestis CO92, however, we found that Hfq stimulates biofilm production in a microtus strain of Y. pestis with the typical plasmids, including pCD1. When we cured pCD1 from this strain, the biofilm phenotype was in accordance with that reported by Bellows et al., indicating that the unknown pCD1-associated factors modulating the regulatory pathways of Y. pestis biofilm formation. Further gene regulation experiments using relevant pCD1+ Y. pestis strains disclose that Hfq positively regulates the expression of hmsHFRS and hmsT encoding a diguanylate cyclase while negatively regulates the expression of hmsP encoding the sole phosphodiesterase. However, Hfq has no regulatory effect on the expression of hmsCDE at the mRNA and protein levels. Our results suggest that we should be cautious to make conclusion from results based on the pCD1-cured Y. pestis.

  17. 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

  18. 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.

  19. Culture and emotion regulation.

    Science.gov (United States)

    Ford, Brett Q; Mauss, Iris B

    2015-06-01

    While anthropological research has long emphasized cultural differences in whether emotions are viewed as beneficial versus harmful, psychological science has only recently begun to systematically examine those differences and their implications for emotion regulation and well-being. Underscoring the pervasive role of culture in people's emotions, we summarize research that has examined links between culture, emotion regulation, and well-being. Specifically, we focus on two questions. First, how does culture lead individuals to regulate their emotions? And second, how does culture modulate the link between emotion regulation and well-being? We finish by suggesting directions for future research to advance the study of culture and emotion regulation.

  20. Radiation Control Regulation 1993

    International Nuclear Information System (INIS)

    1993-01-01

    This Regulation (No. 434-1993) was made in pursuance of the Radiation Control Act 1990 and replaces the Active Substances Regulations 1959 repealed by the Act. It entered into force on 1 September 1993. The Regulation specifies that the technical radiation protection definitions have the same meaning as in the 1990 recommendations. The Regulation provides for the licensing of persons to use radioactive substances and radiation apparatus. It prescribes activities which may only be carried out by an accredited radiation expert and regulates the use of radiation apparatus and radioactive substances as well as the disposal and transport of radiation apparatus and radioactive substances. (NEA)

  1. Load regulating expansion fixture

    International Nuclear Information System (INIS)

    Wagner, L.M.; Strum, M.J.

    1998-01-01

    A free standing self contained device for bonding ultra thin metallic films, such as 0.001 inch beryllium foils is disclosed. The device will regulate to a predetermined load for solid state bonding when heated to a bonding temperature. The device includes a load regulating feature, whereby the expansion stresses generated for bonding are regulated and self adjusting. The load regulator comprises a pair of friction isolators with a plurality of annealed copper members located there between. The device, with the load regulator, will adjust to and maintain a stress level needed to successfully and economically complete a leak tight bond without damaging thin foils or other delicate components. 1 fig

  2. Volume regulation in epithelia

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Hoffmann, Else Kay

    2016-01-01

    to amphibian skin and mammalian cortical collecting tubule of low and intermediate osmotic permeability. Crosstalk between entrance and exit mechanisms interferes with volume regulation both at aniso-osmotic and iso-osmotic volume perturbations. It has been proposed that cell volume regulation is an intrinsic...... regulation are cloned. The volume-regulated anion channel (VRAC) exhibiting specific electrophysiological characteristics seems exclusive to serve cell volume regulation. This is contrary to K+ channels as well as cotransporters and exchange mechanisms that may serve both transepithelial transport and cell...... volume regulation. In the same cell, these functions may be maintained by different ion pathways that are separately regulated. RVD is often preceded by increase in cytosolic free Ca2+, probably via influx through TRP channels, but Ca2+ release from intracellular stores has also been observed. Cell...

  3. Voltage regulator for generator

    Energy Technology Data Exchange (ETDEWEB)

    Naoi, K

    1989-01-17

    It is an object of this invention to provide a voltage regulator for a generator charging a battery, wherein even if the ambient temperature at the voltage regulator rises abnormally high, possible thermal breakage of the semiconductor elements constituting the voltage regulator can be avoided. A feature of this invention is that the semiconductor elements can be protected from thermal breakage, even at an abnormal ambient temperature rise at the voltage regulator for the battery charging generator, by controlling a maximum conduction ratio of a power transistor in the voltage regulator in accordance with the temperature at the voltage regulator. This is achieved through a switching device connected in series to the field coil of the generator and adapted to be controlled in accordance with an output voltage of the generator and the ambient temperature at the voltage regulator. 6 figs.

  4.  The discovery of neuromedin U and its pivotal role in the central regulation of energy homeostasis

    Directory of Open Access Journals (Sweden)

    Katarzyna Kirsz

    2012-04-01

    Full Text Available  Neuromedin U (NMU is a structurally highly conserved neuropeptide and has been paired with the G-protein-coupled receptors (GPCRs NMUR1 and NMUR2, which were formerly classified in the orphan receptor family. Activation of the G protein Gq/11 subunit causes a pertussis toxin (PTX-insensitive activation of both phospholipase C and mitogen-activated protein kinase (MAP, and activation of the Go subunit causes a PTX-sensitive inhibition of adenyl cyclase. Additionally, NMU selectively inhibits L-type high-voltage-gated Ca2 channels in mouse hippocampus, as well as low-voltage-activated T-type Ca2 channels in mouse dorsal root ganglia (DRG. NMU peptide and its receptors are predominantly expressed in the gastrointestinal tract and specific structures within the brain, reflecting its major role in the regulation of energy homeostasis. A novel neuropeptide, neuromedin S (NMS, is structurally related to NMU. They share a C-terminal core structure and both have been implicated in the regulation of food intake, as well as the circadian rhythms. The acute anorectic and weight-reducing effects of NMU and NMS are mediated by NMUR2. This suggests that NMUR2-selective agonists may be useful for the treatment of obesity.

  5. 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.

  6. (S)Pot on Mitochondria: Cannabinoids Disrupt Cellular Respiration to Limit Neuronal Activity.

    Science.gov (United States)

    Harkany, Tibor; Horvath, Tamas L

    2017-01-10

    Classical views posit G protein-coupled cannabinoid receptor 1s (CB1Rs) at the cell surface with cytosolic Giα-mediated signal transduction. Hebert-Chatelain et al. (2016) instead place CB 1 Rs at mitochondria limiting neuronal respiration by soluble adenylyl cyclase-dependent modulation of complex I activity. Thus, neuronal bioenergetics link to synaptic plasticity and, globally, learning and memory. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

    OpenAIRE

    Ping, P; Gelzer-Bell, R; Roth, D A; Kiel, D; Insel, P A; Hammond, H K

    1995-01-01

    To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling as...

  8. Photodynamics of BLUF domain proteins: a new class of the biological blue-light photoreceptors

    OpenAIRE

    Zirak Yousefabadi, Peyman

    2008-01-01

    BLUF domains are light sensors of many microorganisms. They are present in the multi-domain proteins e.g. AppA from the phototrophic proteobacterium Rhodobacter sphaeroides, YcgF from Escherichia coli, PAC (photoactive adenylyl cyclase) from the unicellular flagellate Euglena gracilis and single domain proteins e.g. BlrB from Rhodobacter sphaeroides, Slr1694 from cyanobacterium Synechocystis sp.PCC6803, and Tll0078 of the thermophilic unicellular cyanobacterium Thermosynechococcus elongates B...

  9. TOWARD MORE EFFECTIVE REGULATION

    Energy Technology Data Exchange (ETDEWEB)

    J. GRAF

    2000-06-01

    This paper proposes a model relationship between the operator engaged in a hazardous activity, the regulator of that activity, and the general public. The roles and responsibilities of each entity are described in a way that allows effective communication flow. The role of the regulator is developed using the steam boiler as an example of a hazard subject to regulation; however, the model applies to any regulated activity. In this model the safety analyst has the extremely important role of communicating sometimes difficult technical information to the regulator in a way that the regulator can provide credible assurance to the general public as to the adequacy of the control of the hazardous activity. The conclusion asserts that acceptance of the model, understanding of the roles and responsibilities and definition of who communicates what information to whom will mitigate frustration on the part of each of the three entities.

  10. The development of regulations

    International Nuclear Information System (INIS)

    Slokan Dusic, D.; Levstek, M.F.; Stritar, A.

    2003-01-01

    In October 2002, The Act on Protection Against Ionising Radiation and Nuclear Safety which regulates all aspects of protection against ionising radiation and nuclear safety entered into force in Slovenia. The Slovenian government and its responsible ministries shall issue several governmental and ministerial regulations to support the above - mentioned act. The Slovenian Nuclear Safety Administration (SNSA) which acts within the Ministry of the Environment, Spatial Planing and Energy takes an active part in drafting the regulations which are defined in the act. Due to a very comprehensive and pretentious task, that is to be completed in a relatively short period of time, taking into consideration the involvement of stakeholders and all competent ministries, the SNSA within the Quality Management System developed a special procedure that insures the systematic approach to the preparation of regulations. The article will briefly represent the process that: defines the preparation, development, harmonisation, review, approval and issue of regulations and uniforms the format of developed regulations. (author)

  11. Regulating hedge funds.

    OpenAIRE

    Daníelsson, J.; Zigrand, JP.

    2007-01-01

    Due to the ever-increasing amounts under management and their unregulated and opaque nature, hedge funds have emerged as a key concern for policymakers. While until now, hedge funds have been left essentially unregulated, we are seeing increasing calls for regulation for both microprudential and macroprudential reasons. In our view, most calls for the regulation of hedge funds are based on a misperception of the effectiveness of financial regulations, perhaps coupled with a lack of understand...

  12. Here, there and everywhere: Resistin-like molecules in infection, inflammation, and metabolic disorders.

    Science.gov (United States)

    Pine, Gabrielle M; Batugedara, Hashini M; Nair, Meera G

    2018-06-01

    The Resistin-Like Molecules (RELM) α, β, and γ and their namesake, resistin, share structural and sequence homology but exhibit significant diversity in expression and function within their mammalian host. RELM proteins are expressed in a wide range of diseases, such as: microbial infections (eg. bacterial and helminth), inflammatory diseases (eg. asthma, fibrosis) and metabolic disorders (eg. diabetes). While the expression pattern and molecular regulation of RELM proteins are well characterized, much controversy remains over their proposed functions, with evidence of host-protective and pathogenic roles. Moreover, the receptors for RELM proteins are unclear, although three receptors for resistin, decorin, adenylyl cyclase-associated protein 1 (CAP1), and Toll-like Receptor 4 (TLR4) have recently been proposed. In this review, we will first summarize the molecular regulation of the RELM gene family, including transcription regulation and tissue expression in humans and mouse disease models. Second, we will outline the function and receptor-mediated signaling associated with RELM proteins. Finally, we will discuss recent studies suggesting that, despite early misconceptions that these proteins are pathogenic, RELM proteins have a more nuanced and potentially beneficial role for the host in certain disease settings. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Regulating household financial advice

    Directory of Open Access Journals (Sweden)

    Benjamin F. Cummings

    2012-08-01

    Full Text Available This paper reviews economic theory related to investment advice. This theory explains 1 why financial advisors need to be carefully regulated for the benefit of both the investment advice industry and for consumers, 2 why principles-based regulation (e.g., a fiduciary standard is more efficient than rules-based regulation, 3 why dual regulation of financial professionals providing investment or insurance advice is inefficient and inequitable policy, and 4 why the application of a universal and uniform fiduciary standard will be difficult to implement.

  14. The regulation of hunting

    DEFF Research Database (Denmark)

    Abildtrup, Jens; Jensen, Frank

    Within hunting, wildlife populations are estimated to be too high in many countries which is assumed to be due to the market failure, that each hunter harvests too little compared to what the regulator wants. This may be due to the existing regulation which, among other things, requires knowledge...... by an individual, variable tax rate. The variable tax rate is, among other things, based on the difference in marginal value of the population between the hunter and the regulator. The paper shows that the population tax/subsidy secures a first-best optimum. Thus, the population tax is a good alternative...... to the existing regulation....

  15. Nuclear safety and regulation

    International Nuclear Information System (INIS)

    Kim, Hho Jung

    2000-03-01

    This book contains 12 chapters, which are atom and radiation, nuclear reactor and kinds of nuclear power plant, safeguard actuation system and stability evaluation for rock foundation of nuclear power plant, nuclear safety and principle, safety analysis and classification of incident, probabilistic safety assessment and major incident, nuclear safety regulation, system of nuclear safety regulation, main function and subject of safety regulation in nuclear facilities, regulation of fuel cycle and a nuclear dump site, protection of radiation and, safety supervision and, safety supervision and measurement of environmental radioactivity.

  16. Second messenger production in avian medullary nephron segments in response to peptide hormones.

    Science.gov (United States)

    Goldstein, D L; Reddy, V; Plaga, K

    1999-03-01

    We examined the sites of peptide hormone activation within medullary nephron segments of the house sparrow (Passer domesticus) kidney by measuring rates of hormone-induced generation of cyclic nucleotide second messenger. Thin descending limbs, thick ascending limbs, and collecting ducts had baseline activity of adenylyl cyclase that resulted in cAMP accumulation of 207 +/- 56, 147 +/- 31, and 151 +/- 41 fmol. mm-1. 30 min-1, respectively. In all segments, this activity increased 10- to 20-fold in response to forskolin. Activity of adenylyl cyclase in the thin descending limb was stimulated approximately twofold by parathyroid hormone (PTH) but not by any of the other hormones tested [arginine vasotocin (AVT), glucagon, atrial natriuretic peptide (ANP), or isoproterenol, each at 10(-6) M]. Thick ascending limb was stimulated two- to threefold by both AVT and PTH; however, glucagon and isoproterenol had no effect, and ANP stimulated neither cAMP nor cGMP accumulation. Adenylyl cyclase activity in the collecting duct was stimulated fourfold by AVT but not by the other hormones; likewise, ANP did not stimulate cGMP accumulation in this segment. These data support a tubular action of AVT and PTH in the avian renal medulla.

  17. Sympathetic Neurotransmitters Modulate Osteoclastogenesis and Osteoclast Activity in the Context of Collagen-Induced Arthritis

    Science.gov (United States)

    Muschter, Dominique; Schäfer, Nicole; Stangl, Hubert; Straub, Rainer H.; Grässel, Susanne

    2015-01-01

    Excessive synovial osteoclastogenesis is a hallmark of rheumatoid arthritis (RA). Concomitantly, local synovial changes comprise neuronal components of the peripheral sympathetic nervous system. Here, we wanted to analyze if collagen-induced arthritis (CIA) alters bone marrow-derived macrophage (BMM) osteoclastogenesis and osteoclast activity, and how sympathetic neurotransmitters participate in this process. Therefore, BMMs from Dark Agouti rats at different CIA stages were differentiated into osteoclasts in vitro and osteoclast number, cathepsin K activity, matrix resorption and apoptosis were analyzed in the presence of acetylcholine (ACh), noradrenaline (NA) vasoactive intestinal peptide (VIP) and assay-dependent, adenylyl cyclase activator NKH477. We observed modulation of neurotransmitter receptor mRNA expression in CIA osteoclasts without affecting protein level. CIA stage-dependently altered marker gene expression associated with osteoclast differentiation and activity without affecting osteoclast number or activity. Neurotransmitter stimulation modulated osteoclast differentiation, apoptosis and activity. VIP, NA and adenylyl cyclase activator NKH477 inhibited cathepsin K activity and osteoclastogenesis (NKH477, 10-6M NA) whereas ACh mostly acted pro-osteoclastogenic. We conclude that CIA alone does not affect metabolism of in vitro generated osteoclasts whereas stimulation with NA, VIP plus specific activation of adenylyl cyclase induced anti-resorptive effects probably mediated via cAMP signaling. Contrary, we suggest pro-osteoclastogenic and pro-resorptive properties of ACh mediated via muscarinic receptors. PMID:26431344

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

    Science.gov (United States)

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

    2014-12-01

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

  19. Structure of the Cmr2 Subunit of the CRISPR-Cas RNA Silencing Complex

    Energy Technology Data Exchange (ETDEWEB)

    Cocozaki, Alexis I.; Ramia, Nancy F.; Shao, Yaming; Hale, Caryn R.; Terns, Rebecca M.; Terns, Michael P.; Li, Hong (FSU); (Georgia)

    2012-08-10

    Cmr2 is the largest and an essential subunit of a CRISPR RNA-Cas protein complex (the Cmr complex) that cleaves foreign RNA to protect prokaryotes from invading genetic elements. Cmr2 is thought to be the catalytic subunit of the effector complex because of its N-terminal HD nuclease domain. Here, however, we report that the HD domain of Cmr2 is not required for cleavage by the complex in vitro. The 2.3 {angstrom} crystal structure of Pyrococcus furiosus Cmr2 (lacking the HD domain) reveals two adenylyl cyclase-like and two {alpha}-helical domains. The adenylyl cyclase-like domains are arranged as in homodimeric adenylyl cyclases and bind ADP and divalent metals. However, mutagenesis studies show that the metal- and ADP-coordinating residues of Cmr2 are also not critical for cleavage by the complex. Our findings suggest that another component provides the catalytic function and that the essential role by Cmr2 does not require the identified ADP- or metal-binding or HD domains in vitro.

  20. Functional β2-adrenoceptors in rat left atria: effect of foot-shock stress.

    Science.gov (United States)

    Moura, André Luiz de; Hyslop, Stephen; Grassi-Kassisse, Dora M; Spadari, Regina C

    2017-09-01

    Altered sensitivity to the chronotropic effect of catecholamines and a reduction in the β 1 /β 2 -adrenoceptor ratio have previously been reported in right atria of stressed rats, human failing heart, and aging. In this report, we investigated whether left atrial inotropism was affected by foot-shock stress. Male rats were submitted to 3 foot-shock sessions and the left atrial inotropic response, adenylyl cyclase activity, and β-adrenoceptor expression were investigated. Left atria of stressed rats were supersensitive to isoprenaline when compared with control rats and this effect was abolished by ICI118,551, a selective β 2 -receptor antagonist. Schild plot slopes for the antagonism between CGP20712A (a selective β 1 -receptor antagonist) and isoprenaline differed from unity in atria of stressed but not control rats. Atrial sensitivity to norepinephrine, as well as basal and forskolin- or isoprenaline-stimulated adenylyl cyclase activities were not altered by stress. The effect of isoprenaline on adenylyl cyclase stimulation was partially blocked by ICI118,551 in atrial membranes of stressed rats. These findings indicate that foot-shock stress equally affects inotropism and chronotropism and that β 2 -adrenoceptor upregulation contributes to the enhanced inotropic response to isoprenaline.