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Sample records for adenylate cyclase toxin

  1. Suppression of Platelet Aggregation by Bordetella pertussis Adenylate Cyclase Toxin

    Iwaki, Masaaki; Kamachi, Kazunari; Heveker, Nikolaus; Konda, Toshifumi

    1999-01-01

    The effect of Bordetella pertussis adenylate cyclase toxin (ACT) on platelet aggregation was investigated. This cell-invasive adenylate cyclase completely suppressed ADP (10 μM)-induced aggregation of rabbit platelets at 3 μg/ml and strongly suppressed thrombin (0.2 U/ml)-induced aggregation at 10 μg/ml. The suppression was accompanied by marked increase in platelet intracellular cyclic AMP (cAMP) content and was diminished by the anti-ACT monoclonal antibody B7E11. A catalytically inactive p...

  2. Adenylate cyclase toxin-hemolysin relevance for pertussis vaccines

    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

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

    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

  4. Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

    Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Devoisselle, Jean-Marie; Ladant, Daniel; Chopineau, Joel

    2013-01-01

    Many bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, although how they pass through a lipid bilayer remains largely unknown. Bordetella pertussis adenylate cyclase (CyaA) toxin delivers its catalytic domain directly across the cell membrane. To characterize this unique translocation process, we designed an in vitro assay based on a tethered lipid bilayer assembled over a biosensor surface derivatized with calmodulin, a natural activator of the toxin. C...

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

    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

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

    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: 3.169, year: 2014

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

    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: 9.322, year: 2014

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

    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: 2.403, year: 2014

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

    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 ostatní: 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

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

    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

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

    Radovan Fiser

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

  12. Synthesis of acyclic nucleoside phosphonates bearing (N-methyl)anthraniloyl substituent as potential inhibitors of adenylate cyclase toxin from Bordetella Pertussis

    Břehová, Petra; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Janeba, Zlatko

    Praha: Czech Chemical Society, 2015. s. 61. [Liblice 2015. Advances in Organic , Bioorganic and Pharmaceutical Chemistry /50./. 06.11.2015-08.11.2015, Olomouc] R&D Projects: GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * adenylate cyclase toxin * prodrugs Subject RIV: CC - Organic Chemistry

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

    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: 2.968, year: 2014

  14. Bordetella adenylate cyclase toxin mobilizes its beta2 integrin receptor into lipid rafts to accomplish translocation across target cell membrane in two steps.

    Ladislav Bumba

    2010-05-01

    Full Text Available Bordetella adenylate cyclase toxin (CyaA binds the alpha(Mbeta(2 integrin (CD11b/CD18, Mac-1, or CR3 of myeloid phagocytes and delivers into their cytosol an adenylate cyclase (AC enzyme that converts ATP into the key signaling molecule cAMP. We show that penetration of the AC domain across cell membrane proceeds in two steps. It starts by membrane insertion of a toxin 'translocation intermediate', which can be 'locked' in the membrane by the 3D1 antibody blocking AC domain translocation. Insertion of the 'intermediate' permeabilizes cells for influx of extracellular calcium ions and thus activates calpain-mediated cleavage of the talin tether. Recruitment of the integrin-CyaA complex into lipid rafts follows and the cholesterol-rich lipid environment promotes translocation of the AC domain across cell membrane. AC translocation into cells was inhibited upon raft disruption by cholesterol depletion, or when CyaA mobilization into rafts was blocked by inhibition of talin processing. Furthermore, CyaA mutants unable to mobilize calcium into cells failed to relocate into lipid rafts, and failed to translocate the AC domain across cell membrane, unless rescued by Ca(2+ influx promoted in trans by ionomycin or another CyaA protein. Hence, by mobilizing calcium ions into phagocytes, the 'translocation intermediate' promotes toxin piggybacking on integrin into lipid rafts and enables AC enzyme delivery into host cytosol.

  15. Regulation of brain adenylate cyclase by calmodulin

    This thesis examined the interaction between the Ca2+-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-[125I]-CaM-diazopyruvamide (125I-CAM-DAP) behaved like native CaM with respect to Ca2+-enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and Ca2+-dependent stimulation of adenylate cyclase. 125I-CaM-DAP cross-linked to CaM-binding proteins in a Ca2+-dependent, concentration-dependent, and CaM-specific manner. Photolysis of 125I-CaM-DAP and forskolin-agarose purified CaM-sensitive adenylate cyclase produced an adduct with a molecular weight of 140,000

  16. Molecular cloning and expression of the Bacillus anthracis edema factor toxin gene: a calmodulin-dependent adenylate cyclase.

    Tippetts, M T; Robertson, D L

    1988-01-01

    The Bacillus anthracis exotoxin is composed of a lethal factor, a protective antigen, and an edema factor (EF). EF is a calmodulin-dependent adenylate cyclase which elevates cyclic AMP levels within cells. The entire EF gene (cya) has been cloned in Escherichia coli, but EF gene expression by its own B. anthracis promoter could not be detected in E. coli. However, when the EF gene was placed downstream from the lac or the T7 promoter, enzymatically active EF was produced. The EF gene, like th...

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

    Masin, Jiri; Osickova, Adriana; Sukova, Anna; Fiser, Radovan; Halada, Petr; Bumba, Ladislav; Linhartova, Irena; Osicka, Radim; Sebo, Peter

    2016-01-01

    The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The ‘AC to Hly-linking segment’ thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins. PMID:27581058

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

    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 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ 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 Ca2+ 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 Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating

  19. Molecular cloning and amplification of the adenylate cyclase gene.

    Wang, J Y; Clegg, D O; Koshland, D E

    1981-01-01

    A segment of DNA containing cya, the gene for adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], has been isolated from Salmonella typhimurium. The phage lambda gt4 was used as a cloning vector and adenylate cyclase-positive hybrid phages were isolated that complemented adenylate cyclase-negative bacteria. The cloned DNA fragment encodes a polypeptide of molecular weight 81,000 that gives rise to adenylate cyclase activity. This protein represents a functional mutant of the ...

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

    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.

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

    Ahmad, Jawid Nazir; Cerny, Ondrej; Linhartova, Irena; Masin, Jiri; Osicka, Radim; Sebo, Peter

    2016-03-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) plays a key role in virulence of Bordetella pertussis. CyaA penetrates myeloid cells expressing the complement receptor 3 (αM β2 integrin CD11b/CD18) and subverts bactericidal capacities of neutrophils and macrophages by catalysing unregulated conversion of cytosolic ATP to the key signalling molecule adenosine 3',5'-cyclic monophosphate (cAMP). We show that the signalling of CyaA-produced cAMP hijacks, by an as yet unknown mechanism, the activity of the tyrosine phosphatase SHP-1 and activates the pro-apoptotic BimEL-Bax cascade. Mitochondrial hyperpolarization occurred in human THP-1 macrophages within 10 min of exposure to low CyaA concentrations (e.g. 20 ng ml(-1) ) and was accompanied by accumulation of BimEL and association of the pro-apoptotic factor Bax with mitochondria. BimEL accumulation required cAMP/protein kinase A signalling, depended on SHP-1 activity and was selectively inhibited upon small interfering RNA knockdown of SHP-1 but not of the SHP-2 phosphatase. Moreover, signalling of CyaA-produced cAMP inhibited the AKT/protein kinase B pro-survival cascade, enhancing activity of the FoxO3a transcription factor and inducing Bim transcription. Synergy of FoxO3a activation with SHP-1 hijacking thus enables the toxin to rapidly trigger a persistent accumulation of BimEL, thereby activating the pro-apoptotic programme of macrophages and subverting the innate immunity of the host. PMID:26334669

  2. Pituitary adenylate cyclase activating polypeptide and migraine

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

    2014-01-01

    Pituitary adenylate cyclase activating peptide (PACAP) is found in human trigeminocervical complex and can trigger migraine. PACAP levels were measured using a sensitive radioimmunoassay. Stimulation of the superior sagittal sinus (SSS) in cat elevated PACAP levels in cranial blood. Patients with...... moderate or severe migraine headache had elevated PACAP in the external jugular vein during headache (n = 15), that was reduced 1 h after treatment with sumatriptan 6 mg (n = 11), and further reduced interictally (n = 9). The data suggest PACAP, or its receptors, are a promising target for migraine...

  3. Glucagon and adenylate cyclase: binding studies and requirements for activation.

    Levey, G S; Fletcher, M A; Klein, I

    1975-01-01

    Solubilization of myocardial adenylate cyclase abolished responsiveness to glucagon and catecholamines, two of the hormones which activate the membrane-bound enzyme. Adenylate cyclase freed of detergent by DEAE-cellulose chromatography continues to remain unresponsive to hormone stimulation. However, adding purified bovine brain phospholipids--phosphotidylserine and monophosphatidylinositol--restored responsiveness to glucagon and catecholamines, respectively. 125-i-glucagon binding appeared to be independent of phospholipid, since equal binding was observed in the presence or absence of detergent and in the presence or absence of phospholipids. Chromatography of the solubilized preparation on Sephadex G-100 WAS CHARACTERIZED BY 125-I-glucagon binding and fluoride-stimulatable adenylate cyclase activity appearing in the fractions consistent with the void volume, suggesting a molecular weight greater than 100,000 for the receptor-adenylate cyclase complex. Prior incubation of the binding peak with 125-I-glucagon and rechromatography of the bound glucagon on Sephadex G-100 shifted its elution to a later fraction consistent with a smaller-molecular-weight peak. The molecular weight of this material was 24,000 to 28,000, as determined by SDS polyacrylamide gel electrophoresis. The latter findings are consistent with a dissociable receptor site for glucagon on myocardial adenylate cyclase. PMID:165684

  4. 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.

    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.

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

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

  6. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    Ho, L.T.; Nie, Z.M.; Mende, T.J.; Richardson, S.; Chavan, A.; Kolaczkowska, E.; Watt, D.S.; Haley, B.E.; Ho, R.J. (Univ. of Miami School of Medicine, FL (USA))

    1989-01-01

    Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by (125I)PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that (a) no other AC-regulatory proteins are known to be of this size, (b) the catalytic unit of bovine brain enzyme is in the same range and (c) this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.

  7. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking [125I]PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by [125I]PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that [a] no other AC-regulatory proteins are known to be of this size, [b] the catalytic unit of bovine brain enzyme is in the same range and [c] this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase

  8. Effects of cadmium on canine renal cortical adenylate cyclase

    The present studies examine the effects of cadmium (Cd2+) on adenylate cyclase activity in basolateral renal cortical membranes from normal dogs. Cd2+, in the dose range of 1 to 200 μM caused a dose-dependent inhibition of adenylate cyclase activity due to competitive inhibition with respect to the allosteric activator Mg2+. In addition, increasing Cd2+ concentrations from 0 to 25 μM resulted in a purely competitive inhibition with respect to ATP. In the absence of other divalent cations Cd2+ was a potent stimulator of basal adenylate cyclase activity, far more potent than the physiological activator of the system Mg2+. It is concluded that Cd2+ behaves as a partial agonist in this system, due to its ability to form a new enzymatic substrate complex: Cd-ATP, which competes with the physiological substrate Mg-ATP at the catalytic site of the enzyme. In addition, Cd2+ in the absence of other divalent cation stimulates basal enzyme activity, presumably through interaction at an additional site, closely related to the allosteric metal regulatory site of this enzyme system

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

    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

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

    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.

  11. Adenylate cyclase regulates elongation of mammalian primary cilia

    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.

  12. Adenylate cyclase regulates elongation of mammalian primary cilia

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

    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, [125I]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 [125I]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

  14. Glucose Repression of Fbp1 Transcription in Schizosaccharomyces Pombe Is Partially Regulated by Adenylate Cyclase Activation by a G Protein α Subunit Encoded by Gpa2 (Git8)

    Nocero, M.; Isshiki, T.; Yamamoto, M.; Hoffman, C. S.

    1994-01-01

    In the fission yeast Schizosaccharomyces pombe, genetic studies have identified genes that are required for glucose repression of fbp1 transcription. The git2 gene, also known as cyr1, encodes adenylate cyclase. Adenylate cyclase converts ATP into the second messenger cAMP as part of many eukaryotic signal transduction pathways. The git1, git3, git5, git7, git8 and git10 genes act upstream of adenylate cyclase, presumably encoding an adenylate cyclase activation pathway. In mammalian cells, a...

  15. Cooperative phenomena in binding and activation of Bordetella pertussis adenylate cyclase by calmodulin.

    Bouhss, A; Krin, E; Munier, H; Gilles, A M; Danchin, A; Glaser, P; Bârzu, O

    1993-01-25

    The catalytic domain of Bordetella pertussis adenylate cyclase located within the first 400 amino acids of the protein can be cleaved by trypsin in two subdomains (T25 and T18) corresponding to ATP-(T25) and calmodulin (CaM)-(T18) binding sites. Reassociation of subdomains by CaM is a cooperative process, which is a unique case among CaM-activated enzymes. To understand better the molecular basis of this phenomenon, we used several approaches such as partial deletions of the adenylate cyclase gene, isolation of peptides of various size, and site-directed mutagenesis experiments. We found that a stretch of 72 amino acid residues overlapping the carboxyl terminus of T25 and the amino terminus of T18 accounts for 90% of the binding energy of adenylate cyclase-CaM complex. The hydrophobic "side" of the helical region situated around Trp242 plays a major role in the interaction of adenylate cyclase with CaM, whereas basic residues that alternate with acidic residues in bacterial enzyme play a much less important role. The amino-terminal half of the catalytic domain of adenylate cyclase contributes only 10% to the binding energy of CaM, whereas the last 130 amino acid residues are not at all involved in binding. However, these segments of adenylate cyclase might affect protein/protein interaction and catalysis by propagating conformational changes to the CaM-binding sequence which is located in the middle of the catalytic domain of bacterial enzyme. PMID:8420945

  16. Dependence of the hormonal stimulation of adenylate cyclase on the fraction of the plasma membrane accessible for lateral displacement of proteins of the adenylate cyclase complex

    Hormonal activation of the adenylate cyclase complex is associated with lateral displacement in the membrane of the proteins that constitute this complex. In this work an experimental investigation was made of the changes in the interaction of the proteins of the adenylate cyclase complex with the changing fraction of fluid lipids in the cell membrane. A decrease in the fraction of fluid lipids of rat reticulocyte membranes led to a decrease (all the way down to a total suppression) of the interaction of the β-adrenoreceptors with the regulatory N-proteins. The interaction of the N-proteins with the catalytic proteins was also suppressed. On the other hand, an increase in the fraction of fluid lipids led to more effective interaction. It was shown that in this case the functional intactness of the interacting proteins is unimpaired. An analysis of the results obtained, performed on the basis of the percolation theory, suggests the conclusion that the hormonal stimulation of adenylate cyclase depends on the fraction of fluid lipids in the membrane, and the proteins are displaced during interaction over distances comparable with the size of the membrane itself. It was also shown that characteristic activity of the β-agonist 1-isoproterenol varies from 1.0 to 0, depending on the fraction of fluid lipids in the membrane. The data obtained suggest that in the absence of guanylic nucleotides in the membrane in vitro there are no preexisting complexes with a high affinity for the agonist

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

    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 [125I]iodocyanopindolol. Binding sites had the characteristics of mixed β1- and β2-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 β1-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

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

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. (Veterans Administration Medical Center, Los Angeles, CA (USA) Univ. of California, Los Angeles (USA))

    1987-12-01

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

  19. Six git genes encode a glucose-induced adenylate cyclase activation pathway in the fission yeast Schizosaccharomyces pombe

    Susan M. Byrne; Hoffman, Charles S.

    1993-01-01

    An important eukaryotic signal transduction pathway involves the regulation of the effector enzyme adenylate cyclase, which produces the second messenger, cAMP. Previous genetic analyses demonstrated that glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene requires the function of adenylate cyclase, encoded by the git2 gene. As mutations in git2 and in six additional git genes are suppressed by exogenous cAMP, these ‘upstream’ git genes were proposed to act to produ...

  20. Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

    Glaser, P; Elmaoglou-Lazaridou, A; Krin, E.; Ladant, D.; Bârzu, O; Danchin, A

    1989-01-01

    In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture sup...

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

    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.

  2. Action of radioprotectors - venoms of Central Asian snakes and radiation on the adenylate cyclase system

    Action of venoms of Central Asian snakes (Maja oxiana and Vipera labertina turahica) as radioprotectors on 3'-5'-AMP content and activity of adenylate cyclase and phosphodiesterase in homogenates of liver and spleen of rats 1 and 24 hours after irradiation (800 R) has been studied. c-AMP content and adenylate cyclase activity have been shown to decrease drastically in the organs under study after the action of ionizing radiation. Preventive administration of venoms of cobra (150 μ g/kg) and (700 μ g/kg) one hour before irradiation restores the activity of the enzyme and c - AMP content of the spleen up to 53% and of the liver, to 30%. Phosphodiesterase activity increased markedly after irradiation being practically unaffected by the protector

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

    Koichi Tanda; Norihito Shintani; Akemichi Baba; Hitoshi Hashimoto; Tsuyoshi Miyakawa

    2012-01-01

    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 backcrosse...

  4. Reconstitution of the GTP-dependent adenylate cyclase from products of the yeast CYR1 and RAS2 genes in Escherichia coli.

    Uno, I.; Mitsuzawa, H.; Matsumoto, K.; Tanaka, K; Oshima, T.; Ishikawa, T

    1985-01-01

    Plasmids carrying the CYR1 gene of yeast Saccharomyces cerevisiae, which encodes adenylate cyclase, were introduced into the cya mutant strain of Escherichia coli. The transformants had a GTP-independent adenylate cyclase activity but did not produce cAMP. The E. coli transformant carrying the yeast RAS2 or RAS2val19 gene had no adenylate cyclase activity. Transformant cells carrying both CYR1 and RAS2 produced GTP-dependent adenylate cyclase and cAMP, and those carrying CYR1 and RAS2val19 pr...

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

    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.

  6. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    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

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

    The incorporation of [14C] 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.)

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

    Adrenalectomy caused a large increase in the number of β-adrenergic binding sites on liver plasma membranes as measured by 125I-iodocyanopindolol (22 and 102 fmol/mg protein for control and adrenalectomized (ADX) rats). Concomitantly an increase in the number of binding sites for 3H-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 α2-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

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

    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)

  10. BIOTIC STRESS IMPACT ON ACTIVITY OF VARIOUS FORMS OF ADENYLATE CYCLASE IN ORGANELLES OF POTATO PLANT CELLS

    Lomovatskaya L.A.

    2006-12-01

    Full Text Available Notwithstanding significant interest towards study of adenylate cyclase plant signal system, there is still no complete picture of functioning and regulation mechanisms of this signal system in plants under biotic stress. With this in view, our study was aimed at identification of various forms of adenylate cyclase (transmembrane and “soluble” in the nucleus and chloroplasts of potato cells and modulation of their activity under the impact of exopolysaсcharides ofpotato ring rot pathogen. The investigations conducted allowed to conclude that two forms of adenylate cyclase function in nuclei and chloroplasts of potato plants: transmembrane and “soluble”. Activity of these forms of the enzyme extracted from plant cells of the two potato varieties contrasted by resistance to potato ring rot pathogen Clavibacter michiganensis subsp. sepedonicus, changed in the reverse manner with the mediated impact of exopolysaсcharides secreted by virulent and mucinous strain of bacterial pathogen: in the plants of resistant сultivar it increased, in the plants of sensitive сultivar it was oppressed. It was concluded that activity of both forms of adenylate cyclase directly depended on the degree of resistance of a particular potato variety to given pathogen.

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

    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 beta2-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

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

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

    1986-03-01

    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 (/sup 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/sup -5/ M) suggesting predominate beta/sub 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-/sub 2/-type BAR coupled to adenylate cyclase in rat brown fat.

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

    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.

  14. Pituitary adenylate cyclase-activating polypeptide protects rat cerebellar granule neurons against ethanol-induced apoptotic cell death

    Vaudry, David; Rousselle, Cécile; Basille, Magali; Falluel-Morel, Anthony; Pamantung, Tommy F.; Fontaine, Marc; Fournier, Alain; Vaudry, Hubert; Gonzalez, Bruno J

    2002-01-01

    Alcohol exposure during development can cause brain malformations and neurobehavioral abnormalities. In view of the teratogenicity of ethanol, identification of molecules that could counteract the neurotoxic effects of alcohol deserves high priority. Here, we report that pituitary adenylate cyclase-activating polypeptide (PACAP) can prevent the deleterious effect of ethanol on neuronal precursors. Exposure of cultured cerebellar granule cells to ethanol inhibited neurite outgrowth and provoke...

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

    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

  16. Fine control of adenylate cyclase by the phosphoenolpyruvate:sugar phosphotransferase systems in Escherichia coli and Salmonella typhimurium.

    Feucht, B U; Saier, M H

    1980-01-01

    Inhibition of cellular adenylate cyclase activity by sugar substrates of the phosphoenolpyruvate-dependent phosphotransferase system was reliant on the activities of the protein components of this enzyme system and on a gene designated crrA. In bacterial strains containing very low enzyme I activity, inhibition could be elicited by nanomolar concentrations of sugar. An antagonistic effect between methyl alpha-glucoside and phosphoenolpyruvate was observed in permeabilized Escherichia coli cel...

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

    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 [3H]-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 [3H]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

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

    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.

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

    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-HT1C 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 [3H]serotonin, [3H]lysergic acid diethylamide or [3H]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

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

    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

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

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

    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.

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

    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

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

    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

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

    Hurley, Matthew M; Maunze, Brian; Block, Megan E; Frenkel, Mogen M; Reilly, Michael J; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    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. PMID:27597817

  6. The fission yeast git5 gene encodes a Gbeta subunit required for glucose-triggered adenylate cyclase activation.

    Landry, S; Pettit, M T; Apolinario, E; Hoffman, C. S.

    2000-01-01

    Fission yeast adenylate cyclase is activated by the gpa2 Galpha subunit of a heterotrimeric guanine-nucleotide binding protein (G protein). We show that the git5 gene, also required for this activation, encodes a Gbeta subunit. In contrast to another study, we show that git5 is not a negative regulator of the gpa1 Galpha involved in the pheromone response pathway. While 43% identical to mammalian Gbeta's, the git5 protein lacks the amino-terminal coiled-coil found in other Gbeta subunits, yet...

  7. Hypoxia and glucose independently regulate the beta-adrenergic receptor-adenylate cyclase system in cardiac myocytes.

    Rocha-Singh, K J; Honbo, N Y; Karliner, J S

    1991-01-01

    We explored the effects of two components of ischemia, hypoxia and glucose deprivation, on the beta-adrenergic receptor (beta AR)-adenylate cyclase system in a model of hypoxic injury in cultured neonatal rat ventricular myocytes. After 2 h of hypoxia in the presence of 5 mM glucose, cell surface beta AR density (3H-CGP-12177) decreased from 54.8 +/- 8.4 to 39 +/- 6.3 (SE) fmol/mg protein (n = 10, P less than 0.025), while cytosolic beta AR density (125I-iodocyanopindolol [ICYP]) increased by...

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

    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. PMID:12845765

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

    Several classes of psychoactive compounds have been investigated for their effects on histamine-sensitive adenylate cyclase in cell-free preparations from the guinea-pig cerebral cortex. Their inhibitory actions on this enzyme system have been compared with their abilities to displace [3H]pyrilamine and [3H]cimetidine from histamine H1- and H2-receptor sites, respectively. The results of these studies show that compounds which inhibited the histamine-sensitive cyclase were also displacers of either [3H]pyrilamine or [3H]cimetidine or both 3H-ligands from their binding sites. In spite of the lack of a correlation between binding and cyclase antagonism it was observed that compounds that displace both ligands showed greater inhibition of the cyclase than those that have affinities for sites labeled by one or the other ligand. It was concluded that antihistamines, the antipsychotics and the antidepressants share a common property through their antagonism of H1-receptors and that may be responsible for their sedative side effect. (Auth.)

  10. Bordetella protein toxins

    Mašín, Jiří; Šebo, Peter; Locht, C.

    New York : Elsevier, Academic Press, 2006, s. 291-309. ISBN 978-0-12-088445-2 R&D Projects: GA AV ČR IAA5020406; GA MŠk 1M0506 Institutional research plan: CEZ:AV0Z50200510 Keywords : pertussis toxin * adenylate cyclase toxin * dermonecrotic toxin Subject RIV: EE - Microbiology, Virology

  11. Peculiarities of functional state of myocardium adenylate cyclase system in euthyroid and hyperthyroid rats after staying in the region of radioactive contamination

    The purpose of the investigation was the analysis of effects of low dose irradiation in conditions of radioactive contamination on the activity of adenylate cyclase system of cardio myocytes of normal and hyperthyroid rats. 4-5 months age female rats staying for 1 month on the territory of water-meadow of Pripyat' river (exposed dose rate 1,0 mR/hour) received absorbed radiation dose 1,1 mSv. Hyperthyroid state was achieved by injection of 0,15 mg of thyroxine per kg of body weight with food. Decrease of stimulatory effects of catecholamines and fluorine ions on adenylate cyclase of animals from radio contaminated region was found. This indicates the inhibition of receptor- and G-protein-mediated enzyme activation. Hyperthyroidism was the reason of increasing of catecholamine-dependent and decreasing of fluorine ions-dependent stimulation of adenylate cyclase. Only the changes of adenylate cyclase activity regulated by G-protein in hyperthyroid rats from radio contaminated regions were observed

  12. The adenylate cyclase gene MaAC is required for virulence and multi-stress tolerance of Metarhizium acridum

    Liu Shuyang

    2012-08-01

    Full Text Available Abstract Background The efficacy of entomopathogenic fungi in pest control is mainly affected by various adverse environmental factors, such as heat shock and UV-B radiation, and by responses of the host insect, such as oxidative stress, osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC was cloned from the locust-specific entomopathogenic fungus, Metarhizium acridum, which is homologous to various fungal adenylate cyclase genes. RNA silencing was adapted to analyze the role of MaAC in virulence and tolerance to adverse environmental and host insect factors. Results Compared with the wild type, the vegetative growth of the RNAi mutant was decreased in PD (potato dextrose medium, Czapek-dox and PDA plates, respectively, demonstrating that MaAC affected vegetative growth. The cAMP levels were also reduced in PD liquid culture, and exogenous cAMP restored the growth of RNAi mutants. These findings suggested that MaAC is involved in cAMP synthesis. The knockdown of MaAC by RNAi led to a reduction in virulence after injection or topical inoculation. Furthermore, the RNAi mutant grew much slower than the wild type in the haemolymph of locust in vitro and in vivo, thus demonstrating that MaAC affects the virulence of M. acridum via fungal growth inside the host locust. A plate assay indicated that the tolerances of the MaAC RNAi mutant under oxidative stress, osmotic stress, heat shock and UV-B radiation was decreased compared with the wild type. Conclusion MaAC is required for virulence and tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects fungal virulence via vegetative growth inside the insect and tolerance against oxidative stress, osmotic stress and locust fever.

  13. Multistress resistance of Saccharomyces cerevisiae is generated by insertion of retrotransposon Ty into the 5' coding region of the adenylate cyclase gene

    Heat shock-resistant mutants, which were isolated by their ability to withstand lethal heat treatment, were characterized. Resistance was demonstrated to be a consequence of insertion of retrotransposon Ty into either the 5' coding or noncoding region, close to the putative initiation codon of the adenylate cyclase gene CYR1 (or CDC35). These heat shock-resistant mutants contained about threefold lower adenylate cyclase activity than wild-type strains. The mutants were also observed to be resistant to other stresses such as UV light and ethanol. These results demonstrate that multistress resistance, which may confer a survival advantage to yeast cells, can be generated by transposition of a Ty element into CYR1

  14. The influence of low-level radiation and gangliosides on adenylate cyclase activity in thymus and thyroid glands of chicks in ontogenesis

    Adenylate cyclase (AC) activity was studied in thymus and thyroid gland of intact chick embryos and those irradiated with a dose of 0.029 Gy prior to incubation, and newly hatched chocks in the presence of total ganglioside fractions extracted from the same organs. Gangliosides were shown to increase the enzyme activity of thymocytes and thyreocytes during the postnatal development. It is suggested that small radiation doses potentiate the stimulatory effect of ganglioside fractions on AC

  15. The Role of Vasoactive Intestinal Polypeptide and Pituitary Adenylate Cyclase-Activating Polypeptide in the Neural Pathways Controlling the Lower Urinary Tract

    Yoshiyama, Mitsuharu; de Groat, William C.

    2008-01-01

    Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are expressed in the neural pathways regulating the lower urinary tract. VIP-immunoreactivity (IR) is present in afferent and autonomic efferent neurons innervating the bladder and urethra, whereas PACAP-IR is present primarily in afferent neurons. Exogenously applied VIP relaxes bladder and urethral smooth muscle and excites parasympathetic neurons in bladder ganglia. PACAP relaxes bladder ...

  16. Pituitary Adenylate Cyclase-Activating Peptide in the Central Amygdala Causes Anorexia and Body Weight Loss via the Melanocortin and the TrkB Systems

    Iemolo, Attilio; Ferragud, Antonio; Cottone, Pietro; Sabino, Valentina

    2015-01-01

    Growing evidence suggests that the pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1 receptor system represents one of the main regulators of the behavioral, endocrine, and autonomic responses to stress. Although induction of anorexia is a well-documented effect of PACAP, the central sites underlying this phenomenon are poorly understood. The present studies addressed this question by examining the neuroanatomical, behavioral, and pharmacological mechanisms mediating the anorexi...

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

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

    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 cho...

  18. Neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) slows down Alzheimer's disease-like pathology in amyloid precursor protein-transgenic mice

    Rat, Dorothea; Schmitt, Ulrich; Tippmann, Frank; Dewachter, Ilse; Theunis, Clara; Wieczerzak, Ewa; Postina, Rolf; Van Leuven, Fred Van; Fahrenholz, Falk; Kojro, Elzbieta

    2011-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) has neuroprotective and neurotrophic properties and is a potent alpha-secretase activator. As PACAP peptides and their specific receptor PAC1 are localized in central nervous system areas affected by Alzheimer's disease (AD), this study aims to examine the role of the natural peptide PACAP as a valuable approach in AD therapy. We investigated the effect of PACAP in the brain of an AD transgenic mouse model. The long-term intranasal da...

  19. Impairment of mossy fiber long-term potentiation and associative learning in pituitary adenylate cyclase activating polypeptide type I receptor-deficient Mice

    Otto, Christiane; Kovalchuk, Yury; Wolfer, David Paul; Gass, Peter; Mart??n, Miguel; Zuschratter, Werner; Gr??ne, Hermann Josef; Kellendonk, Christoph; Tronche, Fran??ois; Maldonado, Rafael; Lipp, Hans-Peter; Konnerth, Arthur; Sch??tz, G??nter

    2001-01-01

    The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor (PAC1) is a G-protein-coupled receptor binding the strongly conserved neuropeptide PACAP with 1000-fold higher affinity than the related peptide vasoactive intestinal peptide. PAC1-mediated signaling has been implicated in neuronal differentiation and synaptic plasticity. To gain further insight into the biological significance of PAC1-mediated signaling in vivo, we generated two different mutant mouse strains, har...

  20. (/sup 3/H)forskolin- and (/sup 3/H)dihydroalprenolol-binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

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

    1988-03-01

    The characteristics of the cardiac adenylate cyclase system were studied in rats fed diets containing fish oil (menhaden oil) and other oils. Adenylate cyclase activity generally was higher in cardiac homogenates and membranes of rats fed diet containing 10% menhaden oil than in the other oils. The increase in enzyme activity, especially in forskolin-stimulated activity, was associated with an increase in the concentration of the (/sup 3/H) forskolin-binding sites in cardiac membranes of rats fed menhaden oil. The beta-adrenergic receptor concentration was not significantly altered although the affinity for (/sup 3/H)dihydroalprenolol-binding was lower in membranes of rats fed menhaden oil than those fed the other oils. omega-3 fatty acids from menhaden oil were incorporated into the cardiac membrane phospholipids. The results suggest that the observed increase in myocardial adenylate cyclase activity of rats fed menhaden oil may be due to an increase in the number of the catalytic subunits of the enzyme or due to a greater availability of the forskolin-binding sites.

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

    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.

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

    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.)

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

    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.

  4. Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

    Kong, Linghai; Albano, Rebecca; Madayag, Aric; Raddatz, Nicholas; Mantsch, John R; Choi, SuJean; Lobner, Doug; Baker, David A

    2016-05-01

    Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity. PMID:26851652

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

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

    1987-09-08

    Preincubation of turkey erythrocytes with isoproterenol is associated with (1) 50-60% attenuation of agonist-stimulated adenylate cyclase activity, (2) altered mobility of the ..beta..-adrenergic receptor on sodium dodecyl sulfate-polyacrylamide gels, and (3) increased phosphorylation of the ..beta..-adrenergic receptor. Using a low-cross-linked polyacrylamide gel, the ..beta..-adrenergic receptor protein from isoproterenol-desensitized cells, labeled with /sup 32/P or with the photoaffinity label /sup 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 ..beta..-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 /sup 32/P-labeled ..beta..-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 ..beta..-adrenergic receptors in turkey erythrocytes occurs by a two-step mechanism.

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

    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 32P or with the photoaffinity label 125I-(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 32P-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

  7. Urinary cyclic adenosine 3',5'-monophosphate response in McCune-Albright syndrome: clinical evidence for altered renal adenylate cyclase activity.

    Zung, A; Chalew, S A; Schwindinger, W F; Levine, M A; Phillip, M; Jara, A; Counts, D R; Kowarski, A A

    1995-12-01

    The recent finding of an activating mutation in the Gs alpha protein, the protein that couples receptors to stimulation of adenylate cyclase, from endocrine and nonendocrine tissues of patients with McCune-Albright syndrome (MAS) suggests that alterations in adenylate cyclase activity may account for the clinical abnormalities in these patients. Many patients with MAS have hypophosphatemia. This may result from the presence of the activating Gs alpha mutation in proximal renal tubules or the elaboration of a phosphaturic factor from fibrous dysplasia. We, therefore, sought to characterize renal cAMP generation and phosphate handling in MAS patients. Intravenous infusion of PTH is a classic clinical test used to evaluate hormonal responsiveness of renal proximal tubule adenylate cyclase and examine PTH-dependent phosphate clearance. We performed PTH infusion in 6 MAS patients, 10 normal subjects, and 6 patients with pseudohypoparathyroidism (PHP). The basal urinary cAMP (UcAMP) level in the MAS group [5.5 +/- 2.6 nmol/dL glomerular filtration (GF)] was elevated (P PHP (1.9 +/- 0.6 nmol/dL GF). However, PTH-stimulated peak UcAMP (15.0 +/- 7.0 nmol/dL GF) and the peak/basal UcAMP ratio (3.1 +/- 1.7) in MAS were significantly lower than the respective values in normal subjects (30.8 +/- 16.9 nmol/dL GF and 9.3 +/- 2.9; P PHP (respectively, 3.1 +/- 1.5 nmol/dL GF and 2.0 +/- 1.7). By contrast, the PTH-induced phosphaturic response in MAS patients was similar to that in the normal subjects. Our study provides clinical evidence that MAS patients have altered renal adenylate cyclase activity, manifested by an elevated basal UcAMP, but a blunted UcAMP response to PTH stimulation. These observations are presumably due to a mutation in the Gs alpha protein in the renal tubules. Despite the blunted UcAMP excretion, the phosphaturic response to PTH in MAS patients is intact. PMID:8530601

  8. Differential role of the carboxy-terminus of the A2B adenosine receptor in stimulation of adenylate cyclase, phospholipase Cβ, and interleukin-8

    Ryzhov, Sergey; Zaynagetdinov, Rinat; Goldstein, Anna E.; Matafonov, Anton; Biaggioni, Italo; Feoktistov, Igor

    2009-01-01

    In human mast cells and microvascular endothelial cells, the A2B adenosine receptor controls at least three independent signaling pathways, i.e., Gs-mediated stimulation of adenylate cyclase, Gq-mediated stimulation of phospholipase Cβ, and Gs/Gq-independent upregulation of IL-8. Functional analysis of cells transfected with full-length and truncated receptor constructs revealed that the A2B receptor C-terminus is important for coupling to Gs and Gq proteins. Removal of the entire cytoplasmic...

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

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

    2013-09-01

    The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr-Tyr cross-linking (o,o‧-ditysosine formation) and partial flavin cofactor reduction.

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

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

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

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

  12. The mechanisms of disturbances of hormonal regulation of adenylate cyclase in in-vitro irradiated liver plasma membranes of rats in early ontogenesis

    Using 20-days fetus of the Wistar rat possible causes of the previously established inhibiting action of gamma-irradiation (1-1000 Gy) of isolated plasmatic membranes of rat fetus liver on basal and hormonstimulating activity of adenylate cyclase (AC) have been elucidated. Irradiation of isolated membranes has been performed in the 300 and 500 Gy. AC β-adrenoreceptors state through which isoproterenol effect is realized by the criterion of binding the labelled antagonist of 3-dihydroal prenolol is evaluated. It is concluded that radiation suppression of AC stimulation by isoproterenol can be bound with the decrease of a number of adrenoreceptors, damaging catalytic and guanylnucleotide binding component of the AC system. An idea is expressed on radiation changes of membranes lipid phase state in which complex interactions of the AC system components occur

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

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

    2009-01-01

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

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

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

    2010-01-01

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

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

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

    2001-01-01

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

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

    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.

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

    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

  18. Effects of Yulangsan polysaccharide on monoamine neurotransmitters, adenylate cyclase activity and brain-derived neurotrophic factor expression in a mouse model of depression induced by unpredictable chronic mild stress

    Shuang Liang; Renbin Huang; Xing Lin; Jianchun Huang; Zhongshi Huang; Huagang Liu

    2012-01-01

    The present study established a mouse model of depression induced by unpredictable chronic mild stress. The model mice were treated with Yulangsan polysaccharide (YLSPS; 150, 300 and 600 mg/kg) for 21 days, and compared with fluoxetine-treated and normal control groups. Enzyme-linked immunosorbent assay, radioimmunity and immunohistochemical staining showed that following treatment with YLSPS (300 and 600 mg/kg), monoamine neurotransmitter levels, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression were significantly elevated, and depression-like behaviors were improved. Open-field and novelty-suppressed feeding tests showed that mouse activity levels were increased and feeding latency was shortened following treatment. Our results indicate that YLSPS inhibits depression by upregulating monoamine neurotransmitters, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression.

  19. Effect of peptides corresponding to extracellular domains of serotonin 1B/1D receptors and melanocortin 3 and 4 receptors on hormonal regulation of adenylate cyclase in rat brain.

    Shpakova, E A; Derkach, K V; Shpakov, A O

    2014-03-01

    The ligand-recognizing part of G protein-coupled receptors consists of their extracellular loops and N-terminal domain. Identification of these sites is essential for receptor mapping and for the development and testing of new hormone system regulators. The peptides corresponding by their structure to extracellular loop 2 of serotonin 1B/1D receptor (peptide 1), extracellular loop 3 of melanocortin 3 receptor (peptide 2), and N-terminal domain of melanocortin 4 (peptide 3) were synthesized by the solid-phase method. In synaptosomal membranes isolated from rat brain, peptide 1 (10(-5)-10(-4) M) attenuated the effects of 5-nonyloxytryptamine (selective agonist of serotonin 1B/1D receptor) and to a lesser extent serotonin and 5-methoxy-N,N-dimethyltryptamine acting on all the subtypes of serotonin receptor 1. Peptide 2 (10(-5)-10(-4) M) significantly reduced the adenylate cyclase-stimulating effect of γ-melanocyte-stimulating hormone (agonist of melanocortin receptor 3), but had no effect on the adenylate cyclase effect of THIQ (agonist melanocortin receptor 4). Peptide 3 reduced the adenylate cyclase-stimulating effects of THIQ and α-melanocyte-stimulating hormone (non-selective agonist of melanocortin receptors 3 and 4), but did not modulate the effect of γ-melanocyte-stimulating hormone. The effect of peptide 3 was weaker: it was observed at peptide 3 concentration of 10(-4) M. Peptides 1-3 did no change the adenylate cyclase-modulating effects of hormones acting through non-homologous receptors. Thus, the synthesized peptides specifically inhibited the regulatory effects of hormones acting through homologous receptors. This suggests that the corresponding extracellular domains are involved in ligand recognition and binding and determine functional activity of the receptor. PMID:24770752

  20. Effects of Yulangsan polysaccharide on monoamine neurotransmitters, adenylate cyclase activity and brain-derived neurotrophic factor expression in a mouse model of depression induced by unpredictable chronic mild stress☆

    Liang, Shuang; Huang, Renbin; Lin, Xing; Huang, Jianchun; Huang, Zhongshi; Liu, Huagang

    2012-01-01

    The present study established a mouse model of depression induced by unpredictable chronic mild stress. The model mice were treated with Yulangsan polysaccharide (YLSPS; 150, 300 and 600 mg/kg) for 21 days, and compared with fluoxetine-treated and normal control groups. Enzyme-linked immunosorbent assay, radioimmunity and immunohistochemical staining showed that following treatment with YLSPS (300 and 600 mg/kg), monoamine neurotransmitter levels, prefrontal cortex adenylate cyclase activity ...

  1. Pituitary Adenylate Cyclase Activating Peptide (1-38 and its analog (Acetyl-[Ala15, Ala20] PACAP 38-polyamide reverse methacholine airway hyperresponsiveness in rats

    Mounira Tlili

    2015-09-01

    Full Text Available The aim of this study was to investigate both functionally and structurally bronchodilator effects of Pituitary adenylate cyclase activating peptide (PACAP38 and acetyl-[Ala15, Ala20] PACAP38-polyamide, a potent PACAP38 analog, in rats challenged by methacholine (MeCh. Male Wistar rats were divided randomly into five groups. Groups 1 and 2 inhaled respectively aerosols of saline or increasing doses of MeCh (0.5, 1, 2.12, 4.25, 8.5, 17, 34 and 68mg/L. The other groups received terbutaline (Terb (250 µg/rat (10-6 M, PACAP38 (50 µg/rat (0.1 mM or PACAP38 analog (50 µg/rat associated to MeCh from the dose of 4.25 mg/L. Total lung resistances (RL were recorded before and 2 min after MeCh administration by pneumomultitest equipment. MeCh administration induced a significant and a dose-dependent increase (p<0.05 of RL compared to control rats. Terb, PACAP38 and PACAP38 analog reversed significantly the MeCh-induced bronchial constriction, smooth muscle (SM layer thickness and bronchial lumen mucus abundance. PACAP38 analog prevents effectively bronchial smooth muscle layer thickness, mucus hypersecretion and lumen decrease. Therefore, it may constitute a potent therapeutic bronchodilator.

  2. A direct pyrophosphatase-coupled assay provides new insights into the activation of the secreted adenylate cyclase from Bordetella pertussis by calmodulin.

    Lawrence, Anthony J; Coote, John G; Kazi, Yasmin F; Lawrence, Paul D; MacDonald-Fyall, Julia; Orr, Barbara M; Parton, Roger; Riehle, Mathis; Sinclair, James; Young, John; Price, Nicholas C

    2002-06-21

    Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC ) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained. Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase. The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k(on) by about 30-fold. The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium. The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated. PMID:11934879

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

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

    2016-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP, encoded by adcyap1) plays an important role in ectodermal development. However, the involvement of PACAP in the development of other germ layers is still unclear. This study assessed the expression of a PACAP-specific receptor (PAC1) gene and protein in mouse bone marrow (BM). Cells strongly expressing PAC1(+) were large in size, had oval nuclei, and merged with CD34(+) cells, suggesting that the former were hematopoietic progenitor cells (HPCs). Compared with wild-type mice, adcyap1(-/-) mice exhibited lower multiple potential progenitor cell populations and cell frequency in the S-phase of the cell cycle. Exogenous PACAP38 significantly increased the numbers of colony forming unit-granulocyte/macrophage progenitor cells (CFU-GM) with two peaks in semi-solid culture. PACAP also increased the expression of cyclinD1 and Ki67 mRNAs. These increases were completely and partially inhibited by the PACAP receptor antagonists, PACAP6-38 and VIP6-28, respectively. Little or no adcyap1 was expressed in BM and the number of CFU-GM colonies was similar in adcyap1(-/-) and wild-type mice. However, PACAP mRNA and protein were expressed in paravertebral sympathetic ganglia, which innervate tibial BM, and in the sympathetic fibers of BM cavity. These results suggested that sympathetic nerve innervation may be responsible for PACAP-regulated hematopoiesis in BM, mainly via PAC1. PMID:26925806

  4. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Regulates the Hypothalamo-Pituitary-Thyroid (HPT) Axis via Type 2 Deiodinase in Male Mice.

    Egri, P; Fekete, C; Dénes, Á; Reglődi, D; Hashimoto, H; Fülöp, B D; Gereben, Balázs

    2016-06-01

    The hypothalamic activation of thyroid hormones by type 2 deiodinase (D2), catalyzing the conversion of thyroxine to T3, is critical for the proper function of the hypothalamo-pituitary-thyroid (HPT) axis. Regulation of D2 expression in tanycytes alters the activity of the HPT axis. However, signals that regulate D2 expression in tanycytes are poorly understood. The pituitary adenylate cyclase-activating polypeptide (PACAP) increases intracellular cAMP level, a second messenger known to stimulate the DIO2 gene; however, its importance in tanycytes is not completely characterized. Therefore, we tested whether this ubiquitously expressed neuropeptide regulates the HPT axis through stimulation of D2 in tanycytes. PACAP increased the activity of human DIO2 promoter in luciferase reporter assay that was abolished by mutation of cAMP-response element. Furthermore, PAC1R receptor immunoreactivity was identified in hypothalamic tanycytes, suggesting that these D2-expressing cells could be regulated by PACAP. Intracerebroventricular PACAP administration resulted in increased D2 activity in the mediobasal hypothalamus, suppressed Trh expression in the hypothalamic paraventricular nucleus, and decreased Tshb expression in the pituitary demonstrating that PACAP affects the D2-mediated control of the HPT axis. To understand the role of endogenous PACAP in the regulation of HPT axis, the effect of decreased PACAP expression was studied in heterozygous Adcyap1 (PACAP) knockout mice. These animals were hypothyroid that may be the consequence of altered hypothalamic T3 degradation during set-point formation of the HPT axis. In conclusion, PACAP is an endogenous regulator of the HPT axis by affecting T3-mediated negative feedback via cAMP-induced D2 expression of tanycytes. PMID:27046436

  5. Characteristics of muscarinic receptors that selectively couple to inhibition of adenylate cyclase or stimulation of phospholipase C on NG108-15 and 1321N1 cells

    The purpose of this dissertation was to establish whether different muscarinic receptor proteins selectively couple to different second messenger response system. Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma x glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with (3H)Propylbenzilylcholine mustard ((3H)PBCM) and the mobilities of the (3H)PBCM-labelled species of both cells were compared by SDS-PAGE. 1321N1 and NG108-15 cells each primarily expressed a single (3H)PBCM-labelled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. (3H)PBCM labelling was completely inhibited by 1 μM atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the (3H)PBCM-labelled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Another approach for determining differences in the muscarinic receptors of 2 cells lines was to study agonist-induced alteration of muscarinic receptor number. Exposure of both cell types to agonists resulted in rapid loss of muscarinic receptors from cell surface without change of total cellular muscarinic receptors followed by subsequently loss of receptors from cells. Muscarinic receptors on both cell lines were regulated by agonist with similar properties

  6. Pituitary Adenylate Cyclase-Activating Peptide in the Central Amygdala Causes Anorexia and Body Weight Loss via the Melanocortin and the TrkB Systems.

    Iemolo, Attilio; Ferragud, Antonio; Cottone, Pietro; Sabino, Valentina

    2015-07-01

    Growing evidence suggests that the pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1 receptor system represents one of the main regulators of the behavioral, endocrine, and autonomic responses to stress. Although induction of anorexia is a well-documented effect of PACAP, the central sites underlying this phenomenon are poorly understood. The present studies addressed this question by examining the neuroanatomical, behavioral, and pharmacological mechanisms mediating the anorexia produced by PACAP in the central nucleus of the amygdala (CeA), a limbic structure implicated in the emotional components of ingestive behavior. Male rats were microinfused with PACAP (0-1 μg per rat) into the CeA and home-cage food intake, body weight change, microstructural analysis of food intake, and locomotor activity were assessed. Intra-CeA (but not intra-basolateral amygdala) PACAP dose-dependently induced anorexia and body weight loss without affecting locomotor activity. PACAP-treated rats ate smaller meals of normal duration, revealing that PACAP slowed feeding within meals by decreasing the regularity and maintenance of feeding from pellet-to-pellet; postprandial satiety was unaffected. Intra-CeA PACAP-induced anorexia was blocked by coinfusion of either the melanocortin receptor 3/4 antagonist SHU 9119 or the tyrosine kinase B (TrKB) inhibitor k-252a, but not the CRF receptor antagonist D-Phe-CRF(12-41). These results indicate that the CeA is one of the brain areas through which the PACAP system promotes anorexia and that PACAP preferentially lessens the maintenance of feeding in rats, effects opposite to those of palatable food. We also demonstrate that PACAP in the CeA exerts its anorectic effects via local melanocortin and the TrKB systems, and independently from CRF. PMID:25649277

  7. Structural and functional identification of the pituitary adenylate cyclase-activating polypeptide receptor VPAC2 from the frog Rana tigrina rugulosa.

    Hoo, R L; Alexandre, D; Chan, S M; Anouar, Y; Pang, R T; Vaudry, H; Chow, B K

    2001-10-01

    Recently, a frog pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) receptor (fPVR) has been characterized, and interestingly, this receptor exhibits characteristics of both mammalian PACAP type II receptors VPAC(1)R and VPAC(2)R. In order to investigate the receptors responsible for mediating the actions of VIP and PACAP in amphibians, in this report, a frog VPAC(2) receptor (fVPAC(2)R) cDNA was isolated. fVPAC(2)R shares 47.7, 46.9 and 62.5% amino acid sequence identity with fPVR, human VPAC(1)R and human VPAC(2)R respectively. Functionally, fVPAC(2)R, when expressed in CHO cells, was responsive to both frog peptides including VIP, PACAP38 and PACAP27 where the EC(50) values of these peptides in intracellular cAMP production were 0.15, 0.18 and 0.16 microM respectively. The pharmacological profiles of human peptides (VIP, PACAP38 and peptide histidine methionine) to stimulate frog and human VPAC(2)Rs were compared, and it was found that these peptides could only activate the frog receptor at micromolar concentrations. fVPAC(2)R was found to be widely distributed in various peripheral tissues as well as several regions of the brain. The presence of the receptor transcripts suggests the functional roles of the receptor in mediating the actions of PACAP and/or VIP in these tissues. As VIP and particularly PACAP27 are highly conserved peptides in vertebrate evolution, comparative studies of these peptides and their receptors in non-mammalian vertebrates should provide clues to better understand the physiology of these important peptides in human and other vertebrates. PMID:11564605

  8. Saturated high-fat diet-induced obesity increases adenylate cyclase of myocardial β-adrenergic system and does not compromise cardiac function.

    Vileigas, Danielle F; de Deus, Adriana F; da Silva, Danielle C T; de Tomasi, Loreta C; de Campos, Dijon H S; Adorni, Caroline S; de Oliveira, Scarlet M; Sant'Ana, Paula G; Okoshi, Katashi; Padovani, Carlos R; Cicogna, Antonio C

    2016-09-01

    Obesity is a worldwide pandemic associated with high incidence of cardiovascular disease. The mechanisms by which the obesity leads cardiac dysfunction are not fully elucidated and few studies have evaluated the relationship between obesity and proteins involved in myocardial β-adrenergic (βA) system. The purpose of this study was to evaluate the cardiac function and βA pathway components in myocardium of obese rats. Male Wistar rats were distributed into two groups: control (n = 17; standard diet) and obese (n = 17; saturated high-fat diet) fed for 33 weeks. Nutritional profile and comorbidities were assessed. Cardiac structure and function was evaluated by macroscopic postmortem, echocardiographic and isolated papillary muscle analyzes. Myocardial protein expression of β1- and β2-adrenergic receptors, Gαs protein, adenylate cyclase (AC) and protein kinase A (PKA) was performed by Western blot. Cardiac cyclic adenosine monophosphate (cAMP) levels and PKA activity were assessed by ELISA Obese rats showed increased adiposity index (P < 0.001) and several comorbidities as hypertension, glucose intolerance, insulin resistance, and dyslipidemia compared with control rats. Echocardiographic assessment revealed increased left atrium diameter (C: 4.98 ± 0.38 vs. Ob: 5.47 ± 0.53, P = 0.024) and posterior wall shortening velocity (C: 37.1 ± 3.6 vs. Ob: 41.8 ± 3.8, P = 0.007) in obese group. Papillary muscle evaluation indicated that baseline data and myocardial responsiveness to isoproterenol stimulation were similar between the groups. Protein expression of myocardial AC was higher in obese group than in the control (C: 1.00 ± 0.21 vs. Ob: 1.25 ± 0.10, P = 0.025), whereas the other components were unchanged. These results suggest that saturated high-fat diet-induced obesity was not effective in triggering cardiac dysfunction and impair the beta-adrenergic signaling. PMID:27582064

  9. A new recombinant pituitary adenylate cyclase-activating peptide-derived peptide efficiently promotes glucose uptake and glucose-dependent insulin secretion

    Yi Ma; Tianjie Luo; Wenna Xu; Zulu Ye; An Hong

    2012-01-01

    The recombinant peptide,DBAYL,a promising therapeutic peptide for type 2 diabetes,is a new,potent,and highly selective agonist for VPAC2 generated through sitedirected mutagenesis based on sequence alignments of pituitary adenylate cyclase-activating peptide (PACAP),vasoactive intestinal peptide (VIP),and related analogs.The recombinant DBAYL was used to evaluate its effect and mechanism in blood glucose metabolism and utilization.As much as 28.9 mg recombinant DBAYL peptide with purity over 98% can be obtained from 1 I of Luria-Bertani medium culture by the method established in this study and the prepared DBAYL with four mutations (N10Q,V18L,N29Q,and M added to the N-terminal)were much more stable than BAY55-9837.The half-life of recombinant DBAYL was about 25 folds compared with that of BAY55-9837 in vitro.The bioactivity assay of DBAYL showed that it displaced [125I]PACAP38 and [125I]VIP from VPAC2 with a half-maximal inhibitory concentration of 48.4 ± 6.9 and 47.1 ± 4.9 nM,respectively,which were significantly lower than that of BAY55-9837,one established VPAC2 agonists.DBAYL enhances the cAMP accumulation in CHO cells expressing human VPAC2 with a half-maximal stimulatory concentration (EC5o) of 0.68 nM,whereas the receptor potency of DBAYL at human VPAC1 (ECso of 737 nM) was only 1/1083of that at human VPAC2,and DBAYL had no activity toward human PAC1 receptor.Western blot analysis of the key proteins of insulin receptor signaling pathway:insulin receptor substrate 1 (IRS-1) and glucose transporter 4(GLUT4) indicated that the DBAYL could significantly induce the insulin-stimulated IRS-1 and GLUT4 expression more efficiently than BAY55-9837 and VIP in adipocytes.Compared with BAY55-9837 and PACAP38,the recombinant peptide DBAYL can more efficiently promote insulin release and decrease plasma glucose level in Institute of Cancer Research (ICR) mice.These results suggested that DBAYL could efficiently improve glucose uptake and glucose-dependent insulin

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

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

    2006-01-01

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

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

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

    2001-01-01

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

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

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

    2004-01-01

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

  13. Changes in brain mRNA levels of gonadotropin-releasing hormone, pituitary adenylate cyclase activating polypeptide, and somatostatin during ovulatory luteinizing hormone and growth hormone surges in goldfish.

    Canosa, Luis Fabián; Stacey, Norm; Peter, Richard Ector

    2008-12-01

    In goldfish, circulating LH and growth hormone (GH) levels surge at the time of ovulation. In the present study, changes in gene expression of salmon gonadotropin-releasing hormone (sGnRH), chicken GnRH-II (cGnRH-II), somatostatin (SS) and pituitary adenylate cyclase activating polypeptide (PACAP) were analyzed during temperature- and spawning substrate-induced ovulation in goldfish. The results demonstrated that increases in PACAP gene expression during ovulation are best correlated with the GH secretion profile. These results suggest that PACAP, instead of GnRH, is involved in the control of GH secretion during ovulation. Increases of two of the SS transcripts during ovulation are interpreted as the activation of a negative feedback mechanism triggered by high GH levels. The results showed a differential regulation of sGnRH and cGnRH-II gene expression during ovulation, suggesting that sGnRH controls LH secretion, whereas cGnRH-II correlates best with spawning behavior. This conclusion is further supported by the finding that nonovulated fish induced to perform spawning behavior by prostaglandin F2alpha treatment increased cGnRH-II expression in both forebrain and midbrain, but decreased sGnRH expression in the forebrain. PMID:18815210

  14. Characterizations of a synthetic pituitary adenylate cyclase-activating polypeptide analog displaying potent neuroprotective activity and reduced in vivo cardiovascular side effects in a Parkinson's disease model.

    Lamine, Asma; Létourneau, Myriam; Doan, Ngoc Duc; Maucotel, Julie; Couvineau, Alain; Vaudry, Hubert; Chatenet, David; Vaudry, David; Fournier, Alain

    2016-09-01

    Parkinson's disease (PD) is characterized by a steady loss of dopamine neurons through apoptotic, inflammatory and oxidative stress processes. In that line of view, the pituitary adenylate cyclase-activating polypeptide (PACAP), with its ability to cross the blood-brain barrier and its anti-apoptotic, anti-inflammatory and anti-oxidative properties, has proven to offer potent neuroprotection in various PD models. Nonetheless, its peripheral actions, paired with low metabolic stability, hampered its clinical use. We have developed Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) as an improved PACAP-derived neuroprotective compound. In vitro, this analog stimulated cAMP production, maintained mitochondrial potential and protected SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium (MPP(+)) toxicity, as potently as PACAP. Furthermore, contrasting with PACAP, it is stable in human plasma and against dipeptidyl peptidase IV activity. When injected intravenously to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, PACAP and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) restored tyrosine hydoxylase expression into the substantia nigra and modulated the inflammatory response. Albeit falls of mean arterial pressure (MAP) were observed with both PACAP- and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27)-treated mice, the intensity of the decrease as well as its duration were significantly less marked after iv injections of the analog than after those of the native polypeptide. Moreover, no significant changes in heart rate were measured with the animals for both compounds. Thus, Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) appears as a promising lead molecule for the development of PACAP-derived drugs potentially useful for the treatment of PD or other neurodegenerative diseases. PMID:26006268

  15. Contributions of Histamine, Prostanoids, and Neurokinins to Edema Elicited by Edema Toxin from Bacillus anthracis▿

    Tessier, Jeffrey; Green, Candace; Padgett, Diana; Zhao, Wei; Schwartz, Lawrence; Hughes, Molly; Hewlett, Erik

    2007-01-01

    Bacillus anthracis edema toxin (ET), composed of protective antigen and an adenylate cyclase edema factor (EF), elicits edema in host tissues, but the target cells and events leading from EF-mediated cyclic-AMP production to edema are unknown. We evaluated the direct effect of ET on several cell types in vitro and tested the possibility that mediators of vascular leakage, such as histamine, contribute to edema in rabbits given intradermal ET. ET increased the transendothelial electrical resis...

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

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

    2014-01-01

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

  17. Fatty acylation of recombinant adenylate cyclase toxin (ACT): identification of lysine 983 with C16:0 and cis-C16:1 _9

    Yi, E. C.; Havlíček, Vladimír; Halada, Petr; Higgins, L.; Basar, T.; Kim, K.; Hewlett, E.; Šebo, Peter; Hackett, M.

    Dallas : ASMS, 1999. s. -. [ASMS Conference on Mass Spectrometry and Allied Topics /47./. 13.06.1999-17.06.1999, Dallas] Institutional research plan: CEZ:A53/98:Z5-020-9ii Subject RIV: EE - Microbiology, Virology

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

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

    2015-01-01

    Roč. 194, č. 10 (2015), s. 4901-4913. ISSN 0022-1767 R&D Projects: GA ČR GAP302/12/0460; GA ČR GA13-14547S Institutional support: RVO:61388971 Keywords : CYCLIC-AMP * MURINE MACROPHAGES * IFN-GAMMA Subject RIV: EE - Microbiology, Virology Impact factor: 4.922, year: 2014

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

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

    2010-01-01

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

  20. REGULATION OF POSTNATAL B-ADRENERGIC RECEPTOR/ADENYLATE CYCLASE DEVELOPMENT BY PRENATAL AGONIST STIMULATION AND STEROIDS: ALTERATIONS IN RAT KIDNEY AND LUNG AFTER EXPOSURE TO TERBUTALINE OR DEXAMETHASONE

    Glucocorticoids and adrenergic stimulation are both thought to control the development of adrenergic receptors/responses. n the current study, rats were exposed to dexamethasone or terbutaline during late gestation and the development of B-binding capabilities and adenylate cycla...

  1. Toxins

    Toxins are substances created by plants and animals that are poisonous to humans. Toxins also include some medicines that are helpful in small doses, but poisonous in large amounts. Most toxins that ...

  2. Synthetic peptides with antigenic specificity for bacterial toxins.

    Sela, M; Arnon, R; Jacob, C O

    1986-01-01

    The attachment of a diphtheria toxin-specific synthetic antigenic determinant and a synthetic adjuvant to a synthetic polymeric carrier led to production of a totally synthetic macromolecule which provoked protective antibodies against diphtheria when administered in aqueous solution. When peptides related to the B subunit of cholera toxin were synthesized and attached to tetanus toxoid, antibodies produced against the conjugate reacted in some but not all cases with intact cholera toxin and (especially with peptide CTP 3, residues 50-64) neutralized toxin reactivity, as tested by permeability in rabbit skin, fluid accumulation in ligated small intestinal loops and adenylate cyclase activation. Polymerization of the peptide without any external carrier, or conjugation with the dipalmityl lysine group, had as good an effect in enhancing the immune response as its attachment to tetanus toxoid. Prior exposure to the carrier suppressed the immune response to the epitope attached to it, whereas prior exposure to the synthetic peptide had a good priming effect when the intact toxin was given; when two different peptides were attached to the same carrier, both were expressed. Antisera against peptide CTP 3 were highly cross-reactive with the heat-labile toxin of Escherichia coli and neutralized it to the same extent as cholera toxin, which is not surprising in view of the great homology between the two proteins. A synthetic oligonucleotide coding for CTP 3 has been used to express the peptide in a form suitable for immunization. It led to a priming effect against the intact cholera toxin. PMID:2426052

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

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

  4. Acylation of conserved lysine 983 is sufficient for activity of Bordetella adenylate cyclase toxin: substitutions of alanine 140 in the acyltransferase CyaC modulate the selection of toxin acylation sites

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

    2000-01-01

    Roč. 94, č. 8 (2000), s. 568. ISSN 0009-2770. [Biochemický sjezd /17./. 07.09.2000-10.09.2000, Prague] R&D Projects: GA ČR GA310/98/0432; GA AV ČR IAA5020907; GA MŠk VS96149; GA MŠk ME 167 Subject RIV: CE - Biochemistry

  5. The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

    Marletta Michael A

    2008-10-01

    Full Text Available Abstract Background Soluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases. Results We have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 Å resolution, and show that it is a dimeric molecule. Conclusion Comparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.

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

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

  7. Effect of Baixiangdan Capsule on Expression of GABABR and Adenylate Cyclase of Frontal Cortex in Rats with Anxiety Emotion%白香丹胶囊对焦虑情绪模型大鼠额区皮层γ-氨基丁酸B受体和腺苷酸环化酶表达的影响

    蔡洪信; 许莉莉; 殷慧敏; 张惠云

    2012-01-01

    目的:检测焦虑情绪模型大鼠额区皮层γ-氨基丁酸(GABA)B受体两个亚基GABABR1,GABABR2和腺苷酸环化酶(AC)表达的变化,初步探讨白香丹胶囊对焦虑情绪的干预机制.方法:白香丹胶囊由芍药苷、香附挥发油和丹皮酚配伍组成.大鼠随机分为正常组、模型组、白香丹胶囊组(200 mg·kg-1)、巴氯芬组(8 mg·kg-1),采用“孤养加异种大鼠入侵”方法,大鼠孤养2周,然后异种大鼠居住入侵刺激2周,制备焦虑情绪大鼠模型.从居住入侵第2周开始,药物组按相应剂量ig给药7d,每日1次.通过糖水偏好实验、旷场实验和高架十字迷宫实验进行模型评价,用免疫荧光技术检测各组大鼠额区皮层GABABR1,CABABR2和AC的表达变化.结果:与正常组比较,模型组糖水偏好系数降低,旷场实验得分增高(P<0.05),高架十字迷宫实验进入开放臂次数百分数(OE%)和开放臂停留时间的百分数(OT%)降低(P<0.05),额区皮层GABABR1,GABABR2,AC表达水平降低(P<0.05).与模型组比较,白香丹胶囊组和巴氯芬组旷场实验得分降低(P<0.05),OE%和OT%值升高(P<0.05),额区皮层GABABR1,GABABR2,AC表达水平升高(P<0.05).结论:大鼠额区皮层GABABR1,GABABR2,AC表达下凋可能与焦虑情绪的产生有关,白香丹胶囊抗焦虑作用的中枢机制可能与其恢复额区皮层的GABABR的表达和功能有关.%Objective: To detect the expression of γ-aminobutyric acid B receptor (GABABR) and adenylate cyclase ( AC) of forbral cortex in rats with anxiety emotion and to explore the antianxiety mechanism of baixiangdan capsule. Method; Paeoniflorin, cyperolone and paeonolum were main ingredients of baixiangdan capsule. Wistar rats were divided into normal group, model group, baixiangdan capsule group (200 mg·kg-1 ) and baclofen group (8 mg·kg-1 ). The rats were administrated with relevant drugs intragastrically once a day for 7 days. The anxiety emotion rat models were

  8. Bacterial terpene cyclases.

    Dickschat, Jeroen S

    2016-01-01

    Covering: up to 2015. This review summarises the accumulated knowledge about characterised bacterial terpene cyclases. The structures of identified products and of crystallised enzymes are included, and the obtained insights into enzyme mechanisms are discussed. After a summary of mono-, sesqui- and diterpene cyclases the special cases of the geosmin and 2-methylisoborneol synthases that are both particularly widespread in bacteria will be presented. A total number of 63 enzymes that have been characterised so far is presented, with 132 cited references. PMID:26563452

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

    Nguyen Chinh

    2012-01-01

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

  10. A Continuous Kinetic Assay for Adenylation Enzyme Activity and Inhibition

    Daniel J. Wilson; Aldrich, Courtney C.

    2010-01-01

    Adenylation/adenylate-forming enzymes catalyze the activation of a carboxylic acid at the expense of ATP to form an acyl-adenylate intermediate and pyrophosphate (PPi). In a second half-reaction, adenylation enzymes catalyze the transfer of the acyl moiety of the acyl-adenylate onto an acceptor molecule, which can be either a protein or a small molecule. We describe the design, development, and validation of a coupled continuous spectrophotometric assay for adenylation enzymes that employs hy...

  11. Botulinum toxin type A induces changes in the chemical coding of substance P-immunoreactive dorsal root ganglia sensory neurons supplying the porcine urinary bladder.

    Bossowska, Agnieszka; Lepiarczyk, Ewa; Mazur, Urszula; Janikiewicz, Paweł; Markiewicz, Włodzimierz

    2015-11-01

    Botulinum toxin (BTX) is a potent neurotoxin which blocks acetylcholine release from nerve terminals, and therefore leads to cessation of somatic motor and/or parasympathetic transmission. Recently it has been found that BTX also interferes with sensory transmission, thus, the present study was aimed at investigating the neurochemical characterization of substance P-immunoreactive (SP-IR) bladder-projecting sensory neurons (BPSN) after the toxin treatment. Investigated neurons were visualized with retrograde tracing method and their chemical profile was disclosed with double-labelling immunohistochemistry using antibodies against SP, calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase activating polypeptide (PACAP), neuronal nitric oxide synthase (nNOS), galanin (GAL), calbindin (CB), and somatostatin (SOM). In the control group (n = 6), 45% of the total population of BPSN were SP-IR. Nearly half of these neurons co-expressed PACAP or CGRP (45% and 35%, respectively), while co-localization of SP with GAL, nNOS, SOM or CB was found less frequently (3.7%, 1.8%, 1.2%, and 0.7%, respectively). In BTX-treated pigs (n = 6), toxin-injections caused a decrease in the number of SP-IR cells containing CGRP, SOM or CB (16.2%, 0.5%, and 0%, respectively) and a distinct increase in these nerve cells immunopositive to GAL (27.2%). The present study demonstrates that BTX significantly modifies the chemical phenotypes of SP-IR BPSN. PMID:26580655

  12. Role of Toxin Functional Domains in Anthrax Pathogenesis

    Brossier, Fabien; Weber-Levy, Martine; Mock, Michele; SIRARD, Jean-Claude

    2000-01-01

    We investigated the role of the functional domains of anthrax toxins during infection. Three proteins produced by Bacillus anthracis, the protective antigen (PA), the lethal factor (LF), and the edema factor (EF), combine in pairs to produce the lethal (PA+LF) and edema (PA+EF) toxins. A genetic strategy was developed to introduce by allelic exchange specific point mutations or in-frame deletions into B. anthracis toxin genes, thereby impairing either LF metalloprotease or EF adenylate cyclas...

  13. Uridylation and adenylation of RNAs.

    Song, JianBo; Song, Jun; Mo, BeiXin; Chen, XueMei

    2015-11-01

    The posttranscriptional addition of nontemplated nucleotides to the 3' ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3' ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone mRNAs, mRNA fragments, U6 snRNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area. PMID:26563174

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

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

    1995-01-01

    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...

  15. Intein-mediated Rapid Purification of Recombinant Human Pituitary Adenylate Cyclase Activating Polypeptide

    Rong-jie YU; An HONG; Yun DAI; Yuan GAO

    2004-01-01

    In order to obtain the recombinant human PACAP efficiently by intein-mediated single column purification, a gene encoding human PACAP was synthesized and cloned into Escherichia coli expression vector pKYB. The recombinant vector pKY-PAC was transferred into E. coli ER2566 cells and the target protein was over-expressed as a fusion to the N-terminus of a self-cleavable affinity tag. After the PACAPintein-CBD fusion protein was purified by chitin-affinity chromatography, the self-cleavage activity of the intein was induced by DTT and the rhPACAP was released from the chitin-bound intein tag. The activity of the rhPACAP to stimulate cyclic AMP accumulation was detected using the human pancreas carcinoma cells SW1990. Twenty-two milligrams of rhPACAP with the purity over 98% was obtained by single column purification from 1 liter of induced culture. The preliminary biological assay indicated that the rhPACAP, which has an extra Met at its N-terminus compared with the native human PACAP, had the similar activity of stimulating cAMP accumulation with the standard PACAP38 in the SW1990 cells. A new efficient production procedure of the active recombinant human PACAP was established.

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

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

    2005-01-01

    strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at...... neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition. Udgivelsesdato: 2005-Jun...... least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel...

  17. GABAB receptor modulation of adenylate cyclase activity in rat brain slices.

    Hill, D R

    1985-01-01

    An investigation of the effects of gamma-aminobutyric acid (GABA) and the selective GABAB receptor agonist, baclofen, on basal and stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in slices of rat cerebral cortex has been carried out. Neither GABA nor baclofen produced any significant change in basal cyclic AMP levels. By contrast noradrenaline and forskolin both produced dose-dependent increases in cellular cyclic AMP accumulation. GABA (in the presence of nipecotic acid) ...

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

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

    2012-01-01

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

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

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

    2003-01-01

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

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

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

    2014-01-01

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

  1. Inhibition of heat-labile cholera and Escherichia coli enterotoxins by brefeldin A.

    Donta, S T; Beristain, S; Tomicic, T K

    1993-08-01

    Cholera enterotoxin and the related heat-labile enterotoxins of Escherichia coli enter their target cells through noncoated vesicles, but how the toxins are processed intracellularly and how they get to their targeted enzyme, adenylate cyclase, remain to be defined. Brefeldin A, an inhibitor of the trans-Golgi network, is shown herein to transiently block the morphologic and enzymatic effects of the toxin at a step distal to the initial binding process but prior to activation of adenylate cyclase by the toxin. It is likely, therefore, that these toxins are processed by the Golgi apparatus before trafficking to the membrane adenylate cyclase. PMID:8392970

  2. Cytosolic adenylate changes during exercise in prawn muscle

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

  3. Structural studies of Schistosoma mansoni adenylate kinases

    Marques, I.A. [Universidade Federal de Goias (UFG), Goiania, GO (Brazil); Pereira, H.M.; Garrat, R.C. [Universidade de Sao Paulo (USP-SC), Sao Carlos, SP (Brazil)

    2012-07-01

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

  4. Polyamine toxins

    Strømgaard, Kristian; Jensen, Lars S; Vogensen, Stine B

    2005-01-01

    Polyamine toxins, isolated from spiders and wasps, have been used as pharmacological tools for the study of ionotropic receptors, but their use have so far been hampered by their lack of selectivity. In this mini-review, we describe how careful synthetic modification of native polyamine toxins ha...

  5. Botulinum toxin.

    Savardekar Preeti

    1989-01-01

    Botulinum toxin, one of the most poisonous biological substances known, is a neurotoxin produced by the bacterium Clostridium botulinum. C. botulinum elaborates eight antigenically distinguishable exotoxins (A, B, C 1 , C 2 , D, E, F and G). All serotypes interfere with neural transmission by blocking the release of acetylcholine, the principal neurotransmitter at the neuromuscular junction, causing muscle paralysis. The weakness induced by injection with botulinum toxin A usually lasts about...

  6. Bicarbonate-Regulated Soluble Adenylyl Cyclase

    Wuttke MS

    2001-07-01

    Full Text Available Soluble adenylyl cyclase (sAC represents a novel form of mammalian adenylyl cyclase structurally, molecularly, and biochemically distinct from the G protein-regulated, transmembrane adenylyl cyclases (tmACs. sAC possesses no transmembrane domains and is insensitive to classic modulators of tmACs, such as heterotrimeric G proteins and P site ligands. Thus, sAC defines an independently regulated cAMP signaling system within mammalian cells. sAC is directly stimulated by bicarbonate ion both in vivo in heterologously expressing cells and in vitro using purified protein. sAC appears to be the predominant form of adenylyl cyclase (AC in mammalian sperm, and its direct activation by bicarbonate provides a mechanism for generating the cAMP required to complete the bicarbonate-induced processes necessary for fertilization, including hyperactivated motility, capacitation, and the acrosome reaction. Immunolocalization studies reveal sAC is also abundantly expressed in other tissues which respond to bicarbonate or carbon dioxide levels suggesting it may function as a general bicarbonate/CO(2 sensor throughout the body.

  7. Pituitary adenylate cyclase-activating polypeptide (PACAP): a regulator of the innate and acquired immune functions in juvenile fish.

    Lugo, Juana Maria; Carpio, Yamila; Oliva, Aymé; Morales, Antonio; Estrada, Mario Pablo

    2010-09-01

    To date, published in-vivo studies on the action of the PACAP in fish are few and these are concerned with reproduction, brain development and feeding behavior. Recently, we demonstrated for the first time that PACAP, apart from its neuroendocrine role, influences immune functions in fish larvae. In this work, we have evaluated the effects of recombinant Clarias gariepinus PACAP administration by intraperitoneal injection on important immune parameters in juvenile fish. We observed that a single injection of the recombinant peptide (0.1 microg per g of body weight) was able to increase the nitric oxide synthase-derived metabolites (NOS) and total immunoglobulin M (IgM) concentration in serum of juvenile catfish C. gariepinus and tilapia Orechromis niloticus respectively, after 24 h of its administration. In addition, our results showed that recombinant PACAP increases IgM, NOS and lysozyme in serum correlated with its ability to enhance growth performance in juvenile fish. Finally, the PACAP mRNA expression and PACAP immunoreactivity detected in peripheral blood leucocytes from juvenile catfish suggest a direct autocrine or/and paracrine mechanism of regulation of this peptide to mediate immune functions in fish. PMID:20510368

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

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

    2005-01-01

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

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

    Amin, Faisal Mohammad; Hougaard, Anders; Schytz, Henrik W; Asghar, Mohammad S; Lundholm, Elisabet; Parvaiz, Arushma I; de Koning, Patrick J H; Andersen, Malene R; Larsson, Henrik B W; Fahrenkrug, Jan; Olesen, Jes; Ashina, Messoud

    samples (plasma PACAP38 and vasoactive intestinal polypeptide and serum tryptase), and vital signs (blood pressure, heart rate, respiratory frequency, and end-tidal pressure of CO2) was recorded before and up to 5 h after infusion. Twenty-two patients [mean age 24 years (range 19-36)] completed the study...

  10. Properties of rat anterior pituitary vasopressin receptors: relation to adenylate cyclase and the effect of corticotropin-releasing factor.

    Gaillard, R C; Schoenenberg, P; Favrod-Coune, C A; Muller, A F; Marie, J. (ed.); Bockaert, J.; Jard, S

    1984-01-01

    Crude plasma membrane fractions were prepared from female Wistar rat anterior pituitaries. These fractions contained a single population of specific 3H-labeled [8-lysine]vasopressin [( 3H]vasopressin) binding sites with a dissociation of constant (Kd) of 8 +/- 2 X 10(-9) M and maximal binding capacity of 244 +/- 45 fmol/mg of protein. The Kd values for a series of vasopressin structural analogues with selective vasopressor or antidiuretic activities were determined together with the correspon...

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

    GilLevkowitz

    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.

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

    Aakerlund, L; Gether, U; Fuhlendorff, J; Schwartz, T W; Thastrup, Ole

    1990-01-01

    a rapid and transient increase in the concentration of free calcium in the cytoplasm as measured by the fluorescent probe, Fura-2. The effect of both peptides was independent of extracellular calcium as addition of EGTA or manganese neither changed the size nor the shape of the calcium response. The...... calcium response to NPY was abolished by pretreatment with thapsigargin, which can selectively deplete a calcium store in the endoplasmic reticulum. Y1 receptor stimulation, by both NPY and [Leu31,Pro34]NPY, also inhibited the forskolin-stimulated cAMP production with an EC50 of 3.5 nM. There was a close...... relation between the receptor binding and the cellular effects as half-maximal displacement of [125I-Tyr36]monoiodoNPY from the receptor was obtained with 2.1 nM NPY. The Y2-specific ligand NPY(16-36)peptide had no effect on either intracellular calcium or cAMP levels in the SK-N-MC cells. It is concluded...

  13. Botox (Botulinum Toxin)

    ... and rashes clinical tools newsletter | contact Share | Botox (Botulinum Toxin) A A A BEFORE: Crow's feet before Botox ... wrinkles. One such procedure involves the use of botulinum toxin injections. Botulinum toxin is produced by the fermentation ...

  14. Quantitative Mass Spectrometry for Bacterial Protein Toxins — A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis

    Suzanne Kalb

    2011-03-01

    Full Text Available Matrix-assisted laser-desorption time-of-flight (MALDI-TOF mass spectrometry (MS is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA which combines with lethal factor (LF and edema factor (EF, forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase.

  15. Correlated inter-domain motions in adenylate kinase.

    Santiago Esteban-Martín

    2014-07-01

    Full Text Available Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK, using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs measured under steric alignment by nuclear magnetic resonance (NMR. We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.

  16. Action of ''Bipenst'' preparation and dimethylsulfoxide on the adenyl nucleotide content in liver of irradiated animals

    Action of parenteral administration of a biostimulator ''Bipenst'' and a 10; dimethylsulfoxide solution on the level of adenyl nucleotides in the liver of rats subjected to a single whole-body irradiation (243 R) has been studied. It has been found that the level of adenyl nucleotides in the liver of irradiated animals decreases, and adenyl nucleotide content normalizes under the action of the preparations under study

  17. Functional non-nucleoside adenylyl cyclase inhibitors.

    Lelle, Marco; Hameed, Abdul; Ackermann, Lisa-Maria; Kaloyanova, Stefka; Wagner, Manfred; Berisha, Filip; Nikolaev, Viacheslav O; Peneva, Kalina

    2015-05-01

    In this study, we describe the synthesis of novel functional non-nucleoside adenylyl cyclase inhibitors, which can be easily modified with thiol containing biomolecules such as tumour targeting structures. The linkage between inhibitor and biomolecule contains cleavable bonds to enable efficient intracellular delivery in the reductive milieu of the cytosol as well as in the acidic environment within endosomes and lysosomes. The suitability of this synthetic approach was shown by the successful bioconjugation of a poor cell-permeable inhibitor with a cell-penetrating peptide. Additionally, we have demonstrated the excellent inhibitory effect of the compounds presented here in a live-cell Förster resonance energy transfer-based assay in human embryonic kidney cells. PMID:25319071

  18. Anthrax edema toxin differentially regulates lipopolysaccharide-induced monocyte production of tumor necrosis factor alpha and interleukin-6 by increasing intracellular cyclic AMP.

    Hoover, D L; Friedlander, A M; Rogers, L.C.; Yoon, I. K.; Warren, R L; Cross, A S

    1994-01-01

    Bacillus anthracis exotoxins mediate most of the symptomatology of severe anthrax. In addition to a clinical syndrome reminiscent of septic shock, which may be mediated by cytokines produced by macrophages stimulated with lethal toxin, infected patients show profound edema at sites of infection. Edema is mediated by edema toxin (ET), which comprises of a binding molecule, protective antigen, and an active moiety, edema factor, which possesses intrinsic adenylyl cyclase activity. Intracellular...

  19. *CYANOBACTERIA AND THEIR TOXINS

    Cyanobacteria, or blue-green algae, are naturally-occurring contaminants of surface waters worldwide. These photosynthesizing prokaryotes thrive in warm, shallow, nutrient-rich waters. Many produce potent toxins as secondary metabolites. Cyanobacteria toxins have been document...

  20. Botulinum toxin injection - larynx

    Injection laryngoplasty; Botox-larynx: spasmodic dysphonia-BTX; Essential voice tremor (EVT)-btx; Glottic insufficiency; Percutaneous electromyography-guided botulinum toxin treatment; Percutaneous indirect laryngoscopy-guided botulinum toxin Treatment; ...

  1. Stool C. difficile toxin

    ... page: //medlineplus.gov/ency/article/003590.htm Stool C. difficile toxin To use the sharing features on this page, please enable JavaScript. The stool C. difficile toxin test detects harmful substances produced by ...

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

    Mansouri, Shiva; Lietzau, Grazyna; Lundberg, Mathias; Nathanson, David; Nyström, Thomas; Patrone, Cesare

    2016-01-01

    Hypoglycaemia is a common side-effect of glucose-lowering therapies for type-2 diabetic patients, which may cause cognitive/neurological impairment. Although the effects of hypoglycaemia in the brain have been extensively studied in neurons, how hypoglycaemia impacts the viability of adult neural stem cells (NSCs) has been poorly investigated. In addition, the cellular and molecular mechanisms of how hypoglycaemia regulates NSCs survival have not been characterized. Recent work others and us have shown that the pituitary adenylate cyclase-activating polypeptide (PACAP) and the glucagon-like peptide-1 receptor (GLP-1R) agonist Exendin-4 stimulate NSCs survival against glucolipoapoptosis. The aim of this study was to establish an in vitro system where to study the effects of hypoglycaemia on NSC survival. Furthermore, we determine the potential role of PACAP and Exendin-4 in counteracting the effect of hypoglycaemia. A hypoglycaemic in vitro milieu was mimicked by exposing subventricular zone-derived NSC to low levels of glucose. Moreover, we studied the potential involvement of apoptosis and endoplasmic reticulum stress by quantifying protein levels of Bcl-2, cleaved caspase-3 and mRNA levels of CHOP. We show that PACAP via PAC-1 receptor and PKA activation counteracts impaired NSC viability induced by hypoglycaemia. The protective effect induced by PACAP correlated with endoplasmic reticulum stress, Exendin-4 was ineffective. The results show that hypoglycaemia decreases NSC viability and that this effect can be substantially counteracted by PACAP via PAC-1 receptor activation. The data supports a potential therapeutic role of PAC-1 receptor agonists for the treatment of neurological complications, based on neurogenesis impairment by hypoglycaemia. PMID:27305000

  3. Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

    Kuhn, Michaela

    2016-04-01

    cGMP controls many cellular functions ranging from growth, viability, and differentiation to contractility, secretion, and ion transport. The mammalian genome encodes seven transmembrane guanylyl cyclases (GCs), GC-A to GC-G, which mainly modulate submembrane cGMP microdomains. These GCs share a unique topology comprising an extracellular domain, a short transmembrane region, and an intracellular COOH-terminal catalytic (cGMP synthesizing) region. GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure/volume and energy balance. GC-B is activated by C-type natriuretic peptide, stimulating endochondral ossification in autocrine way. GC-C mediates the paracrine effects of guanylins on intestinal ion transport and epithelial turnover. GC-E and GC-F are expressed in photoreceptor cells of the retina, and their activation by intracellular Ca(2+)-regulated proteins is essential for vision. Finally, in the rodent system two olfactorial GCs, GC-D and GC-G, are activated by low concentrations of CO2and by peptidergic (guanylins) and nonpeptidergic odorants as well as by coolness, which has implications for social behaviors. In the past years advances in human and mouse genetics as well as the development of sensitive biosensors monitoring the spatiotemporal dynamics of cGMP in living cells have provided novel relevant information about this receptor family. This increased our understanding of the mechanisms of signal transduction, regulation, and (dys)function of the membrane GCs, clarified their relevance for genetic and acquired diseases and, importantly, has revealed novel targets for therapies. The present review aims to illustrate these different features of membrane GCs and the main open questions in this field. PMID:27030537

  4. Nucleotidyl cyclase activity of particulate guanylyl cyclase A: comparison with particulate guanylyl cyclases E and F, soluble guanylyl cyclase and bacterial adenylyl cyclases CyaA and edema factor.

    Kerstin Y Beste

    Full Text Available Guanylyl cyclases (GCs regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs and a nitric oxide-activated soluble GC (sGC. Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF from Bacillus anthracis possess nucleotidyl cyclase (NC activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.

  5. Activation of the Pacidamycin PacL Adenylation Domain by MbtH-Like Proteins†

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

    2010-01-01

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (~70 residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated n...

  6. Oligo-2',5'-adenylate synthetase activity in peripheral blood mononuclear leukocytes in various diseases.

    Fujii, N; Kotake, S.; Hirose, S; Ohno, S; Yasuda, I.; Sagawa, A; Ishikawa, K.; Minagawa, T

    1984-01-01

    Interferon induces oligo-2',5'-adenylate synthetase in cells. In various diseases, interferon was detectable in the circulation or was produced spontaneously from peripheral blood mononuclear leukocytes. The oligo-2',5'-adenylate synthetase activity in peripheral blood mononuclear leukocytes was examined in various diseases, including systemic lupus erythematosus, sarcoidosis, Vogt-Koyanagi-Harada disease, and Behcet's disease. The activity of this enzyme was significantly increased in system...

  7. Computational identification of candidate nucleotide cyclases in higher plants

    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.

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

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

    2013-12-22

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

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

    Abir U Igamberdiev

    2015-01-01

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

  10. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Rashna Bhandari; Roy Mathew; K Vijayachandra; Sandhya S Visweswariah

    2000-12-01

    Tyrosine phosphorylation events are key components of several cellular signal transduction pathways. This study describes a novel method for identification of substrates for tyrosine kinases. Co-expression of the tyrosine kinase EphB1 with the intracellular domain of guanylyl cyclase C (GCC) in Escherichia coli cells resulted in tyrosine phosphorylation of GCC, indicating that GCC is a potential substrate for tyrosine kinases. Indeed, GCC expressed in mammalian cells is tyrosine phosphorylated, suggesting that tyrosine phosphorylation may play a role in regulation of GCC signalling. This is the first demonstration of tyrosine phosphorylation of any member of the family of membrane-associated guanylyl cyclases.

  11. Inhibitors of glutaminyl cyclases against Alzheimer´s disease

    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

  12. A Survey of Nucleotide Cyclases in Actinobacteria: Unique Domain Organization and Expansion of the Class III Cyclase Family in Mycobacterium tuberculosis

    Sandhya S. Visweswariah

    2004-01-01

    Full Text Available Cyclic nucleotides are well-known second messengers involved in the regulation of important metabolic pathways or virulence factors. There are six different classes of nucleotide cyclases that can accomplish the task of generating cAMP, and four of these are restricted to the prokaryotes. The role of cAMP has been implicated in the virulence and regulation of secondary metabolites in the phylum Actinobacteria, which contains important pathogens, such as Mycobacterium tuberculosis, M. leprae, M. bovis and Corynebacterium, and industrial organisms from the genus Streptomyces. We have analysed the actinobacterial genome sequences found in current databases for the presence of different classes of nucleotide cyclases, and find that only class III cyclases are present in these organisms. Importantly, prominent members such as M. tuberculosis and M. leprae have 17 and 4 class III cyclases, respectively, encoded in their genomes, some of which display interesting domain fusions seen for the first time. In addition, a pseudogene corresponding to a cyclase from M. avium has been identified as the only cyclase pseudogene in M. tuberculosis and M. bovis. The Corynebacterium and Streptomyces genomes encode only a single adenylyl cyclase each, both of which have corresponding orthologues in M. tuberculosis. A clustering of the cyclase domains in Actinobacteria reveals the presence of typical eukaryote-like, fungi-like and other bacteria-like class III cyclase sequences within this phylum, suggesting that these proteins may have significant roles to play in this important group of organisms.

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

    Andre Terzic

    2009-04-01

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

  14. Understanding malarial toxins.

    Starkl Renar, Katarina; Iskra, Jernej; Križaj, Igor

    2016-09-01

    Recognized since antiquity, malaria is one of the most infamous and widespread infectious diseases in humans and, although the death rate during the last century has been diminishing, it still accounts for more than a half million deaths annually. It is caused by the Plasmodium parasite and typical symptoms include fever, shivering, headache, diaphoresis and nausea, all resulting from an excessive inflammatory response induced by malarial toxins released into the victim's bloodstream. These toxins are hemozoin and glycosylphosphatidylinositols. The former is the final product of the parasite's detoxification of haeme, a by-product of haemoglobin catabolism, while the latter anchor proteins to the Plasmodium cell surface or occur as free molecules. Currently, only two groups of antimalarial toxin drugs exist on the market, quinolines and artemisinins. As we describe, they both target biosynthesis of hemozoin. Other substances, currently in various phases of clinical trials, are directed towards biosynthesis of glycosylphosphatidylinositol, formation of hemozoin, or attenuation of the inflammatory response of the patient. Among the innovative approaches to alleviating the effects of malarial toxins, is the development of antimalarial toxin vaccines. In this review the most important lessons learned from the use of treatments directed against the action of malarial toxins in antimalarial therapy are emphasized and the most relevant and promising directions for future research in obtaining novel antimalarial agents acting on malarial toxins are discussed. PMID:27353131

  15. ADENYLATE ENERGY CHARGE AND ADENINE NUCLEOTIDE MEASUREMENTS AS INDICATORS OF STRESS IN THE MUSSEL, MYTILUS EDULIS, TREATED WITH DREDGED MATERIAL UNDER LABORATORY CONDITIONS

    Adenylate energy charge is an indication of the amount of energy available to an organism from the adenylate pool. t is calculated from measured concentrations of three adenine nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP...

  16. Interconversion of functional motions between mesophilic and thermophilic adenylate kinases.

    Michael D Daily

    2011-07-01

    Full Text Available Dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (AK, for which the open/closed transition limits the rate of catalytic turnover. Here, we compare our previously published coarse-grained (double-well Gō simulation of mesophilic AK from E. coli (AKmeso to simulations of thermophilic AK from Aquifex aeolicus (AKthermo. In AKthermo, as with AKmeso, the LID domain prefers to close before the NMP domain in the presence of ligand, but LID rigid-body flexibility in the open (O ensemble decreases significantly. Backbone foldedness in O and/or transition state (TS ensembles increases significantly relative to AKmeso in some interdomain backbone hinges and within LID. In contact space, the TS of AKthermo has fewer contacts at the CORE-LID interface but a stronger contact network surrounding the CORE-NMP interface than the TS of AKmeso. A "heated" simulation of AKthermo at 375K slightly increases LID rigid-body flexibility in accordance with the "corresponding states" hypothesis. Furthermore, while computational mutation of 7 prolines in AKthermo to their AKmeso counterparts produces similar small perturbations, mutation of these sites, especially positions 8 and 155, to glycine is required to achieve LID rigid-body flexibility and hinge flexibilities comparable to AKmeso. Mutating the 7 sites to proline in AKmeso reduces some hinges' flexibilities, especially hinge 2, but does not reduce LID rigid-body flexibility, suggesting that these two types of motion are decoupled in AKmeso. In conclusion, our results suggest that hinge flexibility and global functional motions alike are correlated with but not exclusively determined by the hinge residues. This mutational framework can inform the rational design of functionally important flexibility and allostery in other proteins toward engineering novel biochemical pathways.

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

    Csati, A; Tajti, J; Kuris, A; Tuka, B; Edvinsson, L; Warfvinge, K

    2012-01-01

    Cranial parasympathetic outflow is mediated through the sphenopalatine ganglion (SPG). The present study was performed to examine the expression of the parasympathetic signaling transmitters and their receptors in human and rat SPG. Indirect immunofluorescence technique was used for the...

  18. Alterations of the membrane-bound adenyl-cyclase enzyme and beta-receptors in developing glia- and neuron tissue cultures

    The development of beta-receptors was investigated on the basis of specific 3H-L-dihydro-alprenolol (DHA) binding. The dependence of the binding on time and concentration the dissociation constant and the number of receptor sites on one cell were determined. It is concluded that the number of 3H-DHA binding sites significantly increases after preincubation with beta-agonists and in mixed glia-neuron cultures. (L.E.)

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

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

    2007-01-01

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

  20. GABAB受体与腺苷酸环化酶偶联环节的脱敏研究%STUDIES ON DESENSITIZATION OF GABAB RECEPTOR COUPLED ADENYLATE CYCLASE

    俞在芳; 程冠军; 胡本荣

    1997-01-01

    将突触体膜与佛波酯(PMA),GABAB受体激动剂巴氯芬(Baclofen,BAL)预孵育一定时间后,BAL对腺苷酸环化酶(AC)基础活性及forskolin刺激的AC活性的抑制率显著降低(脱敏);而forskolin预孵育时,BAL对基础及forskolin刺激的AC活性的抑制率不变,表明GABAB受体与AC偶联环节的脱敏机制涉及蛋白激酶C(PKC)激活,而与蛋白激酶A无关,脱敏时GABAB受体的Kd值增加.本实验提示,可能由于PKC激活导致GABAB受体结构或构象改变,使受体-G蛋白脱偶联而出现脱敏现象.

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

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

    2008-01-01

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

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

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

    2006-01-01

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

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

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

    2005-01-01

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

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

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

    2013-01-01

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions employed by ATP-dependent DNA ligases 1,2 . Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5′-adenylated (5′-AMP) DNA lesions 3–6 (Fig. 1a). Aprataxin (Aptx) reverses DNA-adenylation but the context for dead...

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

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

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

  6. Activation of the Pacidamycin PacL Adenylation Domain by MbtH-Like Proteins†

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

    2010-01-01

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (~70 residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways. PMID:20964365

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

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

    2010-11-23

    Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (∼70-residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways. PMID:20964365

  8. Staphylococcus aureus toxins

    Otto, Michael

    2013-01-01

    Staphylococcus aureus is a dangerous pathogen that causes a variety of severe diseases. The virulence of S. aureus is defined by a large repertoire of virulence factors, among which secreted toxins play a preeminent role. Many S. aureus toxins damage biological membranes, leading to cell death. In particular, S. aureus produces potent hemolysins and leukotoxins. Among the latter, some were recently identified to lyse neutrophils after ingestion, representing an especially powerful weapon agai...

  9. Molecular analysis of adenylyl cyclase: Bacillus anthracis edema factor exotoxin

    Mohammed, Hesham Hamada Taha

    2010-01-01

    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). However, conventional antibiotic treatment is ineffective against either toxemia or antibiotic- resistant strains. Thus, more effective drugs for anthrax treatment are needed. We successfully purified the recombinant full-length EF and EF3(F586A) from E. coli with...

  10. Requirements for the adenylyl cyclases in the development of Dictyostelium.

    Anjard, C; Söderbom, F; Loomis, W F

    2001-09-01

    It has been suggested that all intracellular signaling by cAMP during development of Dictyostelium is mediated by the cAMP-dependent protein kinase, PKA, since cells carrying null mutations in the acaA gene that encodes adenylyl cyclase can develop so as to form fruiting bodies under some conditions if PKA is made constitutive by overexpressing the catalytic subunit. However, a second adenylyl cyclase encoded by acrA has recently been found that functions in a cell autonomous fashion during late development. We have found that expression of a modified acaA gene rescues acrA- mutant cells indicating that the only role played by ACR is to produce cAMP. To determine whether cells lacking both adenylyl cyclase genes can develop when PKA is constitutive we disrupted acrA in a acaA- PKA-C(over) strain. When developed at high cell densities, acrA- acaA- PKA-C(over) cells form mounds, express cell type-specific genes at reduced levels and secrete cellulose coats but do not form fruiting bodies or significant numbers of viable spores. Thus, it appears that synthesis of cAMP is required for spore differentiation in Dictyostelium even if PKA activity is high. PMID:11566867

  11. Labeling of specific proteins in rat ovarian plasma membranes with [γ-32P]GTP

    The authors report evidence that [γ-32P]GTP preferentially labels two proteins in rat ovary and parotid membranes that differ structurally from the proteins that are substrates for ADP-ribosylation by cholera toxin and which are thought to be involved in the regulation of adenylate cyclase by GTP. (Auth.)

  12. Toxins Best Paper Award 2015

    Tesh, Vernon L.

    2015-01-01

    In order to recognize outstanding papers related to biotoxins and toxinology that have been published in Toxins, the Editorial Board established an annual “Toxins Best Paper Award”. We are pleased to announce the first “Toxins Best Paper Award” for 2015. Nominations were selected by the Editorial Board members, with all papers published in 2011 eligible for consideration. Reviews and original research articles were evaluated separately. Following review and voting by the Toxins Best Paper Awa...

  13. Photolabeling of Glu-129 of the S-1 subunit of pertussis toxin with NAD

    Barbieri, J.T.; Mende-Mueller, L.M.; Rappuoli, R.; Collier, R.J. (Medical College of Wisconsin, Milwaukee (USA))

    1989-11-01

    UV irradiation was shown to induce efficient transfer of radiolabel from nicotinamide-labeled NAD to a recombinant protein (C180 peptide) containing the catalytic region of the S-1 subunit of pertussis toxin. Incorporation of label from (3H-nicotinamide)NAD was efficient (0.5 to 0.6 mol/mol of protein) relative to incorporation from (32P-adenylate)NAD (0.2 mol/mol of protein). Label from (3H-nicotinamide)NAD was specifically associated with Glu-129. Replacement of Glu-129 with glycine or aspartic acid made the protein refractory to photolabeling with (3H-nicotinamide)NAD, whereas replacement of a nearby glutamic acid, Glu-139, with serine did not. Photolabeling of the C180 peptide with NAD is similar to that observed with diphtheria toxin and exotoxin A of Pseudomonas aeruginosa, in which the nicotinamide portion of NAD is transferred to Glu-148 and Glu-553, respectively, in the two toxins. These results implicate Glu-129 of the S-1 subunit as an active-site residue and a potentially important site for genetic modification of pertussis toxin for development of an acellular vaccine against Bordetella pertussis.

  14. Toxin plasmids of Clostridium perfringens.

    Li, Jihong; Adams, Vicki; Bannam, Trudi L; Miyamoto, Kazuaki; Garcia, Jorge P; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2013-06-01

    In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  15. Renal Phosphate Wasting in the Absence of Adenylyl Cyclase 6

    Fenton, Robert A; Murray, Fiona; Dominguez Rieg, Jessica A.; Tang, Tong; Levi, Moshe; Rieg, Timo

    2014-01-01

    Parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF-23) enhance phosphate excretion by the proximal tubule of the kidney by retrieval of the sodium-dependent phosphate transporters (Npt2a and Npt2c) from the apical plasma membrane. PTH activates adenylyl cyclase (AC) through PTH 1 receptors and stimulates the cAMP/PKA signaling pathway. However, the precise role and isoform(s) of AC in phosphate homeostasis are not known. We report here that mice lacking AC6 (AC6−/−) have increased...

  16. Mice lacking the ADP ribosyl cyclase CD38 exhibit attenuated renal vasoconstriction to angiotensin II, endothelin-1, and norepinephrine

    Thai, Tiffany L.; Arendshorst, William J.

    2009-01-01

    ADP ribosyl (ADPR) cyclases comprise a family of ectoenzymes recently shown to influence cytosolic Ca2+ concentration in a variety of cell types. At least two ADPR cyclase family members have been identified in mammals: CD38 and CD157. We recently found reduced renal vascular reactivity to angiotensin II (ANG II), endothelin-1 (ET-1), and norepinephrine (NE) in the presence of the broad ADPR cyclase inhibitor nicotinamide. We hypothesized that CD38 mediates effects attributed to ADPR cyclase....

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

    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

  18. Botulinum Toxin Therapy

    ... resources Meet our partners Español Donate Diseases and treatments Acne and rosacea Bumps and growths Color problems Contagious skin diseases ... toxin therapy public SPOT Skin Cancer™ Diseases and treatments Acne and rosacea Bumps and growths Color problems Contagious skin diseases ...

  19. Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

    Sorrentino, Luca; Calogero, Alessandra Maria; Pandini, Vittorio; Vanoni, Maria Antonietta; Sevrioukova, Irina F; Aliverti, Alessandro

    2015-12-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide. PMID:26535916

  20. Toxins Best Paper Award 2015

    Vernon L. Tesh

    2015-03-01

    Full Text Available In order to recognize outstanding papers related to biotoxins and toxinology that have been published in Toxins, the Editorial Board established an annual “Toxins Best Paper Award”. We are pleased to announce the first “Toxins Best Paper Award” for 2015. Nominations were selected by the Editorial Board members, with all papers published in 2011 eligible for consideration. Reviews and original research articles were evaluated separately. Following review and voting by the Toxins Best Paper Award Committee, the following three papers have won Toxins Best Paper Awards for 2015:[...

  1. Autoproteolytic Activation of Bacterial Toxins

    Aimee Shen

    2010-05-01

    Full Text Available Protease domains within toxins typically act as the primary effector domain within target cells. By contrast, the primary function of the cysteine protease domain (CPD in Multifunctional Autoprocessing RTX-like (MARTX and Clostridium sp. glucosylating toxin families is to proteolytically cleave the toxin and release its cognate effector domains. The CPD becomes activated upon binding to the eukaryotic-specific small molecule, inositol hexakisphosphate (InsP6, which is found abundantly in the eukaryotic cytosol. This property allows the CPD to spatially and temporally regulate toxin activation, making it a prime candidate for developing anti-toxin therapeutics. In this review, we summarize recent findings related to defining the regulation of toxin function by the CPD and the development of inhibitors to prevent CPD-mediated activation of bacterial toxins.

  2. Receptor guanylyl cyclases in Inka cells targeted by eclosion hormone.

    Chang, Jer-Cherng; Yang, Ruey-Bing; Adams, Michael E; Lu, Kuang-Hui

    2009-08-11

    A signature of eclosion hormone (EH) action in insect ecdysis is elevation of cGMP in Inka cells, leading to massive release of ecdysis triggering hormone (ETH) and ecdysis initiation. Although this aspect of EH-induced signal transduction is well known, the receptor mediating this process has not been identified. Here, we describe a receptor guanylyl cyclase BdmGC-1 and its isoform BdmGC-1B in the Oriental fruit fly Bactrocera dorsalis that are activated by EH. The B form exhibits the conserved domains and putative N-glycosylation sites found in BdmGC-1, but possesses an additional 46-amino acid insertion in the extracellular domain and lacks the C-terminal tail of BdmGC-1. Combined immunolabeling and in situ hybridization reveal that BdmGC-1 is expressed in Inka cells. Heterologous expression of BdmGC-1 in HEK cells leads to robust increases in cGMP following exposure to low picomolar concentrations of EH. The B-isoform responds only to higher EH concentrations, suggesting different physiological roles of these cyclases. We propose that BdmGC-1 and BdmGC-1B are high- and low-affinity EH receptors, respectively. PMID:19666575

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

    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 P212121. 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 VM = 3.0 Å3 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

  4. The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism*

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-01-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp230 residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  5. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism.

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-11-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

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

    Rouhiainen Leo

    2008-09-01

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

  7. XLF-Cernunnos promotes DNA ligase IV-XRCC4 re-adenylation following ligation.

    Riballo, Enriqueta; Woodbine, Lisa; Stiff, Thomas; Walker, Sarah A; Goodarzi, Aaron A; Jeggo, Penny A

    2009-02-01

    XLF-Cernunnos (XLF) is a component of the DNA ligase IV-XRCC4 (LX) complex, which functions during DNA non-homologous end joining (NHEJ). Here, we use biochemical and cellular approaches to probe the impact of XLF on LX activities. We show that XLF stimulates adenylation of LX complexes de-adenylated by pyrophosphate or following LX decharging during ligation. XLF enhances LX ligation activity in an ATP-independent and dependent manner. ATP-independent stimulation can be attributed to enhanced end-bridging. Whilst ATP alone fails to stimulate LX ligation activity, addition of XLF and ATP promotes ligation in a manner consistent with XLF-stimulated readenylation linked to ligation. We show that XLF is a weakly bound partner of the tightly associated LX complex and, unlike XRCC4, is dispensable for LX stability. 2BN cells, which have little, if any, residual XLF activity, show a 3-fold decreased ability to repair DNA double strand breaks covering a range of complexity. These findings strongly suggest that XLF is not essential for NHEJ but promotes LX adenylation and hence ligation. We propose a model in which XLF, by in situ recharging DNA ligase IV after the first ligation event, promotes double stranded ligation by a single LX complex. PMID:19056826

  8. Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity

    Trung Thanh Thach

    2014-01-01

    Full Text Available Streptococcus pneumoniae (pneumococcus infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S. pneumoniae (SpAdK serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A. Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo.

  9. Toxins and drug discovery.

    Harvey, Alan L

    2014-12-15

    Components from venoms have stimulated many drug discovery projects, with some notable successes. These are briefly reviewed, from captopril to ziconotide. However, there have been many more disappointments on the road from toxin discovery to approval of a new medicine. Drug discovery and development is an inherently risky business, and the main causes of failure during development programmes are outlined in order to highlight steps that might be taken to increase the chances of success with toxin-based drug discovery. These include having a clear focus on unmet therapeutic needs, concentrating on targets that are well-validated in terms of their relevance to the disease in question, making use of phenotypic screening rather than molecular-based assays, and working with development partners with the resources required for the long and expensive development process. PMID:25448391

  10. Asymmetrically acting lycopene beta-cyclases (CrtLm) from non-photosynthetic bacteria.

    Tao, L; Picataggio, S; Rouvière, P E; Cheng, Q

    2004-03-01

    Carotenoids have important functions in photosynthesis, nutrition, and protection against oxidative damage. Some natural carotenoids are asymmetrical molecules that are difficult to produce chemically. Biological production of carotenoids using specific enzymes is a potential alternative to extraction from natural sources. Here we report the isolation of lycopene beta-cyclases that selectively cyclize only one end of lycopene or neurosporene. The crtLm genes encoding the asymmetrically acting lycopene beta-cyclases were isolated from non-photosynthetic bacteria that produced monocyclic carotenoids. Co-expression of these crtLm genes with the crtEIB genes from Pantoea stewartii (responsible for lycopene synthesis) resulted in the production of monocyclic gamma-carotene in Escherichia coli. The asymmetric cyclization activity of CrtLm could be inhibited by the lycopene beta-cyclase inhibitor 2-(4-chlorophenylthio)-triethylamine (CPTA). Phylogenetic analysis suggested that bacterial CrtL-type lycopene beta-cyclases might represent an evolutionary link between the common bacterial CrtY-type of lycopene beta-cyclases and plant lycopene beta- and epsilon-cyclases. These lycopene beta-cyclases may be used for efficient production of high-value asymmetrically cyclized carotenoids. PMID:14740205

  11. Inferring biological functions of guanylyl cyclases with computational methods

    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.

  12. Clostridium difficile binary toxin CDT

    Gerding, Dale N.; Johnson, Stuart; Rupnik, Maja; Aktories, Klaus

    2013-01-01

    Binary toxin (CDT) is frequently observed in Clostridium difficile strains associated with increased severity of C. difficile infection (CDI). CDT belongs to the family of binary ADP-ribosylating toxins consisting of two separate toxin components: CDTa, the enzymatic ADP-ribosyltransferase which modifies actin, and CDTb which binds to host cells and translocates CDTa into the cytosol. CDTb is activated by serine proteases and binds to lipolysis stimulated lipoprotein receptor. ADP-ribosylatio...

  13. Requirements for anthrax toxin entry into cells

    Ryan, Patricia Lynn

    2010-01-01

    Bacillus anthracis secretes a harmful exotoxin called anthrax toxin. Anthrax toxin has deleterious effects on several host cell types and is a significant contributor to anthrax pathogenesis. Toxin-deleted strains of B. anthracis are highly attenuated and many of the symptoms of anthrax can be replicated with anthrax toxin alone. Anthrax toxin is an AB-type toxin with two catalytic A moieties. PA, the B moiety, is responsible for receptor binding, pore formation and translocation of the catal...

  14. Shiga Toxin Detection Methods : A Short Review

    Guerrero, Y. Castaño; González-Aguilar, G.

    2013-01-01

    The Shiga toxins comprise a family of related protein toxins secreted by certain types of bacteria. Shigella dysenteriae, some strain of Escherichia coli and other bacterias can express toxins which caused serious complication during the infection. Shiga toxin and the closely related Shiga-like toxins represent a group of very similar cytotoxins that may play an important role in diarrheal disease and hemolytic-uremic syndrome. The outbreaks caused by this toxin raised serious public health c...

  15. [Soluble guanylate cyclase in the molecular mechanism underlying the therapeutic action of drugs].

    Piatakova, N V; Severina, I S

    2012-01-01

    The influence of ambroxol--a mucolytic drug--on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside and Sin-1) were investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin--an antimalarial drug--on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1-100 microM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylate cyclase with an IC50 value 5.6 microM. Artemisinin (10 microM) also inhibited (by 71 +/- 4.0%) the activation of the enzyme by thiol-dependent NO-donor the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 microM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the sygnalling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin. PMID:22642150

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

    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.

  17. Lymphocyte receptors for pertussis toxin

    We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes

  18. Toxin-Based Therapeutic Approaches

    Itai Benhar

    2010-10-01

    Full Text Available Protein toxins confer a defense against predation/grazing or a superior pathogenic competence upon the producing organism. Such toxins have been perfected through evolution in poisonous animals/plants and pathogenic bacteria. Over the past five decades, a lot of effort has been invested in studying their mechanism of action, the way they contribute to pathogenicity and in the development of antidotes that neutralize their action. In parallel, many research groups turned to explore the pharmaceutical potential of such toxins when they are used to efficiently impair essential cellular processes and/or damage the integrity of their target cells. The following review summarizes major advances in the field of toxin based therapeutics and offers a comprehensive description of the mode of action of each applied toxin.

  19. Conservation of functional domain structure in bicarbonate-regulated “soluble” adenylyl cyclases in bacteria and eukaryotes

    Kobayashi, Mime; Buck, Jochen; Levin, Lonny R.

    2004-01-01

    Soluble adenylyl cyclase (sAC) is an evolutionarily conserved bicarbonate sensor. In mammals, it is responsible for bicarbonate-induced, cAMP-dependent processes in sperm required for fertilization and postulated to be involved in other bicarbonate- and carbon dioxide-dependent functions throughout the body. Among eukaryotes, sAC-like cyclases have been detected in mammals and in the fungi Dictyostelium; these enzymes display extensive similarity extending through two cyclase catalytic domain...

  20. Conventional and Unconventional Mechanisms for Soluble Guanylyl Cyclase Signaling.

    Gao, Yuansheng

    2016-05-01

    Soluble guanylyl cyclase (sGC) is the principal enzyme in mediating the biological actions of nitric oxide. On activation, sGC converts guanosine triphosphate to guanosine 3',5'-cyclic monophosphate (cGMP), which mediates diverse physiological processes including vasodilation, platelet aggregation, and myocardial functions predominantly by acting on cGMP-dependent protein kinases. Cyclic GMP has long been considered as the sole second messenger for sGC action. However, emerging evidence suggests that, in addition to cGMP, other nucleoside 3',5'-cyclic monophosphates (cNMPs) are synthesized by sGC in response to nitric oxide stimulation, and some of these nucleoside 3',5'-cyclic monophosphates are involved in various physiological activities. For example, inosine 3',5'-cyclic monophosphate synthesized by sGC may play a critical role in hypoxic augmentation of vasoconstriction. The involvement of cytidine 3',5'-cyclic monophosphate and uridine 3',5'-cyclic monophosphate in certain cardiovascular activities is also implicated. PMID:26452163

  1. Expression of soluble adenylyl cyclase in acral melanomas.

    Li, H; Kim, S M; Savkovic, V; Jin, S A; Choi, Y D; Yun, S J

    2016-06-01

    Soluble adenylyl cyclase (sAC) regulates melanocytic cells, and is a diagnostic marker for pigmented skin lesions. Because only a few studies on sAC expression in acral melanomas have been performed, we investigated the histopathological significance of sAC expression in 33 cases of acral melanoma, and assessed its diagnostic value in distinguishing melanoma in situ (MIS, n = 17) from acral invasive melanomas (n = 16) and melanocytic naevi (n = 11). Acral melanomas exhibited more marked nuclear immunopositivity compared with acral melanocytic naevi. sAC expression significantly correlated with the nuclear morphology of melanocytes and melanoma cells, namely, hyperchromatic nuclei and prominent nucleoli within vesicular nuclei. sAC expression was predominantly observed in the hyperchromatic nuclei of MIS and the prominent nucleoli invasive melanomas, respectively. In vitro culture models of melanocytes and melanoma cell lines exhibited sAC staining patterns similar to those of acral melanomas. Differentiation induction showed that nuclear and nucleolar expression varied depending on cell morphology. sAC immunostaining may be useful for the differential diagnosis of acral melanocytic lesions, and sAC expressed in the nucleus and nucleolus might be related to cytological and nuclear changes associated with invasion and progression of acral melanomas. PMID:26290224

  2. Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

    Zhang Dapeng

    2012-06-01

    Full Text Available Abstract Background Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX, and the poorly characterized “Photorhabdus virulence cassettes (PVC”, PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of

  3. [Shiga toxin and tetanus toxin as a potential biologic weapon].

    Toczyska, Izabela; Płusa, Tadeusz

    2015-09-01

    Toxins produced by the bacteria are of particular interest as potential cargo combat possible for use in a terrorist attack or war. Shiga toxin is usually produced by shiga toxigenic strains of Escherichia coli (STEC - shigatoxigenic Escherichia coli). To infection occurs mostly after eating contaminated beef. Clinical syndromes associated with Shiga toxin diarrhea, hemorrhagic colitis, hemolytic uremic syndrome (HUS - hemolytic uremic syndrome) or thrombotic thrombocytopenic purpura. Treatment is symptomatic. In HUS, in which mortality during an epidemic reaches 20%, extending the kidney injury dialysis may be necessary. Exposure to tetanus toxin produced by Clostridium tetani, resulting in the most generalized tetanus, characterized by increased muscle tension and painful contractions of individual muscle groups. In the treatment beyond symptomatic behavior (among others spasticity medications, anticonvulsants, muscle relaxants) is used tetanus antitoxin and antibiotics (metronidazole choice). A common complication is acute respiratory failure - then it is necessary to implement mechanical ventilation. PMID:26449578

  4. Path ensembles for conformational transitions in adenylate kinase using weighted--ensemble path sampling

    Bhatt, Divesh

    2009-01-01

    We perform first path sampling simulations of conformational transitions of semi--atomistic protein models. We generate an ensemble of pathways for conformational transitions between open and closed forms of adenylate kinase using weighted ensemble path sampling method. Such an ensemble of pathways is critical in determining the important regions of configuration space sampled during a transition. To different semi--atomistic models are used: one is a pure Go model, whereas the other includes level of residue specificity via use of Miyajawa--Jernigan type interactions and hydrogen bonding. For both the models, we find that the open form of adenylate kinase is more flexible and the the transition from open to close is significantly faster than the reverse transition. We find that the transition occurs via the AMP binding domain snapping shut at a fairly fast time scale. On the other hand, the flexible lid domain fluctuates significantly and the shutting of the AMP binding domain does not depend upon the positi...

  5. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances.

    Howell, Stanley C; Richards, David H; Mitch, William A; Wilson, Corey J

    2015-10-16

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites. PMID:26266833

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

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

  7. Detection of extracellular toxin(s) produced by Vibrio vulnificus.

    Kreger, A; Lockwood, D.

    1981-01-01

    Conditions are described for the production, in high titers, a heat-labile, antigenic, extracellular toxin(s) by Vibrio vulnificus, a recently recognized human pathogen. Bacteriologically sterile culture filtrate preparations obtained from mid-logarithmic-phase cultures of the bacterium possessed cytolytic activity against mammalian erythrocytes, cytotoxic activity for Chinese hamster ovary cells, vascular permeability factor activity in guinea pig skin, and lethal activity for mice. The spec...

  8. Epsilon toxin: a fascinating pore-forming toxin.

    Popoff, Michel R

    2011-12-01

    Epsilon toxin (ETX) is produced by strains of Clostridium perfringens classified as type B or type D. ETX belongs to the heptameric β-pore-forming toxins including aerolysin and Clostridium septicum alpha toxin, which are characterized by the formation of a pore through the plasma membrane of eukaryotic cells consisting in a β-barrel of 14 amphipatic β strands. By contrast to aerolysin and C. septicum alpha toxin, ETX is a much more potent toxin and is responsible for enterotoxemia in animals, mainly sheep. ETX induces perivascular edema in various tissues and accumulates in particular in the kidneys and brain, where it causes edema and necrotic lesions. ETX is able to pass through the blood-brain barrier and stimulate the release of glutamate, which accounts for the symptoms of nervous excitation observed in animal enterotoxemia. At the cellular level, ETX causes rapid swelling followed by cell death involving necrosis. The precise mode of action of ETX remains to be determined. ETX is a powerful toxin, however, it also represents a unique tool with which to vehicle drugs into the central nervous system or target glutamatergic neurons. PMID:21535407

  9. Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains.

    Ishikawa, Fumihiro; Miyamoto, Kengo; Konno, Sho; Kasai, Shota; Kakeya, Hideaki

    2015-12-18

    A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the Escherichia coli expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe-protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the kcat/Km value with the noncognate substrate Sal and a corresponding 48-fold decrease in the kcat/Km value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique

  10. Pertussis toxin modifies the characteristics of both the inhibitory GTP binding proteins and the somatostatin receptor in anterior pituitary tumor cells

    The effects of pertussis toxin treatment on the characteristics of somatostatin receptors in the anterior pituitary tumor cell line AtT-20 were examined. Pertussis toxin selectively catalyzed the ADP ribosylation of the alpha subunits of the inhibitory GTP binding proteins in AtT-20 cells. Toxin treatment abolished somatostatin inhibition of forskolin-stimulated adenylyl cyclase activity and somatostatin stimulation of GTPase activity. To examine the effects of pertussis toxin treatment on the characteristics of the somatostatin receptor, the receptor was labeled by the somatostatin analog [125I]CGP 23996. [125I]CGP 23996 binding to AtT-20 cell membranes was saturable and within a limited concentration range was to a single high affinity site. Pertussis toxin treatment reduced the apparent density of the high affinity [125I]CGP 23996 binding sites in AtT-20 cell membranes. Inhibition of [125I]CGP 23996 binding by a wide concentration range of CGP 23996 revealed the presence of two binding sites. GTP predominantly reduced the level of high affinity sites in control membranes. Pertussis toxin treatment also diminished the amount of high affinity sites. GTP did not affect [125I]CGP 23996 binding in the pertussis toxin-treated membranes. The high affinity somatostatin receptors were covalently labeled with [125I] CGP 23996 and the photoactivated crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. No high affinity somatostatin receptors, covalently bound to [125I]CGP 23996, were detected in the pertussis toxin-treated membranes. These results are most consistent with pertussis toxin uncoupling the inhibitory G proteins from the somatostatin receptor thereby converting the receptor from a mixed population of high and low affinity sites to only low affinity receptors

  11. Molecular identification and functional characterization of an adenylyl cyclase from the honeybee.

    Wachten, Sebastian; Schlenstedt, Jana; Gauss, Renate; Baumann, Arnd

    2006-03-01

    Cyclic AMP (cAMP) serves as an important messenger in virtually all organisms. In the honeybee (Apis mellifera), cAMP-dependent signal transduction has been implicated in behavioural processes as well as in learning and memory. Key components of cAMP-signalling cascades are adenylyl cyclases. However, the molecular identities and biochemical properties of adenylyl cyclases are completely unknown in the honeybee. We have cloned a cDNA (Amac3) from honeybee brain that encodes a membrane-bound adenylyl cyclase. The Amac3 gene is an orthologue of the Drosophila ac39E gene. The corresponding proteins share an overall amino acid similarity of approximately 62%. Phylogenetically, AmAC3 belongs to group 1 adenylyl cyclases. Heterologously expressed AmAC3 displays basal enzymatic activity and efficient coupling to endogenous G protein signalling pathways. Stimulation of beta-adrenergic receptors induces AmAC3 activity with an EC(50) of about 3.1 microm. Enzymatic activity is also increased by forskolin (EC(50) approximately 15 microm), a specific agonist of membrane-bound adenylyl cyclases. Similar to certain biogenic amine receptor genes of the honeybee, Amac3 transcripts are expressed in many somata of the brain, especially in mushroom body neurones. These results suggest that the enzyme serves in biogenic amine signal transduction cascades and in higher brain functions that contribute to learning and memory of the bee. PMID:16464235

  12. Role of soluble guanylate cyclase in the molecular mechanism underlying the physiological effects of nitric oxide.

    Severina, I S

    1998-07-01

    In this review the molecular mechanisms underlying the antihypertensive and antiaggregatory actions of nitric oxide (NO) are discussed. It has been shown that these effects are directly connected with the activation of soluble guanylate cyclase and the accumulation of cyclic 3;,5;-guanosine monophosphate (cGMP). The mechanism of guanylate cyclase activation by NO is analyzed, especially the role and biological significance of the nitrosyl--heme complex formed as a result of interaction of guanylate cyclase heme with NO and the role of sulfhydryl groups of the enzyme in this process. Using new approaches for studying the antihypertensive and antiaggregatory actions of nitric oxide in combination with the newly obtained data on the regulatory role of guanylate cyclase in the platelet aggregation process, the most important results were obtained regarding the molecular bases providing for a directed search for and creation of new effective antihypertensive and antiaggregatory preparations. In studying the molecular mechanism for directed activation of soluble guanylate cyclase by new NO donors, a series of hitherto unknown enzyme activators generating NO and involved in the regulation of hemostasis and vascular tone were revealed. PMID:9721331

  13. Toxin-Based Therapeutic Approaches

    Itai Benhar; Assaf Shapira

    2010-01-01

    Protein toxins confer a defense against predation/grazing or a superior pathogenic competence upon the producing organism. Such toxins have been perfected through evolution in poisonous animals/plants and pathogenic bacteria. Over the past five decades, a lot of effort has been invested in studying their mechanism of action, the way they contribute to pathogenicity and in the development of antidotes that neutralize their action. In parallel, many research groups turned to explore the pharmac...

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

    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. PMID:22426196

  15. Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana

    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

  16. [Toxins of Clostridium perfringens].

    Morris, W E; Fernández-Miyakawa, M E

    2009-01-01

    Clostridium perfringens is an anaerobic gram-positive spore-forming bacillus. It is one of the pathogens with larger distribution in the environment; it can be isolated from soil and water samples, which also belongs to the intestinal flora of animals and humans. However, on some occasions it can act as an opportunistic pathogen, causing diseases such as gas gangrene, enterotoxemia in sheep and goats and lamb dysentery, among others. In human beings, it is associated to diseases such as food poisoning, necrotic enterocolitis of the infant and necrotic enteritis or pigbel in Papua-New Guinea tribes. The renewed interest existing nowadays in the study of C. perfringens as a veterinarian and human pathogen, together with the advance of molecular biology, had enabled science to have deeper knowledge of the biology and pathology of these bacteria. In this review, we discuss and update the principal aspects of C. perfringens intestinal pathology, in terms of the toxins with major medical relevance at present. PMID:20085190

  17. Targeted Toxins in Brain Tumor Therapy

    Walter A. Hall

    2010-11-01

    Full Text Available Targeted toxins, also known as immunotoxins or cytotoxins, are recombinant molecules that specifically bind to cell surface receptors that are overexpressed in cancer and the toxin component kills the cell. These recombinant proteins consist of a specific antibody or ligand coupled to a protein toxin. The targeted toxins bind to a surface antigen or receptor overexpressed in tumors, such as the epidermal growth factor receptor or interleukin-13 receptor. The toxin part of the molecule in all clinically used toxins is modified from bacterial or plant toxins, fused to an antibody or carrier ligand. Targeted toxins are very effective against cancer cells resistant to radiation and chemotherapy. They are far more potent than any known chemotherapy drug. Targeted toxins have shown an acceptable profile of toxicity and safety in early clinical studies and have demonstrated evidence of a tumor response. Currently, clinical trials with some targeted toxins are complete and the final results are pending. This review summarizes the characteristics of targeted toxins and the key findings of the important clinical studies with targeted toxins in malignant brain tumor patients. Obstacles to successful treatment of malignant brain tumors include poor penetration into tumor masses, the immune response to the toxin component and cancer heterogeneity. Strategies to overcome these limitations are being pursued in the current generation of targeted toxins.

  18. Simultaneous stimulation of GABA and beta adrenergic receptors stabilizes isotypes of activated adenylyl cyclase heterocomplex

    Robichon Alain

    2004-06-01

    Full Text Available Abstract Background We investigated how the synthesis of cAMP, stimulated by isoproterenol acting through β-adrenoreceptors and Gs, is strongly amplified by simultaneous incubation with baclofen. Baclofen is an agonist of δ-aminobutyric acid type B receptors [GABAB], known to inhibit adenylyl cyclase via Gi. Because these agents have opposite effects on cAMP levels, the unexpected increase in cAMP synthesis when they are applied simultaneously has been intensively investigated. From previous reports, it appears that cyclase type II contributes most significantly to this phenomenon. Results We found that simultaneous application of isoproterenol and baclofen specifically influences the association/dissociation of molecules involved in the induction and termination of cyclase activity. Beta/gamma from [GABA]B receptor-coupled Gi has a higher affinity for adenylyl cyclase isoform(s when these isoforms are co-associated with Gs. Our data also suggest that, when beta/gamma and Gαs are associated with adenylyl cyclase isoform(s, beta/gamma from [GABA]B receptor-coupled Gi retards the GTPase activity of Gαs from adrenergic receptor. These reciprocal regulations of subunits of the adenylyl cyclase complex might be responsible for the drastic increase of cAMP synthesis in response to the simultaneous signals. Conclusions Simultaneous signals arriving at a particular synapse converge on molecular detectors of coincidence and trigger specific biochemical events. We hypothesize that this phenomenon comes from the complex molecular architectures involved, including scaffolding proteins that make reciprocal interactions between associated molecules possible. The biochemistry of simultaneous signaling is addressed as a key to synaptic function.

  19. Scorpion toxins prefer salt solutions.

    Nikouee, Azadeh; Khabiri, Morteza; Cwiklik, Lukasz

    2015-11-01

    There is a wide variety of ion channel types with various types of blockers, making research in this field very complicated. To reduce this complexity, it is essential to study ion channels and their blockers independently. Scorpion toxins, a major class of blockers, are charged short peptides with high affinities for potassium channels. Their high selectivity and inhibitory properties make them an important pharmacological tool for treating autoimmune or nervous system disorders. Scorpion toxins typically have highly charged surfaces and-like other proteins-an intrinsic ability to bind ions (Friedman J Phys Chem B 115(29):9213-9223, 1996; Baldwin Biophys J 71(4):2056-2063, 1996; Vrbka et al. Proc Natl Acad Sci USA 103(42):15440-15444, 2006a; Vrbka et al. J Phys Chem B 110(13):7036-43, 2006b). Thus, their effects on potassium channels are usually investigated in various ionic solutions. In this work, computer simulations of protein structures were performed to analyze the structural properties of the key residues (i.e., those that are presumably involved in contact with the surfaces of the ion channels) of 12 scorpion toxins. The presence of the two most physiologically abundant cations, Na(+) and K(+), was considered. The results indicated that the ion-binding properties of the toxin residues vary. Overall, all of the investigated toxins had more stable structures in ionic solutions than in water. We found that both the number and length of elements in the secondary structure varied depending on the ionic solution used (i.e., in the presence of NaCl or KCl). This study revealed that the ionic solution should be chosen carefully before performing experiments on these toxins. Similarly, the influence of these ions should be taken into consideration in the design of toxin-based pharmaceuticals. PMID:26475740

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

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

    2005-01-01

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

  1. Role of Guanylate Cyclase Activating Proteins in photoreceptor cells of the retina in health and disease

    López del Hoyo, Natalia

    2014-01-01

    In the last two decades, it has been done a thoroughly research about the role of Guanylate Cyclase Activating Proteins (GCAPs) in photoreceptor cells of the retina as activity regulators of Retinal Guanylate Cyclase (RetGC), which allow to restore cGMP levels to darkness ones when intracellular Ca2+ falls. However, little is known about: a) ¿What determines GCAPs distribution within the cell?, b) ¿Which other functions GCAP proteins, GCAP1 and GCAP2, carry out at other cellular compartm...

  2. Long-Term Exposure to High Corticosterone Levels Inducing a Decrease of Adenylate Kinase 1 Activity

    ZHAO Yu'nan; SHEN Jia; SU Hui; HUANG Yufang; XING Dongming; DU Lijun

    2009-01-01

    Corticosterone, a principal glucocorticoid synthesized in the rodent adrenal cortex, can be cumula-tively toxic to hippocampal neurons, the cause of which is not known. The present study determined whether the cytosol adenylate kinase (AK) system was involved in the neuronal damage induced by long-term exposure to high corticosterone levels. We investigated the effects of long-term exposure to high corticosterone levels on AK1 activity, AK1 mRNA expression, and energy levels in cultured hippocampal neurons. The results show that long-term exposure to high corticosterone levels induces a reduction of the cultured hippocampal neuron viability, significantly reduces energy levels, and causes a time-dependant re-duction of the AK1 activity. These findings indicate that changes in the AK system might be the mechanism underlying neuronal damage induced by long-term exposure to high corticosterone levels.

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

    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 Ca2+ 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 Ca2+ and this interaction may be important for its invasion into animal cells

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

    O'Halloran Damien M

    2006-03-01

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

  5. Botulinum Toxin Injections: A Treatment for Muscle Spasms

    ... A Treatment for Muscle Spasms What is botulinum toxin? Botulinum toxin is a protein that helps stop muscle ... won't have any harmful effects from the toxin. Botulinum toxin has been used safely for a number ...

  6. Sodium Channel Inhibiting Marine Toxins

    Llewellyn, Lyndon E.

    Saxitoxin (STX), tetrodotoxin (TTX) and their many chemical relatives are part of our daily lives. From killing people who eat seafood containing these toxins, to being valuable research tools unveiling the invisible structures of their pharmacological receptor, their global impact is beyond measure. The pharmacological receptor for these toxins is the voltage-gated sodium channel which transports Na ions between the exterior to the interior of cells. The two structurally divergent families of STX and TTX analogues bind at the same location on these Na channels to stop the flow of ions. This can affect nerves, muscles and biological senses of most animals. It is through these and other toxins that we have developed much of our fundamental understanding of the Na channel and its part in generating action potentials in excitable cells.

  7. Entry of Shiga toxin into cells

    Sandvig, Kirsten; van Deurs, Bo

    Cellebiologi, Shiga toxin, receptors, glycolipids, endocytosis, trans-Golgi network, endoplasmic reticulum, retrograde transport......Cellebiologi, Shiga toxin, receptors, glycolipids, endocytosis, trans-Golgi network, endoplasmic reticulum, retrograde transport...

  8. Clostridium difficile and C. difficile Toxin Testing

    ... be limited. Home Visit Global Sites Search Help? Clostridium difficile and C. difficile Toxin Testing Share this page: ... C. diff; C diff antigen; GDH Formal name: Clostridium difficile Culture; C. difficile Toxin, A and B; C. ...

  9. Proteomics analysis of toxins-producing dinoflagellates and toxins-contaminated marine organisms

    蒋析文; Jiang, Xiwen

    2012-01-01

    Paralytic shellfish poisoning (PSP) and ciguatera fish poisoning (CFP) are the two major contributors to illnesses caused by dinoflagellate toxins. Paralytic shellfish poisoning toxins (PSTs) are produced by dinoflagellates in the genera Alexandrium, Gymnodinium, and Pyrodinium while ciguatera fish poisoning toxins, such as ciguatoxins (CTXs), are originated from benthic toxic dinoflagellates (Gambierdiscus, Prorocentrum, Ostreopsis, and Coolia species). These toxins are responsible for human...

  10. Bacterial toxin-antitoxin systems

    Guglielmini, Julien; Van Melderen, Laurence

    2011-01-01

    Toxin-antitoxin (TA) systems are composed of two elements: a toxic protein and an antitoxin which is either an RNA (type I and III) or a protein (type II). Type II systems are abundant in bacterial genomes in which they move via horizontal gene transfer. They are generally composed of two genes organized in an operon, encoding a toxin and a labile antitoxin. When carried by mobile genetic elements, these small modules contribute to their stability by a phenomenon denoted as addiction. Recentl...

  11. Unusual guanylyl cyclases and cGMP signaling in Dictyostelium discoideum

    Veltman, D.M.; Bosgraaf, L.; van Haastert, P. J. M.

    2004-01-01

    cGMP is used as a second messenger in many eukaryotes. cGMP signaling requires at least three components: Guanylyl cyclases synthesize cGMP from GTP. Specific cGMP-binding proteins propagate the signal, usually by phosphorylation of their target Finally, phosphodiesterases terminate the cGMP signal

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

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

    2016-04-01

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

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

    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 intri...

  14. Overexpression of functional human oxidosqualene cyclase in Escherichia coli

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

    2015-01-01

    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...

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

    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

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

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

    2014-01-01

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

  17. Toxin yet not toxic: Botulinum toxin in dentistry.

    Archana, M S

    2016-04-01

    Paracelsus contrasted poisons from nonpoisons, stating that "All things are poisons, and there is nothing that is harmless; the dose alone decides that something is a poison". Living organisms, such as plants, animals, and microorganisms, constitute a huge source of pharmaceutically useful medicines and toxins. Depending on their source, toxins can be categorized as phytotoxins, mycotoxins, or zootoxins, which include venoms and bacterial toxins. Any toxin can be harmful or beneficial. Within the last 100 years, the perception of botulinum neurotoxin (BTX) has evolved from that of a poison to a versatile clinical agent with various uses. BTX plays a key role in the management of many orofacial and dental disorders. Its indications are rapidly expanding, with ongoing trials for further applications. However, despite its clinical use, what BTX specifically does in each condition is still not clear. The main aim of this review is to describe some of the unclear aspects of this potentially useful agent, with a focus on the current research in dentistry. PMID:27486290

  18. Both, toxin A and toxin B, are important in Clostridium difficile infection

    Kuehne, Sarah A; Cartman, Stephen T; Minton, Nigel P.

    2011-01-01

    The bacterium Clostridium difficile is the leading cause of healthcare associated diarrhoea in the developed world and thus presents a major financial burden. The main virulence factors of C. difficile are two large toxins, A and B. Over the years there has been some debate over the respective roles and importance of these two toxins. To address this, we recently constructed stable toxin mutants of C. difficile and found that they were virulent if either toxin A or toxin B was functional. Thi...

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

    Chen Yun-Ru

    2012-09-01

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

  20. Shigella Sonnei and Shiga Toxin

    2016-07-28

    Katherine Lamba, an infectious disease epidemiologist with the California Department of Public Health, discusses Shiga Toxin producing Shigella sonnei.  Created: 7/28/2016 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 7/28/2016.

  1. Risk Assessment of Shellfish Toxins

    Rex Munday

    2013-11-01

    Full Text Available Complex secondary metabolites, some of which are highly toxic to mammals, are produced by many marine organisms. Some of these organisms are important food sources for marine animals and, when ingested, the toxins that they produce may be absorbed and stored in the tissues of the predators, which then become toxic to animals higher up the food chain. This is a particular problem with shellfish, and many cases of poisoning are reported in shellfish consumers each year. At present, there is no practicable means of preventing uptake of the toxins by shellfish or of removing them after harvesting. Assessment of the risk posed by such toxins is therefore required in order to determine levels that are unlikely to cause adverse effects in humans and to permit the establishment of regulatory limits in shellfish for human consumption. In the present review, the basic principles of risk assessment are described, and the progress made toward robust risk assessment of seafood toxins is discussed. While good progress has been made, it is clear that further toxicological studies are required before this goal is fully achieved.

  2. Food irradiation and bacterial toxins

    The authors' findings indicate that irradiation confers no advantage over heat processing in respect of bacterial toxins (clostridium botulinum, neurotoxin A and staphylococcal enterotoxin A). It follows that irradiation at doses less than the ACINF recommended upper limit of 10 kGy could not be used to improve the ambient temperature shelf life on non-acid foods. (author)

  3. hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: Structural and functional studies

    Drakou, Christina E.; Malekkou, Anna; Hayes, Joseph M.; Carsten W Lederer; Leonidas, Demetres D.; Oikonomakos, Nikos G.; Lamond, Angus I.; Santama, Niovi; Zographos, Spyros E.

    2012-01-01

    Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. D...

  4. The RNA Chaperone Hfq Is Required for Virulence of Bordetella pertussis

    Bíbová, Ilona; Škopová, Karolína; Mašín, Jiří; Černý, Ondřej; Hot, D.; Šebo, Peter; Večerek, Branislav

    2013-01-01

    Roč. 81, č. 11 (2013), s. 4081-4090. ISSN 0019-9567 R&D Projects: GA ČR(CZ) GAP302/11/1940; GA ČR GAP302/12/0460 Institutional support: RVO:61388971 Keywords : ADENYLATE-CYCLASE TOXIN * ISLET-ACTIVATING PROTEIN * ESCHERICHIA-COLI HFQ Subject RIV: EC - Immunology Impact factor: 4.156, year: 2013

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

    Katarzyna Kirsz; Dorota A. Zięba

    2012-01-01

     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 inhib...

  6. Down-regulation of adenylate kinase 5 in temporal lobe epilepsy patients and rat model.

    Lai, Yujie; Hu, Xiaotong; Chen, Guojun; Wang, Xuefeng; Zhu, Binglin

    2016-07-15

    Adenylate kinase 5 (AK5) is one member of the AK family and plays a critical role in maintaining cellular homeostasis. Different from the other AKs, AK5 is almost exclusively expressed in the brain. However, its exact biological functions remain unclear. The aim of the present study is to explore the expression pattern of AK5 in patients with refractory epilepsy and in a chronic pilocarpine-induced epileptic rat model. Using Western blot, immunofluorescence and immunoprecipitation analysis, we found that AK5 protein was mainly expressed in neurons, demonstrated by colocalization with the dendritic marker, MAP2, which were similar to the corresponding controls. However, the expression of AK5 decreased remarkably in epileptic patients and experimental rats. Furthermore, immunoprecipitation analysis showed that the interaction of AK5 with copine VI (CPNE6, a brain specific protein) increased in epileptic patients and rat models. Our results are the first to indicate that the expression of AK5 in epileptic brain tissue may play important roles in epilepsy, especially refractory epilepsy. PMID:27288770

  7. Phylogenetic relationships of 18 passerines based on Adenylate Kinase Intron 5 sequences

    GUO Hui-yan; YU Hui-xin; BAI Su-ying; MA Yu-kun

    2008-01-01

    The 18 species of bird studied originally are known to belong to muscicapids, robins and sylviids of passerines, but some disputations are always present in their classification systems. In this experiment, phylogenetic relationships of 18 species of passerines were studied using Adenylate Kinase Intron 5 (AK5) sequences and DNA techniques. Through sequences analysis in comparison with each other, phylogenetic tree figures of 18 species of passerines were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods . The results showed that sylviids should be listed as an independent family, while robins and flycatchers should be listed into Muscicapidae. Since the phylogenetic relationships between long-tailed tits and old world warblers are closer than that between long-tailed tits and parids, the long-tailed tits should be independent of paridae and be categorized into aegithalidae. Muscicapidae and Paridae are known to be two monophylitic families, but Sylviidae is not a monophyletic group. AK5 sequences had better efficacy in resolving close relationships of interspecies among intrageneric groups.

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

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

    2016-02-01

    Quantifying the physiological stress response of chondrichthyans to capture has assisted the development of fishing practices conducive to their survival. However, currently used indicators of stress show significant interspecific and intraspecific variation in species' physiological responses and tolerances to capture. To improve our understanding of chondrichthyan stress physiology and potentially reduce variation when quantifying the stress response, we investigated the use of the adenylate energy charge (AEC); a measure of available metabolic energy. To determine tissues sensitive to metabolic stress, we extracted samples of the brain, heart, liver, white muscle and blood from gummy sharks (Mustelus antarcticus) immediately following gillnet capture and after 3 h recovery under laboratory conditions. Capture caused significant declines in liver, white muscle and blood AEC, whereas no decline was detected in the heart and brain AEC. Following 3 h of recovery from capture, the AEC of the liver and blood returned to "unstressed" levels (control values) whereas white muscle AEC was not significantly different to that immediately after capture. Our results show that the liver is most sensitive to metabolic stress and white muscle offers a practical method to sample animals non-lethally for determination of the AEC. The AEC is a highly informative indicator of stress and unlike current indicators, it can directly measure the change in available energy and thus the metabolic stress experienced by a given tissue. Cellular metabolism is highly conserved across organisms and, therefore, we think the AEC can also provide a standardised form of measuring capture stress in many chondrichthyan species. PMID:26660290

  9. Fluorescence and NMR investigations in the ligand binding properties of adenylate kinases

    A new system for measurement of affinities of adenylate kinases (AK) for substrates and inhibitors is presented. This system is based on the use of the fluorescent ligand α,ω-di[(3' or 2')-O-(N-methyl-anthraniloyl)adenosine-5'] pentaphosphate (MAP5Am), which is an analogue of the bisubstrate inhibitor diadenosine pentaphosphate (AP5A). It allows the determination of dissociation constants for any ligand in the range of 1 x 10-9 to 5 x 10-2 M. Affinities for different bisubstrate inhibitors (AP4A, AP5A, AP6A) and substrates (AMP, ADP, ATP, GTP) were determined in the presence and absence of magnesium. An analysis of the binding of bisubstrate inhibitors is proposed and applied to these data. Temperature denaturation experiments indicate that the mutant enzyme has the same thermal stability as the wild-type enzyme and, as NMR studies indicate, also a very similar structure. Together with the results obtained by Tian et al on the effect of replacement of the conserved His-36 in the cytosolic AK (AK1) from chicken by glutamine and asparagine, this shows that residues 28 of AK from E. coli (AKec) and 36 of AK1 are situated in a comparable environment and are not essential for catalytic activity

  10. Anthrax toxin receptor 2-dependent lethal toxin killing in vivo.

    Heather M Scobie

    2006-10-01

    Full Text Available Anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2 have a related integrin-like inserted (I domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA subunit of anthrax toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by anthrax lethal toxin, providing strong evidence for the physiological importance of ANTXR2 in anthrax disease pathogenesis.

  11. An adenylyl cyclase signalling cascade in rat liver after prenatal gamma-irradiation (6,5 Gy)

    The adenylyl cyclase signalling cascade was tested in the rat liver of males subjected to the following 0.5 Gy prenatal gamma-irradiation: chronic irradiation during the whole time of embryonic development, single dose irradiation at the 9. day, and at the 15. day of embryogenesis. It was found that the prenatal irradiation resulted in the increase of basal and GTP-simulated adenylyl cyclase activity in all three groups of rats. In contrast, the adenylyl cyclase response to glucagon was essentially reduced. These effects were more pronounced in the chronic irradiated animals. From dose-response curves obtained for adenylyl cyclase activators. Mg==, and ATP we concluded that observed changes in glucagon signalling after single dose and chronic irradiation can be caused by alterations in the Gs protein regulatory cycle. (authors)

  12. Immunological cross-reactivity in the absence of DNA homology between Pseudomonas toxin A and diphtheria toxin.

    Sadoff, J C; Buck, G A; Iglewski, B H; Bjorn, M J; Groman, N B

    1982-01-01

    The immunodominant determinant of Pseudomonas toxin A was shown to cross-react with a normally inaccessible determinant in fragment A of diphtheria toxin. Trypsin-treated diphtheria toxin and fragment A of diphtheria toxin inhibited binding of toxin A antibody to whole toxin A, whereas whole diphtheria toxin did not inhibit this reaction. However, even at the lowest stringency no hybridization was detected between diphtheria tox probe and Pseudomonas aeruginosa DNA.

  13. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method.

    J Field; Nikawa, J; Broek, D; MacDonald, B.; Rodgers, L; Wilson, I A; Lerner, R A; Wigler, M

    1988-01-01

    We developed a method for immunoaffinity purification of Saccharomyces cerevisiae adenylyl cyclase based on creating a fusion with a small peptide epitope. Using oligonucleotide technology to encode the peptide epitope we constructed a plasmid that expressed the fusion protein from the S. cerevisiae alcohol dehydrogenase promoter ADH1. A monoclonal antibody previously raised against the peptide was used to purify adenylyl cyclase by affinity chromatography. The purified enzyme appeared to be ...

  14. Why do we study animal toxins?

    Zhang, Yun

    2015-07-18

    Venom (toxins) is an important trait evolved along the evolutionary tree of animals. Our knowledges on venoms, such as their origins and loss, the biological relevance and the coevolutionary patterns with other organisms are greatly helpful in understanding many fundamental biological questions, i.e., the environmental adaptation and survival competition, the evolution shaped development and balance of venoms, and the sophisticated correlations among venom, immunity, body power, intelligence, their genetic basis, inherent association, as well as the cost-benefit and trade-offs of biological economy. Lethal animal envenomation can be found worldwide. However, from foe to friend, toxin studies have led lots of important discoveries and exciting avenues in deciphering and fighting human diseases, including the works awarded the Nobel Prize and lots of key clinic therapeutics. According to our survey, so far, only less than 0.1% of the toxins of the venomous animals in China have been explored. We emphasize on the similarities shared by venom and immune systems, as well as the studies of toxin knowledge-based physiological toxin-like proteins/peptides (TLPs). We propose the natural pairing hypothesis. Evolution links toxins with humans. Our mission is to find out the right natural pairings and interactions of our body elements with toxins, and with endogenous toxin-like molecules. Although, in nature, toxins may endanger human lives, but from a philosophical point of view, knowing them well is an effective way to better understand ourselves. So, this is why we study toxins. PMID:26228472

  15. Exfoliative Toxins of Staphylococcus aureus

    Michal Bukowski

    2010-05-01

    Full Text Available Staphylococcus aureus is an important pathogen of humans and livestock. It causes a diverse array of diseases, ranging from relatively harmless localized skin infections to life-threatening systemic conditions. Among multiple virulence factors, staphylococci secrete several exotoxins directly associated with particular disease symptoms. These include toxic shock syndrome toxin 1 (TSST-1, enterotoxins, and exfoliative toxins (ETs. The latter are particularly interesting as the sole agents responsible for staphylococcal scalded skin syndrome (SSSS, a disease predominantly affecting infants and characterized by the loss of superficial skin layers, dehydration, and secondary infections. The molecular basis of the clinical symptoms of SSSS is well understood. ETs are serine proteases with high substrate specificity, which selectively recognize and hydrolyze desmosomal proteins in the skin. The fascinating road leading to the discovery of ETs as the agents responsible for SSSS and the characterization of the molecular mechanism of their action, including recent advances in the field, are reviewed in this article.

  16. Botulinum toxin: Bioweapon & magic drug

    Dhaked, Ram Kumar; Singh, Manglesh Kumar; Singh, Padma; Gupta, Pallavi

    2010-01-01

    Botulinum neurotoxins, causative agents of botulism in humans, are produced by Clostridium botulinum, an anaerobic spore-former Gram positive bacillus. Botulinum neurotoxin poses a major bioweapon threat because of its extreme potency and lethality; its ease of production, transport, and misuse; and the need for prolonged intensive care among affected persons. A single gram of crystalline toxin, evenly dispersed and inhaled, can kill more than one million people. The basis of the phenomenal p...

  17. Botulinum Toxin in Pediatric Neurology

    Moawad, Eman M. I.; Abdallah, Enas Abdallah Ali

    2015-01-01

    Botulinum neurotoxins are natural molecules produced by anaerobic spore-forming bacteria called Clostradium boltulinum. The toxin has a peculiar mechanism of action by preventing the release of acetylcholine from the presynaptic membrane. Consequently, it has been used in the treatment of various neurological conditions related to muscle hyperactivity and/or spasticity. Also, it has an impact on the autonomic nervous system by acting on smooth muscle, leading to its use in the management of p...

  18. Negative regulation of adenylate cyclase gene (cya) expression by cyclic AMP-cyclic AMP receptor protein in Escherichia coli: studies with cya-lac protein and operon fusion plasmids.

    Kawamukai, M; Kishimoto, J; Utsumi, R; Himeno, M; Komano, T; Aiba, H

    1985-01-01

    We constructed cya-lac protein and operon fusion plasmids in vitro. The effect of cyclic AMP (cAMP) on cya expression was examined by measuring the synthesis of beta-galactosidase in Escherichia coli cells containing fused plasmids. In the cya-lacZ fused protein system, cya expression was strongly repressed by exogenous cAMP. Functional cAMP receptor protein (CRP) was necessary for this effect. On the other hand, in a tet-lacZ fused protein as a control system, tet expression was not affected...

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

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

    2006-01-01

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

  20. Roles for pituitary adenylate cyclase-activating peptide (PACAP) expression and signaling in the bed nucleus of the stria terminalis (BNST) in mediating the behavioral consequences of chronic stress

    Hammack, Sayamwong E.; Roman, Carolyn W.; Lezak, Kimberly R.; Kocho-Shellenberg, Margaret; Grimmig, Bethany; Falls, William A; Braas, Karen; May, Victor

    2010-01-01

    Anxiety disorders are frequently long-lasting and debilitating for more than 40 million American adults. Although stressor exposure plays an important role in the etiology of some anxiety disorders, the mechanisms by which exposure to stressful stimuli alters central circuits that mediate anxiety-like emotional behavior are still unknown. Substantial evidence has implicated regions of the central extended amygdala, including the bed nucleus of the stria terminalis (BNST) and the central nucle...

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

    Markus Otto; Christin Naumann; Wolfgang Brandt; Claus Wasternack; Bettina Hause

    2016-01-01

    Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enz...

  2. An improved technique for the rapid chemical characterisation of bacterial terpene cyclases.

    Dickschat, Jeroen S; Pahirulzaman, Khomaizon A K; Rabe, Patrick; Klapschinski, Tim A

    2014-04-14

    A derivative of the pET28c(+) expression vector was constructed. It contains a yeast replication system (2μ origin of replication) and a yeast selectable marker (URA3), and can be used for gene cloning in yeast by efficient homologous recombination, and for heterologous expression in E. coli. The vector was used for the expression and chemical characterisation of three bacterial terpene cyclases. PMID:24573945

  3. A Comparative Analysis of the Sugar Phosphate Cyclase Superfamily Involved in Primary and Secondary Metabolism

    Wu, Xiumei; Flatt, Patricia M.; Schlörke, Oliver; Zeeck, Axel; Dairi, Tohru; Mahmud, Taifo

    2007-01-01

    Sugar Phosphate Cyclases (SPCs) catalyze the cyclization of sugar phosphates to produce a variety of cyclitol intermediates that serve as the building blocks of many primary metabolites, e.g., aromatic amino acids, and clinically relevant secondary metabolites, e.g., aminocyclitol/aminoglycoside and ansamycin antibiotics. Feeding experiments with isotopically-labeled cyclitols revealed that cetoniacytone A, a unique C7N-aminocyclitol antibiotic isolated from an insect endophytic Actinomyces s...

  4. Stimulation of soluble guanylyl cyclase protects against obesity by recruiting brown adipose tissue

    Hoffmann, Linda S.; Etzrodt, Jennifer; Willkomm, Lena; Sanyal, Abhishek; Scheja, Ludger; Fischer, Alexander W.C.; Stasch, Johannes-Peter; Bloch, Wilhelm; Friebe, Andreas; Heeren, Joerg; Pfeifer, Alexander

    2015-01-01

    Obesity is characterized by a positive energy balance and expansion of white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) combusts energy to produce heat. Here we show that a small molecule stimulator (BAY 41-8543) of soluble guanylyl cyclase (sGC), which produces the second messenger cyclic GMP (cGMP), protects against diet-induced weight gain, induces weight loss in established obesity, and also improves the diabetic phenotype. Mechanistically, the haeme-dependent sGC stimu...

  5. Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo

    Kristen L Lee; Hoey, David A.; Spasic, Milos; Tang, Tong; Hammond, H. Kirk; Jacobs, Christopher R.

    2014-01-01

    Primary cilia are single, nonmotile, antenna-like structures extending from the apical membrane of most mammalian cells. They may mediate mechanotransduction, the conversion of external mechanical stimuli into biochemical intracellular signals. Previously we demonstrated that adenylyl cyclase 6 (AC6), a membrane-bound enzyme enriched in primary cilia of MLO-Y4 osteocyte-like cells, may play a role in a primary cilium-dependent mechanism of osteocyte mechanotransduction in vitro. In this study...

  6. Elevation of lutein content in tomato: a biochemical tug-of-war between lycopene cyclases.

    Giorio, Giovanni; Yildirim, Arzu; Stigliani, Adriana Lucia; D'Ambrosio, Caterina

    2013-11-01

    Lutein is becoming increasingly important in preventive medicine due to its possible role in maintaining good vision and in preventing age-related maculopathy. Average daily lutein intake in developed countries is often below suggested daily consumption levels, and lutein supplementation could be beneficial. Lutein is also valuable in the food and feed industries and is emerging in nutraceutical and pharmaceutical markets. Currently, lutein is obtained at high cost from marigold petals, and synthesis alternatives are thus desirable. Tomato constitutes a promising starting system for production as it naturally accumulates high levels of lycopene. To develop tomato for lutein synthesis, the tomato Red Setter cultivar was transformed with the tomato lycopene ε-cyclase-encoding gene under the control of a constitutive promoter, and the HighDelta (HD) line, characterised by elevated lutein and δ-carotene content in ripe fruits, was selected. HD was crossed to the transgenic HC line and to RS(B) with the aim of converting all residual fruit δ-carotene to lutein. Fruits of both crosses were enriched in lutein and presented unusual carotenoid profiles. The unique genetic background of the crosses used in this study permitted an unprecedented analysis of the role and regulation of the lycopene cyclase enzymes in tomato. A new defined biochemical index, the relative cyclase activity ratio, was used to discern post-transcriptional regulation of cyclases, and will help in the study of carotenoid biosynthesis in photosynthetic plant species and particularly in those, like tomato, that have been domesticated for the production of food, feed or useful by-products. PMID:24141052

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

    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

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

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

    2012-01-01

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

  9. Identifying functional domains within terpene cyclases using a domain-swapping strategy.

    Back, K; Chappell, J.

    1996-01-01

    Cyclic terpenes and terpenoids are found throughout nature. They comprise an especially important class of compounds from plants that mediate plant- environment interactions, and they serve as pharmaceutical agents with antimicrobial and anti-tumor activities. Molecular comparisons of several terpene cyclases, the key enzymes responsible for the multistep cyclization of C10, C15, and C20 allylic diphosphate substrates, have revealed a striking level of sequence similarity and conservation of ...

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

    The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution. In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å

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

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

    2005-01-01

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

  12. [Use of botulinum toxin in strabismus].

    Wabbels, B

    2016-07-01

    Botulinum toxin can be a useful tool for treating acute sixth nerve palsy and excessive eye deviations due to unstable Graves' disease, when surgery is not yet possible. The diagnostic injection for estimation of possible postoperative double vision also makes sense. In convergence spasms, periocular botulinum toxin injections can be a therapeutic option. Botulinum toxin is not a first line option in infantile esotropia without binocularity or in adult horizontal strabismus. Side effects include ptosis and vertical deviations. PMID:27369733

  13. Quantitative microtiter cytotoxicity assay for Shigella toxin.

    Gentry, M. K.; Dalrymple, J M

    1980-01-01

    The cytotoxic activity of Shigella dysenteriae 1 was assayed by exposing HeLa cells in microtiter cultures to dilutions of toxin. Exposure to toxin caused either failure of cells in suspension to attach or detachment of cells from established monolayers. Estimates of toxin potency were made by staining residual cells with crystal violet and visually inspecting the stained plates. Quantitation of the cytotoxic effect was made possible by eluting and spectrophotometrically measuring the stain. ...

  14. Application of Botulinum Toxin in Pain Management

    Sim, Woo Seog

    2011-01-01

    Botulinum toxin has been used for the treatment of many clinical disorders by producing temporary skeletal muscle relaxation. In pain management, botulinum toxin has demonstrated an analgesic effect by reducing muscular hyperactivity, but recent studies suggest this neurotoxin could have direct analgesic mechanisms different from its neuromuscular actions. At the moment, botulinum toxin is widely investigated and used in many painful diseases such as myofascial syndrome, headaches, arthritis,...

  15. Cholera toxin-like toxin released by Salmonella species in the presence of mitomycin C.

    Molina, N C; Peterson, J W

    1980-01-01

    Several serotypes of Salmonella were shown to release increased amounts of a cholera toxin-like toxin during culture in vitro with mitomycin C (MTC). Filter-sterilized culture supernatants containing the toxin caused elongation of Chinese hamster ovary cells, which could be blocked by heating the supernatants at 100 degrees C for 15 min or by adding mixed gangliosides or monospecific cholera antitoxin. When MTC was not added to the Salmonella cultures, little or no toxin was detected in crude...

  16. Synthesis and Biology of Cyclic Imine Toxins, An Emerging Class of Potent, Globally Distributed Marine Toxins

    Stivala, Craig E.; Benoit, Evelyne; Araoz, Romulo; Servent, Denis; Novikov, Alexei; Molgó, Jordi; Zakarian, Armen

    2015-01-01

    From a small group of exotic compounds isolated only two decades ago, Cyclic Imine (CI) toxins have become a major class of marine toxins with global distribution. Their distinct chemical structure, biological mechanism of action, and intricate chemistry ensures that CI toxins will continue to be the subject of fascinating fundamental studies in the broad fields of chemistry, chemical biology, and toxicology. The worldwide occurrence of potent CI toxins in marine environments, their accumulat...

  17. Protein Translocation by Bacterial Toxin Channels: A Comparison of Diphtheria Toxin and Colicin Ia

    Wu, Zhengyan; Jakes, Karen S.; Samelson-Jones, Ben S.; Lai, Bing; Zhao, Gang; London, Erwin; Finkelstein, Alan

    2006-01-01

    Regions of both colicin Ia and diphtheria toxin N-terminal to the channel-forming domains can be translocated across planar phospholipid bilayer membranes. In this article we show that the translocation pathway of diphtheria toxin allows much larger molecules to be translocated than does the translocation pathway of colicin Ia. In particular, the folded A chain of diphtheria toxin is readily translocated by that toxin but is not translocated by colicin Ia. This difference cannot be attributed...

  18. Botulinum Toxin; Bioterror and Biomedicinal Agent

    Jiri Patocka

    2006-04-01

    Full Text Available Botulinum toxin is a group of seven homologous, highly poisonous proteins isolated fromfermentation of the anaerobic bacterium Clostridium botulinum, which naturally occurs in soiland can grow on many meats and vegetables. Botulinum toxin causes neuromuscular disordercalled botulism, which is a potentially lethal disease. There are three types of botulism: Food,wound, and infant botulism. It can lead to death unless appropriate therapy is done. Due to theseverity and potency of botulinum toxin, its importance as a biological weapon is of majorconcern to public health officials. Nevertheless, botulinum toxin is also medicament.

  19. Shiga Toxin (Stx) Classification, Structure, and Function

    Melton-Celsa, Angela R.

    2014-01-01

    Shiga toxin (Stx) is one of the most potent bacterial toxins known. Stx is found in Shigella dysenteriae 1 and in some serogroups of Escherichia coli (called Stx1 in E. coli). In addition to or instead of Stx1, some E. coli strains produce a second type of Stx, Stx2, that has the same mode of action as Stx/Stx1 but that is antigenically distinct. Because subtypes of each toxin have been identified, the prototype toxin for each group is now designated Stx1a or Stx2a. The Stxs consist of two ma...

  20. Alignment-Free Methods for the Detection and Specificity Prediction of Adenylation Domains.

    Agüero-Chapin, Guillermin; Pérez-Machado, Gisselle; Sánchez-Rodríguez, Aminael; Santos, Miguel Machado; Antunes, Agostinho

    2016-01-01

    Identifying adenylation domains (A-domains) and their substrate specificity can aid the detection of nonribosomal peptide synthetases (NRPS) at genome/proteome level and allow inferring the structure of oligopeptides with relevant biological activities. However, that is challenging task due to the high sequence diversity of A-domains (~10-40 % of amino acid identity) and their selectivity for 50 different natural/unnatural amino acids. Altogether these characteristics make their detection and the prediction of their substrate specificity a real challenge when using traditional sequence alignment methods, e.g., BLAST searches. In this chapter we describe two workflows based on alignment-free methods intended for the identification and substrate specificity prediction of A-domains. To identify A-domains we introduce a graphical-numerical method, implemented in TI2BioP version 2.0 (topological indices to biopolymers), which in a first step uses protein four-color maps to represent A-domains. In a second step, simple topological indices (TIs), called spectral moments, are derived from the graphical representations of known A-domains (positive dataset) and of unrelated but well-characterized sequences (negative set). Spectral moments are then used as input predictors for statistical classification techniques to build alignment-free models. Finally, the resulting alignment-free models can be used to explore entire proteomes for unannotated A-domains. In addition, this graphical-numerical methodology works as a sequence-search method that can be ensemble with homology-based tools to deeply explore the A-domain signature and cope with the diversity of this class (Aguero-Chapin et al., PLoS One 8(7):e65926, 2013). The second workflow for the prediction of A-domain's substrate specificity is based on alignment-free models constructed by transductive support vector machines (TSVMs) that incorporate information of uncharacterized A-domains. The construction of the models was

  1. Characterization of Toxin Plasmids in Clostridium perfringens Type C Isolates▿

    Gurjar, Abhijit; Li, Jihong; McClane, Bruce A.

    2010-01-01

    Clostridium perfringens type C isolates cause enteritis necroticans in humans or necrotizing enteritis and enterotoxemia in domestic animals. Type C isolates always produce alpha toxin and beta toxin but often produce additional toxins, e.g., beta2 toxin or enterotoxin. Since plasmid carriage of toxin-encoding genes has not been systematically investigated for type C isolates, the current study used Southern blot hybridization of pulsed-field gels to test whether several toxin genes are plasm...

  2. Botulinum toxin: The Midas touch.

    Shilpa, P S; Kaul, Rachna; Sultana, Nishat; Bhat, Suraksha

    2014-01-01

    Botulinum Toxin (BT) is a natural molecule produced during growth and autolysis of bacterium called Clostridium botulinum. Use of BT for cosmetic purposes has gained popularity over past two decades, and recently, other therapeutic uses of BT has been extensively studied. BT is considered as a minimally invasive agent that can be used in the treatment of various orofacial disorders and improving the quality of life in such patients. The objective of this article is to review the nature, mechanism of action of BT, and its application in various head and neck diseases. PMID:24678189

  3. Clostridial Glucosylating Toxins Enter Cells via Clathrin-Mediated Endocytosis

    Papatheodorou, Panagiotis; Zamboglou, Constantinos; Genisyuerek, Selda; Guttenberg, Gregor; Aktories, Klaus

    2010-01-01

    Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi α-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and α-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an ...

  4. Crystal structures at 2.5 Angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate

    Belrhali, H.; Yaremchuk, A.; Tukalo, M.; Larsen, K.; Berthet-Colominas, C.; Leberman, R.; Beijer, B.; Sproat, B.; Als-Nielsen, J.; Grübel, G.; Legrand, J.-F.; Lehmann, M.; Cusack, S.

    1994-01-01

    crystal from adenosine triphosphate (ATP) and serine hydroxamate, and the second is with a synthetic analog of seryl adenylate (5'-O-[N-(L-seryl)-sulfamoyl]adenosine), which is a strong inhibitor of the enzyme. Both molecules are bound in a similar fashion by a network of hydrogen bond interactions in a......Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 Angstrom resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the...

  5. Plant Insecticidal Toxins in Ecological Networks

    Sébastien Ibanez

    2012-04-01

    Full Text Available Plant secondary metabolites play a key role in plant-insect interactions, whether constitutive or induced, C- or N-based. Anti-herbivore defences against insects can act as repellents, deterrents, growth inhibitors or cause direct mortality. In turn, insects have evolved a variety of strategies to act against plant toxins, e.g., avoidance, excretion, sequestration and degradation of the toxin, eventually leading to a co-evolutionary arms race between insects and plants and to co-diversification. Anti-herbivore defences also negatively impact mutualistic partners, possibly leading to an ecological cost of toxin production. However, in other cases toxins can also be used by plants involved in mutualistic interactions to exclude inadequate partners and to modify the cost/benefit ratio of mutualism to their advantage. When considering the whole community, toxins have an effect at many trophic levels. Aposematic insects sequester toxins to defend themselves against predators. Depending on the ecological context, toxins can either increase insects’ vulnerability to parasitoids and entomopathogens or protect them, eventually leading to self-medication. We conclude that studying the community-level impacts of plant toxins can provide new insights into the synthesis between community and evolutionary ecology.

  6. Toxin-Antitoxin Battle in Bacteria

    Cataudella, Ilaria

    This PhD thesis consists of three research projects revolving around the common thread of investigation of the properties and biological functions of Toxin-Antitoxin loci. Toxin-Antitoxin (TA) loci are transcriptionally regulated via an auto-inhibition mechanism called conditional cooperativity, ...

  7. Target-Driven Evolution of Scorpion Toxins.

    Zhang, Shangfei; Gao, Bin; Zhu, Shunyi

    2015-01-01

    It is long known that peptide neurotoxins derived from a diversity of venomous animals evolve by positive selection following gene duplication, yet a force that drives their adaptive evolution remains a mystery. By using maximum-likelihood models of codon substitution, we analyzed molecular adaptation in scorpion sodium channel toxins from a specific species and found ten positively selected sites, six of which are located at the core-domain of scorpion α-toxins, a region known to interact with two adjacent loops in the voltage-sensor domain (DIV) of sodium channels, as validated by our newly constructed computational model of toxin-channel complex. Despite the lack of positive selection signals in these two loops, they accumulated extensive sequence variations by relaxed purifying selection in prey and predators of scorpions. The evolutionary variability in the toxin-bound regions of sodium channels indicates that accelerated substitutions in the multigene family of scorpion toxins is a consequence of dealing with the target diversity. This work presents an example of atypical co-evolution between animal toxins and their molecular targets, in which toxins suffered from more prominent selective pressure from the channels of their competitors. Our discovery helps explain the evolutionary rationality of gene duplication of toxins in a specific venomous species. PMID:26444071

  8. The Ins and Outs of Anthrax Toxin.

    Friebe, Sarah; van der Goot, F Gisou; Bürgi, Jérôme

    2016-03-01

    Anthrax is a severe, although rather rare, infectious disease that is caused by the Gram-positive, spore-forming bacterium Bacillus anthracis. The infectious form is the spore and the major virulence factors of the bacterium are its poly-γ-D-glutamic acid capsule and the tripartite anthrax toxin. The discovery of the anthrax toxin receptors in the early 2000s has allowed in-depth studies on the mechanisms of anthrax toxin cellular entry and translocation from the endocytic compartment to the cytoplasm. The toxin generally hijacks the endocytic pathway of CMG2 and TEM8, the two anthrax toxin receptors, in order to reach the endosomes. From there, the pore-forming subunit of the toxin inserts into endosomal membranes and enables translocation of the two catalytic subunits. Insertion of the pore-forming unit preferentially occurs in intraluminal vesicles rather than the limiting membrane of the endosome, leading to the translocation of the enzymatic subunits in the lumen of these vesicles. This has important consequences that will be discussed. Ultimately, the toxins reach the cytosol where they act on their respective targets. Target modification has severe consequences on cell behavior, in particular on cells of the immune system, allowing the spread of the bacterium, in severe cases leading to host death. Here we will review the literature on anthrax disease with a focus on the structure of the toxin, how it enters cells and its immunological effects. PMID:26978402

  9. Stealth and mimicry by deadly bacterial toxins

    Yates, S.P.; Jørgensen, Rene; Andersen, Gregers Rom;

    2006-01-01

    Diphtheria toxin and exotoxin A are well-characterized members of the ADP-ribosyltransferase toxin family that serve as virulence factors in the pathogenic bacteria, Corynebacterium diphtheriae and Pseudomonas aeruginosa.  New high-resolution structural data of the Michaelis complex of the...

  10. Brown spider dermonecrotic toxin directly induces nephrotoxicity

    Brown spider (Loxosceles genus) venom can induce dermonecrotic lesions at the bite site and systemic manifestations including fever, vomiting, convulsions, disseminated intravascular coagulation, hemolytic anemia and acute renal failure. The venom is composed of a mixture of proteins with several molecules biochemically and biologically well characterized. The mechanism by which the venom induces renal damage is unknown. By using mice exposed to Loxosceles intermedia recombinant dermonecrotic toxin (LiRecDT), we showed direct induction of renal injuries. Microscopic analysis of renal biopsies from dermonecrotic toxin-treated mice showed histological alterations including glomerular edema and tubular necrosis. Hyalinization of tubules with deposition of proteinaceous material in the tubule lumen, tubule epithelial cell vacuoles, tubular edema and epithelial cell lysis was also observed. Leukocytic infiltration was neither observed in the glomerulus nor the tubules. Renal vessels showed no sign of inflammatory response. Additionally, biochemical analyses showed such toxin-induced changes in renal function as urine alkalinization, hematuria and azotemia with elevation of blood urea nitrogen levels. Immunofluorescence with dermonecrotic toxin antibodies and confocal microscopy analysis showed deposition and direct binding of this toxin to renal intrinsic structures. By immunoblotting with a hyperimmune dermonecrotic toxin antiserum on renal lysates from toxin-treated mice, we detected a positive signal at the region of 33-35 kDa, which strengthens the idea that renal failure is directly induced by dermonecrotic toxin. Immunofluorescence reaction with dermonecrotic toxin antibodies revealed deposition and binding of this toxin directly in MDCK epithelial cells in culture. Similarly, dermonecrotic toxin treatment caused morphological alterations of MDCK cells including cytoplasmic vacuoles, blebs, evoked impaired spreading and detached cells from each other and from

  11. The toxin component of targeted anti-tumor toxins determines their efficacy increase by saponins.

    Weng, Alexander; Thakur, Mayank; Beceren-Braun, Figen; Bachran, Diana; Bachran, Christopher; Riese, Sebastian B; Jenett-Siems, Kristina; Gilabert-Oriol, Roger; Melzig, Matthias F; Fuchs, Hendrik

    2012-06-01

    Tumor-targeting protein toxins are composed of a toxic enzyme coupled to a specific cell binding domain that targets cancer-associated antigens. The anti-tumor treatment by targeted toxins is accompanied by dose-limiting side effects. The future prospects of targeted toxins for therapeutic use in humans will be determined by reduce side effects. Certain plant secondary metabolites (saponins) were shown to increase the efficacy of a particular epidermal growth factor receptor (EGFR)-targeted toxin, paralleled by a tremendous decrease of side effects. This study was conducted in order to investigate the effects of substituting different toxin moieties fused to an EGF ligand binding domain on the augmentative ability of saponins for each against therapeutic potential of the saponin-mediated efficacy increase for different anti-tumor toxins targeting the EGFR. We designed several EGFR-targeted toxins varying in the toxic moiety. Each targeted toxin was used in combination with a purified saponin (SA1641), isolated from the ornamental plant Gypsophila paniculata L. SA1641 was characterized and the SA1641-mediated efficacy increase was investigated on EGFR-transfected NIH-3T3 cells. We observed a high dependency of the SA1641-mediated efficacy increase on the nature of toxin used for the construction of the targeted toxin, indicating high specificity. Structural alignments revealed a high homology between saporin and dianthin-30, the two toxic moieties that benefit most from the combination with SA1641. We further demonstrate that SA1641 did not influence the plasma membrane permeability, indicating an intracellular interaction of SA1641 and the toxin components of targeted toxins. Surface plasmon resonance measurements point to a transient binding of SA1641 to the toxin components of targeted toxins. PMID:22309811

  12. Interplay between toxin transport and flotillin localization

    Pust, Sascha; Dyve, Anne Berit; Torgersen, Maria L; van Deurs, Bo; Sandvig, Kirsten

    2010-01-01

    for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells....... The toxicity of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport......The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we...

  13. Immunotoxins: The Role of the Toxin

    David FitzGerald

    2013-08-01

    Full Text Available Immunotoxins are antibody-toxin bifunctional molecules that rely on intracellular toxin action to kill target cells. Target specificity is determined via the binding attributes of the chosen antibody. Mostly, but not exclusively, immunotoxins are purpose-built to kill cancer cells as part of novel treatment approaches. Other applications for immunotoxins include immune regulation and the treatment of viral or parasitic diseases. Here we discuss the utility of protein toxins, of both bacterial and plant origin, joined to antibodies for targeting cancer cells. Finally, while clinical goals are focused on the development of novel cancer treatments, much has been learned about toxin action and intracellular pathways. Thus toxins are considered both medicines for treating human disease and probes of cellular function.

  14. Botulinum toxin in poststroke spasticity.

    Ozcakir, Suheda; Sivrioglu, Koncuy

    2007-06-01

    Poststroke hemiparesis, together with abnormal muscle tone, is a major cause of morbidity and disability. Although most hemiparetic patients are able to reach different ambulatory levels with rehabilitation efforts, upper and lower limb spasticity can impede activities of daily living, personal hygiene, ambulation and, in some cases, functional improvement. The goals of spasticity management include increasing mobility and range of motion, attaining better hygiene, improving splint wear and other functional activities. Conservative measures, such as positioning, stretching and exercise are essential in spasticity management, but alone often are inadequate to effectively control it. Oral antispastic medications often provide limited effects with short duration and frequent unwanted systemic side effects, such as weakness, sedation and dry mouth. Therefore, neuromuscular blockade by local injections have become the first choice for the treatment of focal spasticity, particularly in stroke patients. Botulinum toxin (BTX), being one of the most potent biological toxins, acts by blocking neuromuscular transmission via inhibiting acetylcholine release. Currently, focal spasticity is being treated successfully with BTX via injecting in the spastic muscles. Two antigenically distinct serotypes of BTX are available on the market as type A and B. Clinical studies of BTX used for spastic hemiplegic patients are reviewed in this article in two major categories, upper and lower limb applications. This review addresses efficacy in terms of outcome measures, such as muscle tone reduction and functional outcome, as well as safety issues. Application modifications of dose, dilutions, site of injections and combination therapies with BTX injections are also discussed. PMID:17607049

  15. Signal-transduction protein P(II) from Synechococcus elongatus PCC 7942 senses low adenylate energy charge in vitro.

    Fokina, Oleksandra; Herrmann, Christina; Forchhammer, Karl

    2011-11-15

    P(II) proteins belong to a family of highly conserved signal-transduction proteins that occurs widely in bacteria, archaea and plants. They respond to the central metabolites ATP, ADP and 2-OG (2-oxoglutarate), and control enzymes, transcription factors and transport proteins involved in nitrogen metabolism. In the present study, we examined the effect of ADP on in vitro P(II)-signalling properties for the cyanobacterium Synechococcus elongatus, a model for oxygenic phototrophic organisms. Different ADP/ATP ratios strongly affected the properties of P(II) signalling. Increasing ADP antagonized the binding of 2-OG and directly affected the interactions of P(II) with its target proteins. The resulting P(II)-signalling properties indicate that, in mixtures of ADP and ATP, P(II) trimers are occupied by mixtures of adenylate nucleotides. Binding and kinetic activation of NAGK (N-acetyl-L-glutamate kinase), the controlling enzyme of arginine biosynthesis, by P(II) was weakened by ADP, but relief from arginine inhibition remained unaffected. On the other hand, ADP enhanced the binding of P(II) to PipX, a co-activator of the transcription factor NtcA and, furthermore, antagonized the inhibitory effect of 2-OG on P(II)-PipX interaction. These results indicate that S. elongatus P(II) directly senses the adenylate energy charge, resulting in target-dependent differential modification of the P(II)-signalling properties. PMID:21774788

  16. Roles of Anthrax Toxin Receptor 2 in Anthrax Toxin Membrane Insertion and Pore Formation

    Jianjun Sun

    2016-01-01

    Full Text Available Interaction between bacterial toxins and cellular surface receptors is an important component of the host-pathogen interaction. Anthrax toxin protective antigen (PA binds to the cell surface receptor, enters the cell through receptor-mediated endocytosis, and forms a pore on the endosomal membrane that translocates toxin enzymes into the cytosol of the host cell. As the major receptor for anthrax toxin in vivo, anthrax toxin receptor 2 (ANTXR2 plays an essential role in anthrax toxin action by providing the toxin with a high-affinity binding anchor on the cell membrane and a path of entry into the host cell. ANTXR2 also acts as a molecular clamp by shifting the pH threshold of PA pore formation to a more acidic pH range, which prevents premature pore formation at neutral pH before the toxin reaches the designated intracellular location. Most recent studies have suggested that the disulfide bond in the immunoglobulin (Ig-like domain of ANTXR2 plays an essential role in anthrax toxin action. Here we will review the roles of ANTXR2 in anthrax toxin action, with an emphasis on newly updated knowledge.

  17. Chemically Induced Cryptic Sesquiterpenoids and Expression of Sesquiterpene Cyclases in Botrytis cinerea Revealed New Sporogenic (+)-4-Epieremophil-9-en-11-ols.

    Pinedo, Cristina; Moraga, Javier; Barua, Javier; González-Rodríguez, Victoria E; Aleu, Josefina; Durán-Patrón, Rosa; Macías-Sánchez, Antonio J; Hanson, James R; Viaud, Muriel; Hernández-Galán, Rosario; Garrido, Carlos; Collado, Isidro G

    2016-05-20

    The sequencing of the genomes of the B05.10 and T4 strains of the fungus Botrytis cinerea revealed an abundance of novel biosynthetic gene clusters, the majority of which were unexpected on the basis of the previous analyses of the fermentation of these and closely related species. By systematic alteration of easy accessible cultivation parameters, using chemical induction with copper sulfate, we have found a cryptic sesquiterpenoid family with new structures related to eremophil-9-ene, which had the basic structure of the sesquiterpene (+)-5-epiaristolochene ((+)-4-epieremophil-9-ene). An expression study of the sesquiterpene cyclase genes present in the Botrytis cinerea genome, under culture conditions, is reported. In general, a 3 day delay and a higher BcSTC genes expression were observed when copper (5 ppm) was fed to the fermentation broth. In addition, to the observed effect on the BcBOT2 (BcSTC1) gene, involved in the biosynthesis of the botrydial toxin, a higher expression level for BcSTC3 and BcSTC4 was observed with respect to the control in the strain B05.10. Interestingly, under copper conditions, the BcSTC4 gene was the most expressed gene in the Botrytis cinerea UCA992 strain. In vitro evaluation of the biological role of these metabolites indicates that they contributed to the conidial development in B. cinerea and appear to be involved in self-regulation of the production of asexual spores. Furthermore, they promoted the formation of complex appressoria or infection cushions. PMID:26900713

  18. Structure of RNA 3′-phosphate cyclase bound to substrate RNA

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

    2014-01-01

    RNA 3′-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3′-phosphate to form a 2′,3′-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA–AMP and RNA(3′)pp(5′)A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3′-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes s...

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

    Xiao-Hong eWen; Dizhoor, Alexander M.; Makino, Clint L.

    2014-01-01

    In vertebrate rods and cones, photon capture by rhodopsin leads to the destruction of cyclic GMP (cGMP) and the subsequent closure of cyclic nucleotide gated (CNG) ion channels in the outer segment plasma membrane. Replenishment of cGMP and reopening of the channels limit the growth of the photon response and are requisite for its recovery. In different vertebrate retinas, there may be as many as four types of membrane guanylyl cyclases (GCs) for cGMP synthesis. Ten neuronal Ca2+ sensor prote...

  20. Inhibition of glutaminyl cyclases for Alzheimer´s disease treatment

    Kolenko, Petr

    Praha: Katedra inženýrství pevných látek, Fakulta jaderná a fyzikálně inženýrská, ČVUT v Praze, 2014. s. 17. [Student Scientific Conference on Solid State Physics /4./. 23.06.2014-27.06.2014, Nové Hrady] R&D Projects: GA MŠk(CZ) EE2.3.30.0029 Institutional support: RVO:61389013 Keywords : glutaminyl cyclase * structure * inhibition Subject RIV: CE - Biochemistry

  1. Structure of the polyketide cyclase SnoaL reveals a novel mechanism for enzymatic aldol condensation

    Sultana, Azmiri; Kallio, Pauli; Jansson, Anna; Wang, Ji-Shu; Niemi, Jarmo; Mäntsälä, Pekka; Schneider, Gunter

    2004-01-01

    SnoaL belongs to a family of small polyketide cyclases, which catalyse ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. Several of these antibiotics are among the most used anti-cancer drugs currently in use. The crystal structure of SnoaL, involved in nogalamycin biosynthesis, with a bound product, has been determined to 1.35 Å resolution. The fold of the subunit can be described as a distorted α+β barrel, and the ligand is bound in the hydrophobic i...

  2. Structure of a Diguanylate Cyclase from Thermotoga maritima: Insights into Activation, Feedback Inhibition and Thermostability

    Deepthi, Angeline; Liew, Chong Wai; Liang, Zhao-Xun; Swaminathan, Kunchithapadam; Lescar, Julien

    2014-01-01

    Large-scale production of bis-3′-5′-cyclic-di-GMP (c-di-GMP) would facilitate biological studies of numerous bacterial signaling pathways and phenotypes controlled by this second messenger molecule, such as virulence and biofilm formation. C-di-GMP constitutes also a potentially interesting molecule as a vaccine adjuvant. Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain) from ...

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

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

    2003-01-01

    Adenosine induces vasoconstriction of renal afferent arterioles through activation of A1 adenosine receptors (A1AR). A1AR are directly coupled to Gi/Go, resulting in inhibition of adenylate cyclase, but the contribution of this signaling pathway to smooth muscle cell activation is unclear. In......-induced vasoconstriction was stable for up to 30 min and was most pronounced in the most distal part of the afferent arterioles. Adenosine did not cause vasoconstriction in arterioles from A1AR-/- mice. Pretreatment with pertussis toxin (PTX) (400 ng/ml) for 2 h blocked the vasoconstricting action of adenosine or N(6......) blocked the constriction responses to both adenosine and angiotensin II. In contrast, the adenylate cyclase inhibitor SQ22536 (10 micro M) and the protein kinase A antagonist KT5720 (0.1 and 1 micro M) did not induce significant vasoconstriction of afferent arterioles. It is concluded that the...

  4. Cyanobacterial toxins: risk management for health protection

    This paper reviews the occurrence and properties of cyanobacterial toxins, with reference to the recognition and management of the human health risks which they may present. Mass populations of toxin-producing cyanobacteria in natural and controlled waterbodies include blooms and scums of planktonic species, and mats and biofilms of benthic species. Toxic cyanobacterial populations have been reported in freshwaters in over 45 countries, and in numerous brackish, coastal, and marine environments. The principal toxigenic genera are listed. Known sources of the families of cyanobacterial toxins (hepato-, neuro-, and cytotoxins, irritants, and gastrointestinal toxins) are briefly discussed. Key procedures in the risk management of cyanobacterial toxins and cells are reviewed, including derivations (where sufficient data are available) of tolerable daily intakes (TDIs) and guideline values (GVs) with reference to the toxins in drinking water, and guideline levels for toxigenic cyanobacteria in bathing waters. Uncertainties and some gaps in knowledge are also discussed, including the importance of exposure media (animal and plant foods), in addition to potable and recreational waters. Finally, we present an outline of steps to develop and implement risk management strategies for cyanobacterial cells and toxins in waterbodies, with recent applications and the integration of Hazard Assessment Critical Control Point (HACCP) principles

  5. Crystal structures at 2.5 Angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate

    Belrhali, H.; Yaremchuk, A.; Tukalo, M.;

    1994-01-01

    Crystal structures of seryl-tRNA synthetase from Thermus thermophilus complexed with two different analogs of seryl adenylate have been determined at 2.5 Angstrom resolution. The first complex is between the enzyme and seryl-hydroxamate-AMP (adenosine monophosphate), produced enzymatically in the...

  6. Comparative analysis of oligonucleotide primers for high-throughput screening of genes encoding adenylation domains of nonribosomal peptide synthetases in actinomycetes

    Bakal, Tomáš; Goo, K.-S.; Najmanová, Lucie; Plháčková, Kamila; Kadlčík, Stanislav; Ulanová, Dana

    2015-01-01

    Roč. 108, č. 5 (2015), s. 1267-1274. ISSN 0003-6072 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : Nonribosomal peptide synthetase * Adenylation domain * Actinomycetes Subject RIV: EE - Microbiology, Virology Impact factor: 1.806, year: 2014

  7. Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals

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

    2014-01-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3 -, and sAC has been confirmed to be a HCO3 - sensor in a variety of mammalian cell types. In addition, sA...

  8. Mechanism of activation of particulate guanylate cyclase by atrial natriuretic peptide as deduced from radiation inactivation analysis

    The interaction between the receptor (Rc) for atrial natriuretic peptide (ANP) and the effector enzyme particulate guanylate cyclase (GC) has been studied by radiation inactivation. Irradiation of bovine lung membranes produced an increase in GC activity at low radiation doses followed by a dose-dependent reduction at higher doses. This deviation from linearity in the inactivation curve disappeared when lung membranes were pretreated with ANP. Essentially identical results were also obtained with adrenal membranes. Based on these radiation inactivation data, the following dissociative mechanism of activation of particulate guanylate cyclase by ANP has been proposed: Rc.GC(inactive) + ANP----Rc.ANP + GC(active)

  9. The Enterotoxicity of Clostridium difficile Toxins

    Hanping Feng; Tor Savidge; Xingmin Sun

    2010-01-01

    The major virulence factors of Clostridium difficile infection (CDI) are two large exotoxins A (TcdA) and B (TcdB). However, our understanding of the specific roles of these toxins in CDI is still evolving. It is now accepted that both toxins are enterotoxic and proinflammatory in the human intestine. Both purified TcdA and TcdB are capable of inducing the pathophysiology of CDI, although most studies have focused on TcdA. C. difficile toxins exert a wide array of biological activities by act...

  10. Retrograde transport of protein toxins through the Golgi apparatus

    Sandvig, Kirsten; Skotland, Tore; van Deurs, Bo; Klokk, Tove Irene

    2013-01-01

    A number of protein toxins from plants and bacteria take advantage of transport through the Golgi apparatus to gain entry into the cytosol where they exert their action. These toxins include the plant toxin ricin, the bacterial Shiga toxins, and cholera toxin. Such toxins bind to lipids or proteins...... at the cell surface, and they are endocytosed both by clathrin-dependent and clathrin-independent mechanisms. Sorting to the Golgi and retrograde transport to the endoplasmic reticulum (ER) are common to these toxins, but the exact mechanisms turn out to be toxin and cell-type dependent. In the ER......, the enzymatically active part is released and then transported into the cytosol, exploiting components of the ER-associated degradation system. In this review, we will discuss transport of different protein toxins, but we will focus on factors involved in entry and sorting of ricin and Shiga toxin...

  11. Binding and uptake of diphtheria toxin by toxin-resistant Chinese hamster ovary and mouse cells.

    Didsbury, J R; Moehring, J M; Moehring, T. J.

    1983-01-01

    We investigated two phenotypically distinct types of diphtheria toxin-resistant mutants of Chinese hamster cells and compared their resistance with that of naturally resistant mouse cells. All are resistant due to a defect in the process of internalization and delivery of toxin to its target in the cytosol, elongation factor 2. By cell hybridization studies, analysis of cross-resistance, and determination of specific binding sites for 125I-labeled diphtheria toxin, we showed that these cell s...

  12. A rhodopsin-guanylyl cyclase gene fusion functions in visual perception in a fungus.

    Avelar, Gabriela M; Schumacher, Robert I; Zaini, Paulo A; Leonard, Guy; Richards, Thomas A; Gomes, Suely L

    2014-06-01

    Sensing light is the fundamental property of visual systems, with vision in animals being based almost exclusively on opsin photopigments [1]. Rhodopsin also acts as a photoreceptor linked to phototaxis in green algae [2, 3] and has been implicated by chemical means as a light sensor in the flagellated swimming zoospores of the fungus Allomyces reticulatus [4]; however, the signaling mechanism in these fungi remains unknown. Here we use a combination of genome sequencing and molecular inhibition experiments with light-sensing phenotype studies to examine the signaling pathway involved in visual perception in the closely related fungus Blastocladiella emersonii. Our data show that in these fungi, light perception is accomplished by the function of a novel gene fusion (BeGC1) of a type I (microbial) rhodopsin domain and guanylyl cyclase catalytic domain. Photobleaching of rhodopsin function prevents accumulation of cGMP levels and phototaxis of fungal zoospores exposed to green light, whereas inhibition of guanylyl cyclase activity negatively affects fungal phototaxis. Immunofluorescence microscopy localizes the BeGC1 protein to the external surface of the zoospore eyespot positioned close to the base of the swimming flagellum [4, 5], demonstrating this is a photoreceptive organelle composed of lipid droplets. Taken together, these data indicate that Blastocladiomycota fungi have a cGMP signaling pathway involved in phototaxis similar to the vertebrate vision-signaling cascade but composed of protein domain components arranged as a novel gene fusion architecture and of distant evolutionary ancestry to type II rhodopsins of animals. PMID:24835457

  13. Neofunctionalization of Chromoplast Specific Lycopene Beta Cyclase Gene (CYC-B) in Tomato Clade

    Mohan, Vijee; Pandey, Arun; Sreelakshmi, Yellamaraju; Sharma, Rameshwar

    2016-01-01

    The ancestor of tomato underwent whole genome triplication ca. 71 Myr ago followed by widespread gene loss. However, few of the triplicated genes are retained in modern day tomato including lycopene beta cyclase that mediates conversion of lycopene to β-carotene. The fruit specific β-carotene formation is mediated by a chromoplast-specific paralog of lycopene beta cyclase (CYC-B) gene. Presently limited information is available about how the variations in CYC-B gene contributed to its neofunctionalization. CYC-B gene in tomato clade contained several SNPs and In-Dels in the coding sequence (33 haplotypes) and promoter region (44 haplotypes). The CYC-B gene coding sequence in tomato appeared to undergo purifying selection. The transit peptide sequence of CYC-B protein was predicted to have a stronger plastid targeting signal than its chloroplast specific paralog indicating a possible neofunctionalization. In promoter of two Bog (Beta old gold) mutants, a NUPT (nuclear plastid) DNA fragment of 256 bp, likely derived from a S. chilense accession, was present. In transient expression assay, this promoter was more efficient than the “Beta type” promoter. CARGATCONSENSUS box sequences are required for the binding of the MADS-box regulatory protein RIPENING INHIBITOR (RIN). The loss of CARGATCONSENSUS box sequence from CYC-B promoter in tomato may be related to attenuation of its efficiency to promote higher accumulation of β-carotene than lycopene during fruit ripening. PMID:27070417

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

    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. PMID:26625288

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

    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. [Botulinum toxin in disabling dermatological diseases].

    Messikh, R; Atallah, L; Aubin, F; Humbert, P

    2009-05-01

    Botulinum toxin could represent nowadays a new treatment modality especially for cutaneous conditions in course of which conventional treatments remain unsuccessful. Besides palmar and plantar hyperhidrosis, botulinum toxin has demonstrated efficacy in different conditions associated with hyperhidrosis, such as dyshidrosis, multiple eccrine hidrocystomas, hidradenitis suppurativa, Frey syndrome, but also in different conditions worsened by hyperhidrosis such as Hailey-Hailey disease, Darier disease, inversed psoriasis, aquagenic palmoplantar keratoderma, pachyonychia congenital. Moreover, different cutaneous conditions associated with sensitive disorders and/or neurological involvements could benefit from botulinum toxin, for example anal fissures, leg ulcers, lichen simplex, notalgia paresthetica, vestibulitis. Endly, a case of cutis laxa was described where the patient was improved by cutaneous injections of botulinum toxin. PMID:19576479

  17. Hemolytic anemia caused by chemicals and toxins

    Anemia - hemolytic - caused by chemicals or toxins ... Possible substances that can cause hemolytic anemia include: Anti-malaria drugs (quinine compounds) Arsenic Dapsone Intravenous water infusion (not half-normal saline or normal saline) Metals (chromium/chromates, ...

  18. NNDSS - Table II. Shiga toxin to Shigellosis

    U.S. Department of Health & Human Services — NNDSS - Table II. Shiga toxin to Shigellosis - 2015. In this Table, provisional cases of selected notifiable diseases (≥1,000 cases reported during the preceding...

  19. NNDSS - Table II. Shiga toxin to Shigellosis

    U.S. Department of Health & Human Services — NNDSS - Table II. Shiga toxin to Shigellosis - 2016. In this Table, provisional* cases of selected†notifiable diseases (≥1,000 cases reported during the...

  20. Anticancer potential of animal venoms and toxins.

    Gomes, Antony; Bhattacharjee, Pushpak; Mishra, Roshnara; Biswas, Ajoy K; Dasgupta, Subir Chandra; Giri, Biplab

    2010-02-01

    Anticancer drug development from natural resources are ventured throughout the world. Animal venoms and toxins a potential bio resource and a therapeutic tool were known to man for centuries through folk and traditional knowledge. The biodiversity of venoms and toxins made it a unique source of leads and structural templates from which new therapeutic agents may be developed. Venoms of several animal species (snake, scorpion, toad, frog etc) and their active components (protein and non protein toxins, peptides, enzymes, etc) have shown therapeutic potential against cancer. In the present review, the anticancer potential of venoms and toxins from snakes, scorpions, toads and frogs has been discussed. Some of these molecules are in the clinical trials and may find their way towards anticancer drug development in the near future. The implications of combination therapy of natural products in cancer have been discussed. PMID:20455317

  1. Updates on tetanus toxin: a fundamental approach

    Md. Ahaduzzaman

    2015-03-01

    Full Text Available Clostridium tetani is an anaerobic bacterium that produces second most poisonous protein toxins than any other bacteria. Tetanus in animals is sporadic in nature but difficult to combat even by using antibiotics and antiserum. It is crucial to understand the fundamental mechanisms and signals that control toxin production for advance research and medicinal uses. This review was intended for better understanding the basic patho-physiology of tetanus and neurotoxins (TeNT among the audience of related field.

  2. Nanoanalysis of the arthropod neuro-toxins

    Nakajima, Terumi

    2006-01-01

    Many kinds of venomous principles modulate physiological responses of mammalian signal transduction systems, on which they act selectively as enhancers, inhibitors or some other kind of effectors. These toxins become useful tools for physiological research. We have employed and characterized paralyzing toxins from the venom of spiders, insects and scorpions with a limited supply. We have developed rapid and sensitive mass spectrometric technology and applied for the identification of these to...

  3. Target-Driven Evolution of Scorpion Toxins

    Shangfei Zhang; Bin Gao; Shunyi Zhu

    2015-01-01

    It is long known that peptide neurotoxins derived from a diversity of venomous animals evolve by positive selection following gene duplication, yet a force that drives their adaptive evolution remains a mystery. By using maximum-likelihood models of codon substitution, we analyzed molecular adaptation in scorpion sodium channel toxins from a specific species and found ten positively selected sites, six of which are located at the core-domain of scorpion α-toxins, a region known to interact wi...

  4. Toxicological Perspective on Climate Change: Aquatic Toxins.

    Botana, Luis M

    2016-04-18

    In recent years, our group and several others have been describing the presence of new, not previously reported, toxins of high toxicity in vectors that may reach the human food chain. These include tetrodotoxin in gastropods in the South of Europe, ciguatoxin in fish in the South of Spain, palytoxin in mussels in the Mediterranean Sea, pinnatoxin all over Europe, and okadaic acid in the south of the U.S. There seem to be new marine toxins appearing in areas that are heavy producers of seafood, and this is a cause of concern as most of these new toxins are not included in current legislation and monitoring programs. Along with the new toxins, new chemical analogues are being reported. The same phenomenom is being recorded in freshwater toxins, such as the wide appearance of cylindrospermopsin and the large worldwide increase of microcystin. The problem that this phenomenon, which may be linked to climate warming, poses for toxicologists is very important not only because there is a lack of chronic studies and an incomplete comprehension of the mechanism driving the production of these toxins but also because the lack of a legal framework for them allows many of these toxins to reach the market. In some cases, it is very difficult to control these toxins because there are not enough standards available, they are not always certified, and there is an insufficient understanding of the toxic equivalency factors of the different analogues in each group. All of these factors have been revealed and grouped through the massive increase in the use of LC-MS as a monitoring tool, legally demanded, creating more toxicological problems. PMID:26958981

  5. Botulinum toxin treatment of hemifacial spasm.

    Elston, J S

    1986-01-01

    Six patients with hemifacial spasm were treated with injections of botulinum toxin A into the orbicularis oculi; the abnormal movements around the eye were relieved for an average of 15 weeks. There were no systemic or significant local side effects, and in view of the risks involved in neurosurgical treatment, a trial of botulinum toxin injections is recommended in the first instance in this condition.

  6. Botulinum Toxin; Bioterror and Biomedicinal Agent

    Jiri Patocka; Kamil Kuca; Daniel Jun

    2006-01-01

    Botulinum toxin is a group of seven homologous, highly poisonous proteins isolated fromfermentation of the anaerobic bacterium Clostridium botulinum, which naturally occurs in soiland can grow on many meats and vegetables. Botulinum toxin causes neuromuscular disordercalled botulism, which is a potentially lethal disease. There are three types of botulism: Food,wound, and infant botulism. It can lead to death unless appropriate therapy is done. Due to theseverity and potency of botulinum toxi...

  7. Normal and Pathologic Concentrations of Uremic Toxins

    Duranton, Flore; Cohen, Gerald; De Smet, Rita; Rodriguez, Mariano; Jankowski, Joachim; Vanholder, Raymond; Argiles, Angel

    2012-01-01

    An updated review of the existing knowledge regarding uremic toxins facilitates the design of experimental studies. We performed a literature search and found 621 articles about uremic toxicity published after a 2003 review of this topic. Eighty-seven records provided serum or blood measurements of one or more solutes in patients with CKD. These records described 32 previously known uremic toxins and 56 newly reported solutes. The articles most frequently reported concentrations of β2-microgl...

  8. Plant Insecticidal Toxins in Ecological Networks

    Sébastien Ibanez; Christiane Gallet; Laurence Després

    2012-01-01

    Plant secondary metabolites play a key role in plant-insect interactions, whether constitutive or induced, C- or N-based. Anti-herbivore defences against insects can act as repellents, deterrents, growth inhibitors or cause direct mortality. In turn, insects have evolved a variety of strategies to act against plant toxins, e.g., avoidance, excretion, sequestration and degradation of the toxin, eventually leading to a co-evolutionary arms race between insects and plants and to co-diversificati...

  9. Lethal effects of Clostridium perfringens epsilon toxin are potentiated by alpha and perfringolysin-O toxins in a mouse model

    Fernandez-Miyakawa, Mariano E.; Jost, B. Helen; Billington, Stephen J; Uzal, Francisco A.

    2007-01-01

    Epsilon-toxin (ETX) is the most important virulence factor of Clostridium perfringens type D. Two other important toxins, alpha-toxin (CPA) and perfringolysin-O (PFO), are encoded and potentially produced by most C. perfringens type D isolates. The biological effects of these toxins are dissimilar although they are all lethal. Since the possible interaction of these toxins during infection is unknown, the effects of CPA and PFO on the lethal activity of ETX were studied in a mouse model. Mice...

  10. A Mitochondrial RNAi Screen Defines Cellular Bioenergetic Determinants and Identifies an Adenylate Kinase as a Key Regulator of ATP Levels

    Nathan J. Lanning

    2014-05-01

    Full Text Available Altered cellular bioenergetics and mitochondrial function are major features of several diseases, including cancer, diabetes, and neurodegenerative disorders. Given this important link to human health, we sought to define proteins within mitochondria that are critical for maintaining homeostatic ATP levels. We screened an RNAi library targeting >1,000 nuclear-encoded genes whose protein products localize to the mitochondria in multiple metabolic conditions in order to examine their effects on cellular ATP levels. We identified a mechanism by which electron transport chain (ETC perturbation under glycolytic conditions increased ATP production through enhanced glycolytic flux, thereby highlighting the cellular potential for metabolic plasticity. Additionally, we identified a mitochondrial adenylate kinase (AK4 that regulates cellular ATP levels and AMPK signaling and whose expression significantly correlates with glioma patient survival. This study maps the bioenergetic landscape of >1,000 mitochondrial proteins in the context of varied metabolic substrates and begins to link key metabolic genes with clinical outcome.

  11. A Multiple-Labeling Strategy for Nonribosomal Peptide Synthetases Using Active-Site-Directed Proteomic Probes for Adenylation Domains.

    Ishikawa, Fumihiro; Suzuki, Takehiro; Dohmae, Naoshi; Kakeya, Hideaki

    2015-12-01

    Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple-labeling technique with active-site-directed probes for adenylation domains. When applied to gramicidin S-producing and -nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high-resolution identification of low-abundance active NRPS enzymes in native proteomic environments. PMID:26467472

  12. Affinity chromatography of tetanus toxin, tetanus toxoid, and botulinum A toxin on synaptosomes, and differentiation of their acceptors

    125I-labelled tetanus toxin and 125I-labelled botulinum A neurotoxin are known to be specifically bound to brain synaptosomes. In order to discriminate between active toxin and inactive admixtures present in the starting material or arising during iodination, synaptosome columns were prepared using bromacetylcellulose and/or kieselgur (Celite) as carriers. Both types of columns adsorb the toxins from low ionic strength medium and release them if the pH and ionic strength are raised. Botulinum toxin was eluted with lower ionic strength than tetanus toxin, and could be freed from nontoxic admixtures. Analysis by affinity chromatography disclosed partially toxoided tetanus toxin in both labelled and unlabelled toxin samples. High concentrations of formaldehyde (0.5%) destroyed both toxicity and affinity to the synaptosomes of tetanus toxin. Low concentrations of formaldehyde (0.05%) yielded a derivative of low toxicity which was still, however less firmly, bound to synaptosomes. Tetanus and botulinum toxin differ by their acceptors. Whereas unlabelled botulinum toxin is unable to compete with labelled tetanus toxin, unlabelled tetanus toxin slightly competes with botulinum toxin. Both labelled toxins display anomalous binding behaviour in that they cannot be displaced completely even with a large excess of unlabelled toxin. (orig.)

  13. Molecular Cloning,Expression,and Characterization of an Adenylyl Cyclase-associated Protein from Gossypium arboreum Fuzzless Mutant

    2008-01-01

    CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in cotton.For the further research,we isolated two CAP homologues from wild type

  14. Clostridium perfringens Delta-Toxin Induces Rapid Cell Necrosis

    Seike, Soshi; Miyamoto, Kazuaki; Kobayashi, Keiko; Takehara, Masaya; Nagahama, Masahiro

    2016-01-01

    Clostridium perfringens delta-toxin is a β-pore-forming toxin and a putative pathogenic agent of C. perfringens types B and C. However, the mechanism of cytotoxicity of delta-toxin remains unclear. Here, we investigated the mechanisms of cell death induced by delta-toxin in five cell lines (A549, A431, MDCK, Vero, and Caco-2). All cell lines were susceptible to delta-toxin. The toxin caused rapid ATP depletion and swelling of the cells. Delta-toxin bound and formed oligomers predominantly in plasma membrane lipid rafts. Destruction of the lipid rafts with methyl β-cyclodextrin inhibited delta-toxin-induced cytotoxicity and ATP depletion. Delta-toxin caused the release of carboxyfluorescein from sphingomyelin-cholesterol liposomes and formed oligomers; toxin binding to the liposomes declined with decreasing cholesterol content in the liposomes. Flow cytometric assays with annexin V and propidium iodide revealed that delta-toxin treatment induced an elevation in the population of annexin V-negative and propidium iodide-positive cells. Delta-toxin did not cause the fragmentation of DNA or caspase-3 activation. Furthermore, delta-toxin caused damage to mitochondrial membrane permeability and cytochrome c release. In the present study, we demonstrate that delta-toxin produces cytotoxic activity through necrosis. PMID:26807591

  15. Clostridium perfringens Delta-Toxin Induces Rapid Cell Necrosis.

    Soshi Seike

    Full Text Available Clostridium perfringens delta-toxin is a β-pore-forming toxin and a putative pathogenic agent of C. perfringens types B and C. However, the mechanism of cytotoxicity of delta-toxin remains unclear. Here, we investigated the mechanisms of cell death induced by delta-toxin in five cell lines (A549, A431, MDCK, Vero, and Caco-2. All cell lines were susceptible to delta-toxin. The toxin caused rapid ATP depletion and swelling of the cells. Delta-toxin bound and formed oligomers predominantly in plasma membrane lipid rafts. Destruction of the lipid rafts with methyl β-cyclodextrin inhibited delta-toxin-induced cytotoxicity and ATP depletion. Delta-toxin caused the release of carboxyfluorescein from sphingomyelin-cholesterol liposomes and formed oligomers; toxin binding to the liposomes declined with decreasing cholesterol content in the liposomes. Flow cytometric assays with annexin V and propidium iodide revealed that delta-toxin treatment induced an elevation in the population of annexin V-negative and propidium iodide-positive cells. Delta-toxin did not cause the fragmentation of DNA or caspase-3 activation. Furthermore, delta-toxin caused damage to mitochondrial membrane permeability and cytochrome c release. In the present study, we demonstrate that delta-toxin produces cytotoxic activity through necrosis.

  16. Toxin from skin of frogs of the genus Atelopus: differentiation from Dendrobatid toxins.

    Fuhrman, F A; Fuhrman, G J; Mosher, H S

    1969-09-26

    A potent, dialyzable toxin (atelopidtoxin) occurs in the skin of frogs of the genus Atelopus. A concentrate of atelopidtoxin from Atelopus zeteki has an LD(50) in mice of 16 micrograms per kilogram. It differs from batrachotoxin, tetrodotoxin, and saxitoxin, the only known nonprotein substances of greater toxicity, as well as from all toxins previously isolated from amphibia. PMID:5807965

  17. Differential activation of yeast adenylyl cyclase by Ras1 and Ras2 depends on the conserved N terminus.

    Hurwitz, N; Segal, M; Marbach, I; Levitzki, A

    1995-11-21

    Although both Ras1 and Ras2 activate adenylyl cyclase in yeast, a number of differences can be observed regarding their function in the cAMP pathway. To explore the relative contribution of conserved and variable domains in determining these differences, chimeric RAS1-RAS2 or RAS2-RAS1 genes were constructed by swapping the sequences encoding the variable C-terminal domains. These constructs were expressed in a cdc25ts ras1 ras2 strain. Biochemical data show that the difference in efficacy of adenylyl cyclase activation between the two Ras proteins resides in the highly conserved N-terminal domain. This finding is supported by the observation that Ras2 delta, in which the C-terminal domain of Ras2 has been deleted, is a more potent activator of the yeast adenylyl cyclase than Ras1 delta, in which the C-terminal domain of Ras1 has been deleted. These observations suggest that amino acid residues other than the highly conserved residues of the effector domain within the N terminus may determine the efficiency of functional interaction with adenylyl cyclase. Similar levels of intracellular cAMP were found in Ras1, Ras1-Ras2, Ras1 delta, Ras2, and Ras2-Ras1 strains throughout the growth curve. This was found to result from the higher expression of Ras1 and Ras1-Ras2, which compensate for their lower efficacy in activating adenylyl cyclase. These results suggest that the difference between the Ras1 and the Ras2 phenotype is not due to their different efficacy in activating the cAMP pathway and that the divergent C-terminal domains are responsible for these differences, through interaction with other regulatory elements. PMID:7479926

  18. Animal Toxins: How is Complexity Represented in Databases?

    Jungo, Florence; Estreicher, Anne; Bairoch, Amos; Bougueleret, Lydie; Xenarios, Ioannis

    2010-02-01

    Peptide toxins synthesized by venomous animals have been extensively studied in the last decades. To be useful to the scientific community, this knowledge has been stored, annotated and made easy to retrieve by several databases. The aim of this article is to present what type of information users can access from each database. ArachnoServer and ConoServer focus on spider toxins and cone snail toxins, respectively. UniProtKB, a generalist protein knowledgebase, has an animal toxin-dedicated annotation program that includes toxins from all venomous animals. Finally, the ATDB metadatabase compiles data and annotations from other databases and provides toxin ontology. PMID:22069583

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

    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.

  20. The roles of cysteines in the heme domain of human soluble guanylate cyclase

    Fang Fang Zhong; Xiao Xiao Liu; Jie Pan; Zhong Xian Huang; Xiang Shi Tan

    2012-01-01

    Soluble guanylate cyclase (sGC) is a critical heme-containing enzyme involved in NO signaling.The dimerization of sGC subunits is necessary for its bioactivity and its mechanism is a striiking and an indistinct issue.The roles of heme domain cysteines of the sGC on the dimerization and heme binding were investigated herein.The site-directed mutations of three conserved cysteines (C78A,C 122A and C 174S) were studied systematically and the three mutants were characterized by gel filtration analysis,UV-vis spectroscopy and heime transfer examination.Cys78 was involved in heme binding but not referred to the dimerization,while Cys174 was demonstrated to be involved in the homodimerization.These results provide new insights into the cysteine-related dimerization regulation of sGC.

  1. Stimulation of soluble guanylyl cyclase protects against obesity by recruiting brown adipose tissue.

    Hoffmann, Linda S; Etzrodt, Jennifer; Willkomm, Lena; Sanyal, Abhishek; Scheja, Ludger; Fischer, Alexander W C; Stasch, Johannes-Peter; Bloch, Wilhelm; Friebe, Andreas; Heeren, Joerg; Pfeifer, Alexander

    2015-01-01

    Obesity is characterized by a positive energy balance and expansion of white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) combusts energy to produce heat. Here we show that a small molecule stimulator (BAY 41-8543) of soluble guanylyl cyclase (sGC), which produces the second messenger cyclic GMP (cGMP), protects against diet-induced weight gain, induces weight loss in established obesity, and also improves the diabetic phenotype. Mechanistically, the haeme-dependent sGC stimulator BAY 41-8543 enhances lipid uptake into BAT and increases whole-body energy expenditure, whereas ablation of the haeme-containing β1-subunit of sGC severely impairs BAT function. Notably, the sGC stimulator enhances differentiation of human brown adipocytes as well as induces 'browning' of primary white adipocytes. Taken together, our data suggest that sGC is a potential pharmacological target for the treatment of obesity and its comorbidities. PMID:26011238

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

    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.

  3. Characterization and expression of soluble guanylate cyclase in skins and melanocytes of sheep.

    Yang, Shanshan; Zhang, Junzhen; Ji, Kaiyuan; Jiao, Dingxing; Fan, Ruiwen; Dong, Changsheng

    2016-04-01

    The study reported the characterization of soluble guanylate cyclase (sGC) with the size of CDS of 1860bp, encoding a protein of 620 amino acids and containing several conserved functional domains including HNOB, HNOBA, and CHD. Quantitative real time PCR analysis of sGC showed that the expression of sGC mRNA is higher (∼5 fold) in white sheep skin relative to black sheep skin with significant difference (Ptyrosinase (TYR), tyrosinase related protein 1(TYRTP1), and tyrosinase related protein 2(TYRP2) both at mRNA and protein level. Moreover, the melanocytes was capable of producing cGMP and cAMP. The observed differential expression and localization of sGC in sheep skins and melanocytes and the capability of producing cGMP and cAMP, which suggested a potential role for this gene in hair color regulation. PMID:26805580

  4. An approach to mimicking the sesquiterpene cyclase phase by nickel-promoted diene/alkyne cooligomerization.

    Holte, Dane; Götz, Daniel C G; Baran, Phil S

    2012-01-20

    Artificially mimicking the cyclase phase of terpene biosynthesis inspires the invention of new methodologies, since working with carbogenic frameworks containing minimal functionality limits the chemist's toolbox of synthetic strategies. For example, the construction of terpene skeletons from five-carbon building blocks would be an exciting pathway to mimic in the laboratory. Nature oligomerizes, cyclizes, and then oxidizes γ,γ-dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) to all of the known terpenes. Starting from isoprene, the goal of this work was to mimic Nature's approach for rapidly building molecular complexity. In principle, the controlled oligomerization of isoprene would drastically simplify the synthesis of terpenes used in the medicine, perfumery, flavor, and materials industries. This article delineates our extensive efforts to cooligomerize isoprene or butadiene with alkynes in a controlled fashion by zerovalent nickel catalysis building off the classic studies by Wilke and co-workers. PMID:22229741

  5. Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase

    Oliver, S.; Tiessen, A.; Fernie, A.; Geigenberger, P.

    2008-01-01

    Adenine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. It was previously reported that decreased expression of plastidial adenylate kinase, catalysing the interconversion of ATP and AMP to ADP, leads to increased adenylate pools and starch content in transgenic potato tubers. However, the underlying mechanisms were not elucidated. Here, it is shown that decreased expression of plastidial ade...

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

    Xiao-Hong eWen

    2014-06-01

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

  7. Isolation and characterization of glutaminyl cyclases from Drosophila: evidence for enzyme forms with different subcellular localization.

    Schilling, Stephan; Lindner, Christiane; Koch, Birgit; Wermann, Michael; Rahfeld, Jens-Ulrich; von Bohlen, Alex; Rudolph, Thomas; Reuter, Gunter; Demuth, Hans-Ulrich

    2007-09-25

    Glutaminyl cyclases (QCs) present in plants and vertebrates catalyze the formation of pyroglutamic acid (pGlu) from N-terminal glutamine. Pyroglutamyl hormones also identified in invertebrates imply the involvement of QC activity during their posttranslational maturation. Database mining led to the identification of two genes in Drosophila, which putatively encode QCs, CG32412 (DromeQC) and CG5976 (isoDromeQC). Analysis of their primary structure suggests different subcellular localizations. While DromeQC appeared to be secreted due to an N-terminal signal peptide, isoDromeQC contains either an N-terminal mitochondrial targeting or a secretion signal due to generation of different transcripts from gene CG5976. According to the prediction, homologous expression of the corresponding cDNAs in S2 cells revealed either secreted protein in the medium or intracellular QC activity. Subcellular fractionation and immunochemistry support export of isoDromeQC into the mitochondrion. For enzymatic characterization, DromeQC and isoDromeQC were expressed heterologously in Pichia pastoris and Escherichia coli, respectively. Compared to mammalian QCs, the specificity constants were about 1 order of magnitude lower for most of the analyzed substrates. The pH dependence of the specificity constant was similar for both enzymes, indicating the necessity of an unprotonated substrate amino group and two protonated groups of the enzyme, resulting in an asymmetric bell-shaped characteristic. The determination of the metal content of DromeQC revealed equimolar protein-bound zinc. These results prove conserved enzymatic mechanisms between QCs from invertebrates and mammals. Drosophila is the first organism for which isoenzymes of glutaminyl cyclase have been isolated. The identification of a mitochondrial QC points toward yet undiscovered physiological functions of these enzymes. PMID:17722885

  8. A truncated diphtheria toxin based recombinant porcine CTLA-4 fusion toxin.

    Peraino, Jaclyn Stromp; Schenk, Marian; Zhang, Huiping; Li, Guoying; Hermanrud, Christina E; Neville, David M; Sachs, David H; Huang, Christene A; Duran-Struuck, Raimon; Wang, Zhirui

    2013-05-31

    Targeted cell therapies are possible through the generation of recombinant fusion proteins that combine a toxin, such as diphtheria toxin (DT), with an antibody or other molecule that confers specificity. Upon binding of the fusion protein to the cell of interest, the diphtheria toxin is internalized which results in protein synthesis inhibition and subsequent cell death. We have recently expressed and purified the recombinant soluble porcine CTLA-4 both with and without N-glycosylation in yeast Pichia pastoris for in vivo use in our preclinical swine model. The glycosylated and non-N-glycosylated versions of this recombinant protein each bind to a porcine CD80 expressing B-cell lymphoma line (LCL13271) with equal affinity (K(D)=13 nM). In this study we have linked each of the glycosylated and non-N-glycosylated soluble porcine CTLA-4 proteins to the truncated diphtheria toxin DT390 through genetic engineering yielding three versions of the porcine CTLA-4 fusion toxins: 1) monovalent glycosylated soluble porcine CTLA-4 fusion toxin; 2) monovalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin and 3) bivalent non-N-glycosylated soluble porcine CTLA-4 fusion toxin. Protein synthesis inhibition analysis demonstrated that while all three fusion toxins are capable of inhibiting protein synthesis in vitro, the non-N-glycosylated porcine CTLA-4 isoforms function most efficiently. Binding analysis using flow cytometry of the porcine CTLA-4 fusion toxins to LCL13271 cells also demonstrated that the non-N-glycosylated porcine CTLA-4 isoforms bind to these cells with higher affinity compared to the glycosylated fusion toxin. The monovalent non-N-glycosylated porcine CTLA-4 fusion toxin was tested in vivo. NSG (NOD/SCID IL-2 receptor γ(-)/(-)) mice were injected with porcine CD80(+) LCL13271 tumor cells. All animals succumbed to tumors and those treated with the monovalent non-N-glycosylated porcine CTLA-4 fusion toxin survived longer based on a symptomatic scoring

  9. Further characterization of Mycobacterium ulcerans toxin.

    Hockmeyer, W T; Krieg, R E; Reich, M; Johnson, R D

    1978-07-01

    Mycobacterium ulcerans produces an exotoxin in culture which, when inoculated into guinea pig skin, causes inflammation, necrosis, edema, and other histopathological changes resembling those in infections of humans. The toxin was resistant to heat and to alkalies and was moderately acid labile. Toxic activity was destroyed by Pronase, phospholipase, lipase, amylase, and glucosidase but not by trypsin, collagenase, cellulase, lysozyme, hyaluronidase, or neuraminidase. Toxic activity was resistant to treatment with 2-mercaptoethanol, urea, guanidine hydrochloride, p-chloromercuribenzoate, ethylenediaminetetraacetate, and sodium deoxycholate but was destroyed by sodium m-periodate and sodium dodecyl sulfate. The toxin was precipitated by a wide range of ammonium sulfate concentrations. Extraction with chlorofrom-methanol or petroleum ether destroyed its activity. Isopycnic density gradient ultracentrifugation in KBr produced a high-density lipoprotein layer with a 24-fold increase in specific activity. The results indicate that this toxin is a high-molecular-weight phospholipoprotein-polysaccharide complex. PMID:30694

  10. ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites*

    Randak, Christoph O.; Dong, Qian; Ver Heul, Amanda R.; Elcock, Adrian H.; Welsh, Michael J.

    2013-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, ...

  11. The glucagon signal transduction through the liver adenylyl cyclase of adult rats irradiated by single dose gamma-irradiation in utero

    It was found that the one-fold (0,5 Gy) prenatal gamma-irradiation on the 9 and the 15 day of embryogenesis (at the beginning and the end of organogenesis, respectively) resulted in the different alterations of glucagon signalling through the receptor/G s-protein/adenylyl cyclase in adult rat liver. The prenatal irradiation on the 9 day enhanced the effect of GTP (the activator of Gs-protein) on the adenylyl cyclase activity. While the gamma-irradiation on the 15 day of embryo development increased the basal, GTP- and glucagon-stimulated adenylyl cyclase activity. The results suggest that the receptor/Gs-protein/ adenylyl cyclase coupling is more sensitive to low doses of gamma-irradiation performed at the end of the organogenesis

  12. Recent advances in the medicinal chemistry of polyamine toxins

    Strømgaard, K; Andersen, K; Krogsgaard-Larsen, P;

    2001-01-01

    This review describes the recent developments in the field of polyamine toxins, with focus on structure activity relationship investigations, including studies of importance of the polyamine moiety for biological activity, photolabeling studies using polyamine toxins as templates, as well as use ...... solid phase methods for the synthesis of polyamine toxins. The review is mainly concerned with effects of polyamine toxins on nicotinic acetylcholine receptors and ionotropic glutamate receptors....

  13. Recent Insights into Clostridium perfringens Beta-Toxin

    Masahiro Nagahama; Sadayuki Ochi; Masataka Oda; Kazuaki Miyamoto; Masaya Takehara; Keiko Kobayashi

    2015-01-01

    Clostridium perfringens beta-toxin is a key mediator of necrotizing enterocolitis and enterotoxemia. It is a pore-forming toxin (PFT) that exerts cytotoxic effect. Experimental investigation using piglet and rabbit intestinal loop models and a mouse infection model apparently showed that beta-toxin is the important pathogenic factor of the organisms. The toxin caused the swelling and disruption of HL-60 cells and formed a functional pore in the lipid raft microdomains of sensitive cells. Thes...

  14. Structural interactions of a voltage sensor toxin with lipid membranes

    Mihailescu, Mihaela; Krepkiy, Dmitriy; Milescu, Mirela; Gawrisch, Klaus; Swartz, Kenton J.; White, Stephen

    2014-01-01

    Tarantula venom contains protein toxins that interact with diverse families of ion channels and alter their activity. A number of tarantula toxins are known to interact with membranes and are thought to bind to ion channel proteins within the lipid bilayer. In the present study, we find that tarantula toxins influence the structure and dynamics of the lipid bilayer, and that the toxin orients itself within membranes to facilitate formation of the toxin–channel complexes. Our results have impl...

  15. Natural Toxins for Use in Pest Management

    Kevin K. Schrader

    2010-07-01

    Full Text Available Natural toxins are a source of new chemical classes of pesticides, as well as environmentally and toxicologically safer molecules than many of the currently used pesticides. Furthermore, they often have molecular target sites that are not exploited by currently marketed pesticides. There are highly successful products based on natural compounds in the major pesticide classes. These include the herbicide glufosinate (synthetic phosphinothricin, the spinosad insecticides, and the strobilurin fungicides. These and other examples of currently marketed natural product-based pesticides, as well as natural toxins that show promise as pesticides from our own research are discussed.

  16. Positive regulation of Clostridium difficile toxins.

    Moncrief, J S; Barroso, L A; Wilkins, T D

    1997-01-01

    The toxigenic element of Clostridium difficile VPI 10463 contains a small open reading frame (ORF) immediately upstream of the toxin B gene (G. A. Hammond and J. L. Johnson, Microb. Pathog. 19:203-213, 1995). The deduced amino acid sequence of the ORF, which we have designated txeR, encodes a 22-kDa protein which contains a helix-turn-helix motif with sequence identity to DNA binding regulatory proteins. We used a DNA fragment containing the C. difficile toxin A repeating units (ARU) as a rep...

  17. Streptococcal toxins: role in pathogenesis and disease.

    Barnett, Timothy C; Cole, Jason N; Rivera-Hernandez, Tania; Henningham, Anna; Paton, James C; Nizet, Victor; Walker, Mark J

    2015-12-01

    Group A Streptococcus (Streptococcus pyogenes), group B Streptococcus (Streptococcus agalactiae) and Streptococcus pneumoniae (pneumococcus) are host-adapted bacterial pathogens among the leading infectious causes of human morbidity and mortality. These microbes and related members of the genus Streptococcus produce an array of toxins that act against human cells or tissues, resulting in impaired immune responses and subversion of host physiological processes to benefit the invading microorganism. This toxin repertoire includes haemolysins, proteases, superantigens and other agents that ultimately enhance colonization and survival within the host and promote dissemination of the pathogen. PMID:26433203

  18. Characterisation of cholera toxin by liquid chromatography - Electrospray mass spectrometry

    Baar, B.L.M. van; Hulst, A.G.; Wils, E.R.J.

    1999-01-01

    Cholera toxin, one of the toxins that may be generated by various strains of the bacterium Vibrio cholerae, can be considered as a substance possibly used in biological warfare. The possibilities of characterising the toxin by liquid chromatography electrospray mass spectrometry (LC-ES-MS) were inve

  19. Pichia acaciae Killer System: Genetic Analysis of Toxin Immunity▿

    Paluszynski, John P.; Klassen, Roland; Meinhardt, Friedhelm

    2007-01-01

    The gene responsible for self-protection in the Pichia acaciae killer plasmid system was identified by heterologous expression in Saccharomyces cerevisiae. Resistance profiling and conditional toxin/immunity coexpression analysis revealed dose-independent protection by pPac1-2 ORF4 and intracellular interference with toxin function, suggesting toxin reinternalization in immune killer cells.

  20. Effect of treatment with botulinum toxin on spasticity.

    Das, T K; Park, D M

    1989-01-01

    Botulinum toxin, a product of Clostridium botulinum, produces presynaptic neuromuscular block by preventing release of acetylcholine from nerve endings. The toxin was injected directly into the skeletal muscles of six patients with severe spasticity due to stroke-related hemiplegia. It produced both subjective and objective improvement. The toxin injections were well tolerated and no significant side effect was reported.

  1. Conserved chloroplast open-reading frame ycf54 is required for activity of the magnesium protoporphyrin monomethylester oxidative cyclase in Synechocystis PCC 6803.

    Hollingshead, Sarah; Kopecná, Jana; Jackson, Philip J; Canniffe, Daniel P; Davison, Paul A; Dickman, Mark J; Sobotka, Roman; Hunter, C Neil

    2012-08-10

    The cyclase step in chlorophyll (Chl) biosynthesis has not been characterized biochemically, although there are some plausible candidates for cyclase subunits. Two of these, Sll1214 and Sll1874 from the cyanobacterium Synechocystis 6803, were FLAG-tagged in vivo and used as bait in separate pulldown experiments. Mass spectrometry identified Ycf54 as an interaction partner in each case, and this interaction was confirmed by a reciprocal pulldown using FLAG-tagged Ycf54 as bait. Inactivation of the ycf54 gene (slr1780) in Synechocystis 6803 resulted in a strain that exhibited significantly reduced Chl levels. A detailed analysis of Chl precursors in the ycf54 mutant revealed accumulation of very high levels of Mg-protoporphyrin IX methyl ester and only traces of protochlorophyllide, the product of the cyclase, were detected. Western blotting demonstrated that levels of the cyclase component Sll1214 and the Chl biosynthesis enzymes Mg-protoporphyrin IX methyltransferase and protochlorophyllide reductase are significantly impaired in the ycf54 mutant. Ycf54 is, therefore, essential for the activity and stability of the oxidative cyclase. We discuss a possible role of Ycf54 as an auxiliary factor essential for the assembly of a cyclase complex or even a large multienzyme catalytic center. PMID:22711541

  2. Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase.

    Selwood, D L; Brummell, D G; Budworth, J; Burtin, G E; Campbell, R O; Chana, S S; Charles, I G; Fernandez, P A; Glen, R C; Goggin, M C; Hobbs, A J; Kling, M R; Liu, Q; Madge, D J; Meillerais, S; Powell, K L; Reynolds, K; Spacey, G D; Stables, J N; Tatlock, M A; Wheeler, K A; Wishart, G; Woo, C K

    2001-01-01

    Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases. PMID:11141091

  3. Staphylococcus hyicus exfoliative toxin: Purification and demonstration of antigenic diversity among toxins from virulent strains

    Andresen, Lars Ole; Bille-Hansen, Vivi; Wegener, Henrik Caspar

    1997-01-01

    hydrophobic interaction chromatography and successively anion exchange chromatography. The purified toxin was tested in a piglet skin assay. Weak epidermal lesions were macroscopically and microscopically similar to lesions caused by (NH4)(2)SO4-precipitated culture supernatant from the same strain. Addition...... of 0.5 mM CuSO4 to the purified toxin resulted in more intense skin alterations comparable to lesions caused by precipitated culture supernatant diluted 1:10. These results indicated that the activity of the exfoliative toxin was dependent on the presence of Cu2+. Polyclonal and monoclonal antibodies....... hyicus. These results showed antigenic diversity among exfoliative toxins produced by different strains of S. hyicus. (C) 1997 Academic Press Limited....

  4. A Cell-Based Fluorescent Assay to Detect the Activity of Shiga Toxin and Other Toxins That Inhibit Protein Synthesis

    Escherichia coli O157:H7, a major cause of food-borne illness, produces Shiga toxins that block protein synthesis by inactivating the ribosome. In this chapter we describe a simple cell-based fluorescent assay to detect Shiga toxins and inhibitors of toxin activity. The assay can also be used to d...

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

    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

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

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

    2002-01-01

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

  7. High adenylyl cyclase activity and in vivo cAMP fluctuations in corals suggest central physiological role

    Barott, K.L.; Helman, Y.; Haramaty, L.; Barron, M. E.; Hess, K.C.; Buck, J.; Levin, L. R.; Tresguerres, M.

    2013-01-01

    Corals are an ecologically and evolutionarily significant group, providing the framework for coral reef biodiversity while representing one of the most basal of metazoan phyla. However, little is known about fundamental signaling pathways in corals. Here we investigate the dynamics of cAMP, a conserved signaling molecule that can regulate virtually every physiological process. Bioinformatics revealed corals have both transmembrane and soluble adenylyl cyclases (AC). Endogenous cAMP levels in ...

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

    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.

  9. The Diurnal Oscillation of MAP Kinase and Adenylyl Cyclase Activities in the Hippocampus Depends on the SCN

    Phan, Trongha; Chan, Guy; Sindreu, Carlos; Eckel-Mahan, Kristin; Storm, Daniel R.

    2011-01-01

    Consolidation of hippocampus dependent memory is dependent on activation of the cAMP/ Erk/MAPK signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadi...

  10. The Receptor Guanylyl Cyclase Type D (GC-D) Ligand Uroguanylin Promotes the Acquisition of Food Preferences in Mice

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

    2013-01-01

    Rodents rely on olfactory stimuli to communicate information between conspecifics that is critical for health and survival. For example, rodents that detect a food odor simultaneously with the social odor carbon disulfide (CS2) will acquire a preference for that food. Disruption of the chemosensory transduction cascade in CS2-sensitive olfactory sensory neurons (OSNs) that express the receptor guanylyl cyclase type D (GC-D; GC-D+ OSNs) will prevent mice from acquiring these preferences. GC-D+...

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

    Kabir Hassan Biswas

    2015-04-01

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

  12. The poly(adenylic acid)-protein complex is restricted to the nonpolysomal messenger ribonucleoprotein of Physarum polycephalum.

    Adams, D S; Noonan, D; Jeffery, W R

    1980-04-29

    The distribution of poly(adenylic acid) [poly(A)]-protein complexes in the polysomal and nonpolysomal messenger ribonucleoprotein (mRNP) fractions of Physarum polycephalum was examined in the present study. Poly-(A)-containing components released from the nonpolysomal mRNP by ribonuclease (RNase) digestion were quantitatively adsorbed to nitrocellulose filters at low ionic strength, were highly resistant to micrococcal nuclease under conditions in which free poly(A) was completely degraded, and sedimented as a 10-15S particle which was disrupted by sodium dodecyl sulfate and protease treatment. These are characteristics of the poly(A)-protein complex. In contrast,poly(A)-containing molecules released from the polysomes by RNase were refractive to nitrocellulose, were completely sensitive to micrococcal nuclease, and sedimented at 2-4 S, identical with the sedimentation exhibited by protein-free poly(A). Examination of the poly(A) sequences present in polysomal and nonpolysomal mRNP by polyacylamide gel electrophoresis showed that the former contained only very short sequences, averaging approximately 15 nucleotides, while the latter exhibited only much longer segments, averaging approximately 65 nucleotides. It is concluded that poly(A)-protein complexes are restricted to the nonpolysomal mRNP of Physarum and that the limiting factor in complex formation may be the length of the available poly(A) binding site. PMID:7378386

  13. A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy

    Hass, Mathias A. S.; Liu, Wei-Min [Leiden University, Leiden Institute of Chemistry (Netherlands); Agafonov, Roman V.; Otten, Renee; Phung, Lien A. [Brandeis University, Department of Biochemistry, Howard Hughes Medical Institute (United States); Schilder, Jesika T. [Leiden University, Leiden Institute of Chemistry (Netherlands); Kern, Dorothee [Brandeis University, Department of Biochemistry, Howard Hughes Medical Institute (United States); Ubbink, Marcellus, E-mail: m.ubbink@chem.leidenuniv.nl [Leiden University, Leiden Institute of Chemistry (Netherlands)

    2015-02-15

    NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements.

  14. A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy

    NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements

  15. Staphylococcus hyicus exfoliative toxin: Purification and demonstration of antigenic diversity among toxins from virulent strains

    Andresen, Lars Ole; Bille-Hansen, Vivi; Wegener, Henrik Caspar

    1997-01-01

    The exfoliative toxin produced by Staphylococcus hyicus strain 1289D-88 was purified as a single protein of approximately 30 kDa. Extracellular proteins of S. hyicus grown under small scale fermentation conditions were precipitated with ammonium sulfate. Separation of proteins was performed by....... hyicus. These results showed antigenic diversity among exfoliative toxins produced by different strains of S. hyicus. (C) 1997 Academic Press Limited....

  16. Toxins and antimicrobial peptides: interactions with membranes

    Schlamadinger, Diana E.; Gable, Jonathan E.; Kim, Judy E.

    2009-08-01

    The innate immunity to pathogenic invasion of organisms in the plant and animal kingdoms relies upon cationic antimicrobial peptides (AMPs) as the first line of defense. In addition to these natural peptide antibiotics, similar cationic peptides, such as the bee venom toxin melittin, act as nonspecific toxins. Molecular details of AMP and peptide toxin action are not known, but the universal function of these peptides to disrupt cell membranes of pathogenic bacteria (AMPs) or a diverse set of eukaryotes and prokaryotes (melittin) is widely accepted. Here, we have utilized spectroscopic techniques to elucidate peptide-membrane interactions of alpha-helical human and mouse AMPs of the cathelicidin family as well as the peptide toxin melittin. The activity of these natural peptides and their engineered analogs was studied on eukaryotic and prokaryotic membrane mimics consisting of melittin and human cathelicidin embedded in bilayer vesicles. Collectively, our results provide clues to the functional structures of the engineered and toxic peptides and may impact the design of synthetic antibiotic peptides that can be used against the growing number of antibiotic-resistant pathogens.

  17. Frey syndrome treatment with botulinum toxin.

    Dulguerov, P; Quinodoz, D; Cosendai, G; Piletta, P; Lehmann, W

    2000-06-01

    The goal of this work is to present our results of the intradermic infiltration with botulinum toxin in patients with Frey syndrome. Sixteen hemifaces in 15 patients were studied. Gustatory stimulation was evoked by sucking on a slice of lemon while measurements were done on both hemifaces, with the normal side being used as a control. Skin temperature and color (erythema) were measured with a digital surface thermometer and a skin chromameter, respectively. Sweat quantity and surface were measured by using the previously described blotting paper and iodine-sublimated paper histogram methods, respectively. Testing was repeated 2 weeks after skin infiltration with botulinum toxin (dilution of 50 U/mL). The interinjection distances were 1 cm, and 0.1 mL (5 U) was infiltrated at each injection site. Frey syndrome complaints disappeared in all patients. Small residual amounts of sweat were measurable. The difference in sweat quantity before and after botulinum toxin infiltration was significant in every patient (P < 0.001). Skin temperature and color measurement gave inconclusive results. In conclusion, Frey syndrome treatment with botulinum toxin is an efficient and well-tolerated technique. Further work should address the optimal injection parameters. PMID:10828793

  18. Okadaic Acid: More than a Diarrheic Toxin

    Josefina Méndez

    2013-10-01

    Full Text Available Okadaic acid (OA is one of the most frequent and worldwide distributed marine toxins. It is easily accumulated by shellfish, mainly bivalve mollusks and fish, and, subsequently, can be consumed by humans causing alimentary intoxications. OA is the main representative diarrheic shellfish poisoning (DSP toxin and its ingestion induces gastrointestinal symptoms, although it is not considered lethal. At the molecular level, OA is a specific inhibitor of several types of serine/threonine protein phosphatases and a tumor promoter in animal carcinogenesis experiments. In the last few decades, the potential toxic effects of OA, beyond its role as a DSP toxin, have been investigated in a number of studies. Alterations in DNA and cellular components, as well as effects on immune and nervous system, and even on embryonic development, have been increasingly reported. In this manuscript, results from all these studies are compiled and reviewed to clarify the role of this toxin not only as a DSP inductor but also as cause of alterations at the cellular and molecular levels, and to highlight the relevance of biomonitoring its effects on human health. Despite further investigations are required to elucidate OA mechanisms of action, toxicokinetics, and harmful effects, there are enough evidences illustrating its toxicity, not related to DSP induction, and, consequently, supporting a revision of the current regulation on OA levels in food.

  19. Future Avenues to Decrease Uremic Toxin Concentration.

    Vanholder, Raymond C; Eloot, Sunny; Glorieux, Griet L R L

    2016-04-01

    In this article, we review approaches for decreasing uremic solute concentrations in chronic kidney disease and in particular, in end-stage renal disease (ESRD). The rationale to do so is the straightforward relation between concentration and biological (toxic) effect for most toxins. The first section is devoted to extracorporeal strategies (kidney replacement therapy). In the context of high-flux hemodialysis and hemodiafiltration, we discuss increasing dialyzer blood and dialysate flows, frequent and/or extended dialysis, adsorption, bioartificial kidney, and changing physical conditions within the dialyzer (especially for protein-bound toxins). The next section focuses on the intestinal generation of uremic toxins, which in return is stimulated by uremic conditions. Therapeutic options are probiotics, prebiotics, synbiotics, and intestinal sorbents. Current data are conflicting, and these issues need further study before useful therapeutic concepts are developed. The following section is devoted to preservation of (residual) kidney function. Although many therapeutic options may overlap with therapies provided before ESRD, we focus on specific aspects of ESRD treatment, such as the risks of too-strict blood pressure and glycemic regulation and hemodynamic changes during dialysis. Finally, some recommendations are given on how research might be organized with regard to uremic toxins and their effects, removal, and impact on outcomes of uremic patients. PMID:26500179

  20. Treatment of Frontal Hyperhidrosis With Botulinum Toxin

    Ayşe Esra Koku Aksu

    Full Text Available Focal hyperhidrosis is usually localized to the axillae, palms and soles. Less frequently, hyperhidrosis may be confined to the forehead and may have negative impact on patient’s quality of life. A 34-year-old man presented to our clinic with the complaint of frontal hyperhidrosis. He was treated with botulinum toxin A. Thirty points were marked over the forehead and at each injection point, 0.15 ml (3U botulinum toxin A were injected intracutaneously. Hyperhidrosis was significantly reduced and the effect lasted for 12 months. Skindex-29, a quality-of-life measure for skin disease, was administered to the patient at the beginning and at the end of second week of botulinum toxin A injection. There was a significant improvement on the Skindex-29 scale at the end of the treatment. There was no any side effect detected during and after the treatment. Botulinum toxin A treatment is considered to be effective and safe for frontal hyperhidrosis.

  1. Natural toxins for use in pest management

    Natural toxins are a source of new chemical classes of pesticides, as well as environmentally and toxicologically safer molecules than many of the currently used pesticides. Furthermore, they often have molecular target sites that are not exploited by currently marketed pesticides. There are highly ...

  2. Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

    Kohno, Kenji (National Institute for Basic Biology, Aichi (Japan)); Hayes, H.; Mekada, Eisuke (Osaka Univ. (Japan)); Uchida, Tsuyoshi (National Institute for Basic Biology, Aichi (Japan) Osaka Univ. (Japan))

    1987-09-01

    A mutant of Chinese hamster ovary cells, GE1, that is highly resistant to diphtheria toxin was isolated. The mutant contains 50% ADP-ribosylatable elongation factor 2, but its protein synthesis was not inhibited by the toxin even at concentrations above 100 {mu}g/ml. {sup 125}I-labeled diphtheria toxin was associated with GE1 cells as well as with the parent cells but did not block protein synthesis of GE1 cells even when the cells were exposed to low pH in the presence or absence of NH{sub 4}Cl. The infections of GE1 cells and the parent cells by vesicular stomatitis virus were similar. GE1 cells were cross-resistant to Pseudomonas aeruginosa exotoxin A and so were about 1,000 times more resistant to this toxin than the parent cells. Hybrids of GE1 cells and the parent cells or mutant cells lacking a functional receptor were more sensitive to diphtheria toxin than GE1 cells. These results suggest that entry of diphtheria toxin into cells requires a cellular factor(s) in addition to those involved in receptor function and acidification of endosomes and that GE1 cells do not express this cellular factor. This character is recessive in GE1 cells.

  3. Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases

    Uzal, F. A.; J. E. Vidal; McClane, B A; Gurjar, A. A.

    2010-01-01

    Clostridium perfringens is a gram-positive anaerobic rod that is classified into 5 toxinotypes (A, B, C, D, and E) according to the production of 4 major toxins, namely alpha (CPA), beta (CPB), epsilon (ETX) and iota (ITX). However, this microorganism can produce up to 16 toxins in various combinations, including lethal toxins such as perfringolysin O (PFO), enterotoxin (CPE), and beta2 toxin (CPB2). Most diseases caused by this microorganism are mediated by one or more of these toxins. The r...

  4. Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice.

    Losada-Eaton, D M; Fernandez-Miyakawa, M E

    2010-12-01

    Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia. PMID:20434186

  5. Preliminary spectroscopic characterization of six toxins from Latin American scorpions.

    Possani, L; Steinmetz, W E; Dent, M A; Alagón, A C; Wüthrich, K

    1981-07-28

    This paper reports on spectroscopic studies of six toxins from the Latin American scorpions Centruroides noxius Hoffmann, Centruroides elegans Thorell and Tityus serrulatus Lutz and Mello. The isolation and purification of five of these toxins was described previously. The preparation of toxin II.9.2.2 from the venom of C. noxius is first described here. Circular dichroism and nuclear magnetic resonance spectra indicate similarities and differences between these scorpion toxins and previously characterized snake toxins. While there is evidence that the toxins from scorpions and snakes both contain extended beta-sheet secondary structures, the spectral properties of the scorpion toxins are overall of a different type from those of snake toxins. Among the six scorpion toxins those from T. serrulatus have spectral properties markedly different from those of the Centruroides species. Furthermore, thermal denaturation and amide proton exchange measurements showed that the globular structures of the Tityus toxins were markedly less stable and less rigid than those of the Centruroides toxins. PMID:7284435

  6. Mode of action of mosquitocidal Bacillus thuringiensis toxins.

    Soberón, Mario; Fernández, Luisa E; Pérez, Claudia; Gill, Sarjeet S; Bravo, Alejandra

    2007-04-01

    Cry toxins from Bacillus thuringiensis (Bt) are used for insect control. Their primary action is to lyse midgut epithelial cells. In lepidopteran insects, Cry1A monomeric toxins interact with a first receptor and this interaction triggers toxin oligomerization. The oligomeric structure interacts then with a second GPI-anchored receptor that induces insertion into membrane microdomains and larvae death. In the case of mosquitocidal Bt strains, two different toxins participate, Cry and Cyt. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin-resistance. We will summarize recent findings on the identification of Cry receptors in mosquitoes and the mechanism of synergism: Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a Cry membrane-bound receptor. PMID:17145072

  7. The molecular mechanism of heme loss from oxidized soluble guanylate cyclase induced by conformational change.

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

    2016-05-01

    Heme oxidation and loss of soluble guanylate cyclase (sGC) is thought to be an important contributor to the development of cardiovascular diseases. Nevertheless, it remains unknown why the heme loses readily in oxidized sGC. In the current study, the conformational change of sGC upon heme oxidation by ODQ was studied based on the fluorescence resonance energy transfer (FRET) between the heme and a fluorophore fluorescein arsenical helix binder (FlAsH-EDT2) labeled at different domains of sGC β1. This study provides an opportunity to monitor the domain movement of sGC relative to the heme. The results indicated that heme oxidation by ODQ in truncated sCC induced the heme-associated αF helix moving away from the heme, the Per/Arnt/Sim domain (PAS) domain moving closer to the heme, but led the helical domain going further from the heme. We proposed that the synergistic effect of these conformational changes of the discrete region upon heme oxidation forces the heme pocket open, and subsequent heme loss readily. Furthermore, the kinetic studies suggested that the heme oxidation was a fast process and the conformational change was a relatively slow process. The kinetics of heme loss from oxidized sGC was monitored by a new method based on the heme group de-quenching the fluorescence of FlAsH-EDT2. PMID:26876536

  8. Soluble Guanylate Cyclase Stimulators: a Novel Treatment Option for Heart Failure Associated with Cardiorenal Syndromes?

    Dubin, Ruth F; Shah, Sanjiv J

    2016-06-01

    Heart failure in the setting of chronic kidney disease (CKD) is an increasingly common scenario and carries a poor prognosis. Clinicians lack tools for primary or secondary heart failure prevention in patients with cardiorenal syndromes. In patients without CKD, angiotensin-converting enzyme inhibitors (ACE-I) or angiotensin receptor blockers (ARB) and statins mitigate cardiovascular risk in large part due to salutary effects on the endothelium. In the setting of CKD, use of these therapies is limited by adverse effects of hyperkalemia in pre-dialysis CKD (ACE-I/ARB), or potential increased risk of stroke in end-stage renal disease (statins). The soluble guanylate cyclase (sGC) stimulators are a novel class of medications that promote endothelial and myocardial function with no known risk of hyperkalemia or stroke. In this review, we discuss the evidence emerging from recent clinical trials of sGC stimulators in pulmonary hypertension and heart failure, the diseased pathways involved in cardiorenal syndromes likely to be restored by sGC stimulators, and several strategies for designing future clinical trials of cardiorenal syndromes that might shorten the timeline for discovery and approval of effective cardiovascular therapies in these high-risk patients. PMID:27118234

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

    Markus Otto

    2016-01-01

    Full Text Available Jasmonates (JAs are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC. Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4 have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 + AOC1 and AOC4 + AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles.

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

    Otto, Markus; Naumann, Christin; Brandt, Wolfgang; Wasternack, Claus; Hause, Bettina

    2016-01-01

    Jasmonates (JAs) are lipid-derived signals in plant stress responses and development. A crucial step in JA biosynthesis is catalyzed by allene oxide cyclase (AOC). Four genes encoding functional AOCs (AOC1, AOC2, AOC3 and AOC4) have been characterized for Arabidopsis thaliana in terms of organ- and tissue-specific expression, mutant phenotypes, promoter activities and initial in vivo protein interaction studies suggesting functional redundancy and diversification, including first hints at enzyme activity control by protein-protein interaction. Here, these analyses were extended by detailed analysis of recombinant proteins produced in Escherichia coli. Treatment of purified AOC2 with SDS at different temperatures, chemical cross-linking experiments and protein structure analysis by molecular modelling approaches were performed. Several salt bridges between monomers and a hydrophobic core within the AOC2 trimer were identified and functionally proven by site-directed mutagenesis. The data obtained showed that AOC2 acts as a trimer. Finally, AOC activity was determined in heteromers formed by pairwise combinations of the four AOC isoforms. The highest activities were found for heteromers containing AOC4 + AOC1 and AOC4 + AOC2, respectively. All data are in line with an enzyme activity control of all four AOCs by heteromerization, thereby supporting a putative fine-tuning in JA formation by various regulatory principles. PMID:27135223

  11. The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit

    Ampomah-Dwamena, Charles; McGhie, Tony; Wibisono, Reginald; Montefiori, Mirco; Hellens, Roger P.; Allan, Andrew C.

    2009-01-01

    The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-β) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-β), and epsilon carotene hydroxylase (CRH-ϵ) showed some variation in gene expression. The LCY-β gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-β gene. PMID:19574250

  12. Modulation of soluble guanylate cyclase for the treatment of erectile dysfunction.

    Lasker, George F; Pankey, Edward A; Kadowitz, Philip J

    2013-07-01

    Nitric oxide (NO) is the principal mediator of penile erection, and PDE-5 inhibitors are the first-line agents used to treat erectile dysfunction (ED). When NO formation or bioavailability is decreased by oxidative stress and PDE-5 inhibitors are no longer effective, a new class of agents called soluble guanylate cyclase (sGC) stimulators like BAY 41-8543 will induce erection. sGC stimulators bind to the normally reduced, NO-sensitive form of sGC to increase cGMP formation and promote erection. The sGC stimulators produce normal erectile responses when NO formation is inhibited and the nerves innervating the corpora cavernosa are damaged. However, with severe oxidative stress, the heme iron on sGC can be oxidized, rendering the enzyme unresponsive to NO or sGC stimulators. In this pathophysiological situation, another newly developed class of agents called sGC activators can increase the catalytic activity of the oxidized enzyme, increase cGMP formation, and promote erection. The use of newer agents that stimulate or activate sGC to promote erection and treat ED is discussed in this brief review article. PMID:23817801

  13. DgcA, a diguanylate cyclase from Xanthomonas oryzae pv. oryzae regulates bacterial pathogenicity on rice

    Su, Jianmei; Zou, Xia; Huang, Liangbo; Bai, Tenglong; Liu, Shu; Yuan, Meng; Chou, Shan-Ho; He, Ya-Wen; Wang, Haihong; He, Jin

    2016-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice blight disease as well as a serious phytopathogen worldwide. It is also one of the model organisms for studying bacteria-plant interactions. Current progress in bacterial signal transduction pathways has identified cyclic di-GMP as a major second messenger molecule in controlling Xanthomonas pathogenicity. However, it still remains largely unclear how c-di-GMP regulates the secretion of bacterial virulence factors in Xoo. In this study, we focused on the important roles played by DgcA (XOO3988), one of our previously identified diguanylate cyclases in Xoo, through further investigating the phenotypes of several dgcA-related mutants, namely, the dgcA-knockout mutant ΔdgcA, the dgcA overexpression strain OdgcA, the dgcA complemented strain CdgcA and the wild-type strain. The results showed that dgcA negatively affected virulence, EPS production, bacterial autoaggregation and motility, but positively triggered biofilm formation via modulating the intracellular c-di-GMP levels. RNA-seq data further identified 349 differentially expressed genes controlled by DgcA, providing a foundation for a more solid understanding of the signal transduction pathways in Xoo. Collectively, the present study highlights DgcA as a major regulator of Xoo virulence, and can serve as a potential target for preventing rice blight diseases. PMID:27193392

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

    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.

  15. The kiwifruit lycopene beta-cyclase plays a significant role in carotenoid accumulation in fruit.

    Ampomah-Dwamena, Charles; McGhie, Tony; Wibisono, Reginald; Montefiori, Mirco; Hellens, Roger P; Allan, Andrew C

    2009-01-01

    The composition of carotenoids, along with anthocyanins and chlorophyll, accounts for the distinctive range of colour found in the Actinidia (kiwifruit) species. Lutein and beta-carotene are the most abundant carotenoids found during fruit development, with beta-carotene concentration increasing rapidly during fruit maturation and ripening. In addition, the accumulation of beta-carotene and lutein is influenced by the temperature at which harvested fruit are stored. Expression analysis of carotenoid biosynthetic genes among different genotypes and fruit developmental stages identified Actinidia lycopene beta-cyclase (LCY-beta) as the gene whose expression pattern appeared to be associated with both total carotenoid and beta-carotene accumulation. Phytoene desaturase (PDS) expression was the least variable among the different genotypes, while zeta carotene desaturase (ZDS), beta-carotene hydroxylase (CRH-beta), and epsilon carotene hydroxylase (CRH-epsilon) showed some variation in gene expression. The LCY-beta gene was functionally tested in bacteria and shown to convert lycopene and delta-carotene to beta-carotene and alpha-carotene respectively. This indicates that the accumulation of beta-carotene, the major carotenoid in these kiwifruit species, appears to be controlled by the level of expression of LCY-beta gene. PMID:19574250

  16. Disruption of Epac1 protects the heart from adenylyl cyclase type 5-mediated cardiac dysfunction.

    Cai, Wenqian; Fujita, Takayuki; Hidaka, Yuko; Jin, Huiling; Suita, Kenji; Prajapati, Rajesh; Liang, Chen; Umemura, Masanari; Yokoyama, Utako; Sato, Motohiko; Okumura, Satoshi; Ishikawa, Yoshihiro

    2016-06-17

    Type 5 adenylyl cyclase (AC5) plays an important role in the development of chronic catecholamine stress-induced heart failure and arrhythmia in mice. Epac (exchange protein activated by cAMP), which is directly activated by cAMP independent of protein kinase A, has been recently identified as a novel mediator of cAMP signaling in the heart. However, the role of Epac in AC5-mediated cardiac dysfunction and arrhythmias remains poorly understood. We therefore generated AC5 transgenic mice (AC5TG) with selective disruption of the Epac1 gene (AC5TG-Epac1KO), and compared their phenotypes with those of AC5TG after chronic isoproterenol (ISO) infusion. Decreased cardiac function as well as increased susceptibility to pacing-induced atrial fibrillation (AF) in response to ISO were significantly attenuated in AC5TG-Epac1KO mice, compared to AC5TG mice. Increased cardiac apoptosis and cardiac fibrosis were also concomitantly attenuated in AC5TG-Epac1KO mice compared to AC5TG mice. These findings indicate that Epac1 plays an important role in AC5-mediated cardiac dysfunction and AF susceptibility. PMID:27117748

  17. The Functional State of Hormone-Sensitive Adenylyl Cyclase Signaling System in Diabetes Mellitus

    Alexander O. Shpakov

    2013-01-01

    Full Text Available Diabetes mellitus (DM induces a large number of diseases of the nervous, cardiovascular, and some other systems of the organism. One of the main causes of the diseases is the changes in the functional activity of hormonal signaling systems which lead to the alterations and abnormalities of the cellular processes and contribute to triggering and developing many DM complications. The key role in the control of physiological and biochemical processes belongs to the adenylyl cyclase (AC signaling system, sensitive to biogenic amines and polypeptide hormones. The review is devoted to the changes in the GPCR-G protein-AC system in the brain, heart, skeletal muscles, liver, and the adipose tissue in experimental and human DM of the types 1 and 2 and also to the role of the changes in AC signaling in the pathogenesis and etiology of DM and its complications. It is shown that the changes of the functional state of hormone-sensitive AC system are dependent to a large extent on the type and duration of DM and in experimental DM on the model of the disease. The degree of alterations and abnormalities of AC signaling pathways correlates very well with the severity of DM and its complications.

  18. DgcA, a diguanylate cyclase from Xanthomonas oryzae pv. oryzae regulates bacterial pathogenicity on rice.

    Su, Jianmei; Zou, Xia; Huang, Liangbo; Bai, Tenglong; Liu, Shu; Yuan, Meng; Chou, Shan-Ho; He, Ya-Wen; Wang, Haihong; He, Jin

    2016-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice blight disease as well as a serious phytopathogen worldwide. It is also one of the model organisms for studying bacteria-plant interactions. Current progress in bacterial signal transduction pathways has identified cyclic di-GMP as a major second messenger molecule in controlling Xanthomonas pathogenicity. However, it still remains largely unclear how c-di-GMP regulates the secretion of bacterial virulence factors in Xoo. In this study, we focused on the important roles played by DgcA (XOO3988), one of our previously identified diguanylate cyclases in Xoo, through further investigating the phenotypes of several dgcA-related mutants, namely, the dgcA-knockout mutant ΔdgcA, the dgcA overexpression strain OdgcA, the dgcA complemented strain CdgcA and the wild-type strain. The results showed that dgcA negatively affected virulence, EPS production, bacterial autoaggregation and motility, but positively triggered biofilm formation via modulating the intracellular c-di-GMP levels. RNA-seq data further identified 349 differentially expressed genes controlled by DgcA, providing a foundation for a more solid understanding of the signal transduction pathways in Xoo. Collectively, the present study highlights DgcA as a major regulator of Xoo virulence, and can serve as a potential target for preventing rice blight diseases. PMID:27193392

  19. Guanylyl cyclase / atrial natriuretic peptide receptor-A: role in the pathophysiology of cardiovascular regulation.

    Pandey, Kailash N

    2011-08-01

    Atrial natriuretic factor (ANF), also known as atrial natriuretic peptide (ANP), is an endogenous and potent hypotensive hormone that elicits natriuretic, diuretic, vasorelaxant, and anti-proliferative effects, which are important in the control of blood pressure and cardiovascular events. One principal locus involved in the regulatory action of ANP and brain natriuretic peptide (BNP) is guanylyl cyclase / natriuretic peptide receptor-A (GC-A/NPRA). Studies on ANP, BNP, and their receptor, GC-A/NPRA, have greatly increased our knowledge of the control of hypertension and cardiovascular disorders. Cellular, biochemical, and molecular studies have helped to delineate the receptor function and signaling mechanisms of NPRA. Gene-targeted and transgenic mouse models have advanced our understanding of the importance of ANP, BNP, and GC-A/NPRA in disease states at the molecular level. Importantly, ANP and BNP are used as critical markers of cardiac events; however, their therapeutic potentials for the diagnosis and treatment of hypertension, heart failure, and stroke have just begun to be realized. We are now just at the initial stage of molecular therapeutics and pharmacogenomic advancement of the natriuretic peptides. More investigations should be undertaken and ongoing ones be extended in this important field. PMID:21815745

  20. Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.

    WANG Sheng; ZHAO Guo-hong; JIA Yin-hua; DU Xiong-ming

    2009-01-01

    The aim of this study was to clone CAP (adenylyl cyclase-associated protein) gene from Gossypium arboreum L. and develop a platform for expressing and purifying CAP protein, which is a base for the construction and function researches of CAP. In this work, a CAP homolog from cotton (DPL971) ovule was identified and cloned. And the cDNA sequence consisted of an open reading frame of 1416 nucleotides encoding a protein of 471 amino acid residues with a calculated molecular weight of 50.6 kDa. To gain insight on the CAP role in cotton fiber development, the cloned CAP cDNA was expressed. A significant higher yield pure protein was obtained with the chromatographic method. Further experiments showed that the purified protein can bind with the actin in vitro indicating that the recombinant cotton CAP is functional. The procedure described here produced high yield pure protein through one chromatographic step, suitable for further structure-function studies.

  1. Adenylyl cyclase 6 mediates loading-induced bone adaptation in vivo.

    Lee, Kristen L; Hoey, David A; Spasic, Milos; Tang, Tong; Hammond, H Kirk; Jacobs, Christopher R

    2014-03-01

    Primary cilia are single, nonmotile, antenna-like structures extending from the apical membrane of most mammalian cells. They may mediate mechanotransduction, the conversion of external mechanical stimuli into biochemical intracellular signals. Previously we demonstrated that adenylyl cyclase 6 (AC6), a membrane-bound enzyme enriched in primary cilia of MLO-Y4 osteocyte-like cells, may play a role in a primary cilium-dependent mechanism of osteocyte mechanotransduction in vitro. In this study, we determined whether AC6 deletion impairs loading-induced bone formation in vivo. Skeletally mature mice with a global knockout of AC6 exhibited normal bone morphology and responded to osteogenic chemical stimuli similar to wild-type mice. Following ulnar loading over 3 consecutive days, bone formation parameters were assessed using dynamic histomorphometry. Mice lacking AC6 formed significantly less bone than control animals (41% lower bone formation rate). Furthermore, there was an attenuated flow-induced increase in COX-2 mRNA expression levels in primary bone cells isolated from AC6 knockout mice compared to controls (1.3±0.1- vs. 2.6±0.2-fold increase). Collectively, these data indicate that AC6 plays a role in loading-induced bone adaptation, and these findings are consistent with our previous studies implicating primary cilia and AC6 in a novel mechanism of osteocyte mechanotransduction. PMID:24277577

  2. Expression of nitric oxide synthase and guanylate cyclase in the human ciliary body and trabecular meshwork

    WU Ren-yi; MA Ning

    2012-01-01

    Background The role played by the nitric oxide (NO) signaling pathway in the aqueous humor dynamics is still unclear.This study was designed to investigate the expression and distribution of NO synthase (NOS) isoforms and guanylate cyclase (GC) in human ciliary body,trabecular meshwork and the Schlemm's canal.Methods Twelve eyes after corneal transplantation were used.Expression of three NOS isoforms (i.e.neuronal NOS (nNOS),inducible NOS (iNOS) and endothelial NOS (eNOS)) and GC were assessed in 10 eyes by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC.Ciliary bodies were dissected free and the total proteins were extracted.Western blotting was performed to confirm the protein expression of 3 NOS isoforms and GC.Results Expression of 3 NOS isoforms and GC were observed in the ciliary epithelium,ciliary muscle,trabecular meshwork and the endothelium of the Schlemm's canal.Immunoreactivity of nNOS was detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and pigmented epithelial (PE) cells.Protein expressions of 3 NOS isoforms and GC were confirmed in isolated human ciliary body by Western blotting.Conclusions The expression of NOS isoforms and GC in human ciliary body suggest the possible involvement of NO and cyclic guanosine monophosphate (cyclic GMP,cGMP) signaling pathway in the ciliary body,and may play a role in both processes of aqueous humor formation and drainage.

  3. Soluble guanylate cyclase stimulation: an emerging option in pulmonary hypertension therapy

    H. A. Ghofrani

    2009-03-01

    Full Text Available The prognosis for patients with pulmonary hypertension remains poor despite recent treatment advances, and there is a need for therapies with new modes of action. Nitric oxide (NO is an endogenous vasodilator, the levels of which are regulated throughout the lung to ensure preferential perfusion of well-ventilated regions. Drugs that act in synergy with endogenous NO would therefore promote pulmonary vasodilation while maintaining optimal gas exchange. Riociguat is an oral stimulator of the NO receptor soluble guanylate cyclase. It synergises with NO and has demonstrated vasodilatory and antiremodelling properties in preclinical studies. Riociguat has been shown to have a favourable safety profile in healthy volunteers and in patients with pulmonary hypertension. Pharmacokinetic analyses have revealed substantial interindividual variation, suggesting that individual dose titration will be required. In a proof-of-concept study of patients with pulmonary arterial hypertension or chronic thromboembolic pulmonary hypertension, riociguat improved cardiopulmonary haemodynamics from baseline. It also caused systemic vasodilation, which was well tolerated but should be monitored in future studies. Dose titration of riociguat should promote pulmonary vasodilation while maintaining control of systemic effects, and has been investigated in a phase-II study of patients with pulmonary arterial hypertension or chronic thromboembolic pulmonary hypertension. Preliminary results indicate that phase-III trials are warranted.

  4. Toxin(s), Other Than Cholera Toxin, Produced by Environmental Non O1 Non O139 Vibrio cholerae

    Kohinur Begum; Chowdhury R. Ahsan; Mohammad Ansaruzzaman; Dilip K. Dutta; Qazi S.Ahmad; Kaisar A. Talukder

    2006-01-01

    A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis.Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin (ST), as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary (CHO) cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin (CT). Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen (CAMP) method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.

  5. Diphtheria toxin translocation across cellular membranes is regulated by sphingolipids

    Diphtheria toxin is translocated across cellular membranes when receptor-bound toxin is exposed to low pH. To study the role of sphingolipids for toxin translocation, both a mutant cell line lacking the first enzyme in de novo sphingolipid synthesis, serine palmitoyltransferase, and a specific inhibitor of the same enzyme, myriocin, were used. The serine palmitoyltransferase-deficient cell line (LY-B) was found to be 10-15 times more sensitive to diphtheria toxin than the genetically complemented cell line (LY-B/cLCB1) and the wild-type cell line (CHO-K1), both when toxin translocation directly across the plasma membrane was induced by exposing cells with surface-bound toxin to low pH, and when the toxin followed its normal route via acidified endosomes into the cytosol. Toxin binding was similar in these three cell lines. Furthermore, inhibition of serine palmitoyltransferase activity by addition of myriocin sensitized the two control cell lines (LY-B/cLCB1 and CHO-K1) to diphtheria toxin, whereas, as expected, no effect was observed in cells lacking serine palmitoyltransferase (LY-B). In conclusion, diphtheria toxin translocation is facilitated by depletion of membrane sphingolipids

  6. Sea Anemone (Cnidaria, Anthozoa, Actiniaria Toxins: An Overview

    Agostinho Antunes

    2012-08-01

    Full Text Available The Cnidaria phylum includes organisms that are among the most venomous animals. The Anthozoa class includes sea anemones, hard corals, soft corals and sea pens. The composition of cnidarian venoms is not known in detail, but they appear to contain a variety of compounds. Currently around 250 of those compounds have been identified (peptides, proteins, enzymes and proteinase inhibitors and non-proteinaceous substances (purines, quaternary ammonium compounds, biogenic amines and betaines, but very few genes encoding toxins were described and only a few related protein three-dimensional structures are available. Toxins are used for prey acquisition, but also to deter potential predators (with neurotoxicity and cardiotoxicity effects and even to fight territorial disputes. Cnidaria toxins have been identified on the nematocysts located on the tentacles, acrorhagi and acontia, and in the mucous coat that covers the animal body. Sea anemone toxins comprise mainly proteins and peptides that are cytolytic or neurotoxic with its potency varying with the structure and site of action and are efficient in targeting different animals, such as insects, crustaceans and vertebrates. Sea anemones toxins include voltage-gated Na+ and K+ channels toxins, acid-sensing ion channel toxins, Cytolysins, toxins with Kunitz-type protease inhibitors activity and toxins with Phospholipase A2 activity. In this review we assessed the phylogentic relationships of sea anemone toxins, characterized such toxins, the genes encoding them and the toxins three-dimensional structures, further providing a state-of-the-art description of the procedures involved in the isolation and purification of bioactive toxins.

  7. Molecular composition of Clostridium botulinum type A progenitor toxins.

    Inoue, K; Fujinaga, Y; Watanabe, T; Ohyama, T; Takeshi, K; Moriishi, K; Nakajima, H; Inoue, K; Oguma, K

    1996-01-01

    The molecular composition of progenitor toxins produced by a Clostridium botulinum type A strain (A-NIH) was analyzed. The strain produced three types of progenitor toxins (19 S, 16 S, and 12 S) as reported previously. Purified 19 S and 16 S toxins demonstrated the same banding profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that they consist of the same protein components. The nontoxic components of the 19 S and 16 S toxins are a nontoxic non-hemagglutinin (HA) (molecular mass, 120 kDa) and HA. HA could be fractionated into five subcomponents with molecular masses of 52, 35, 20, 19, and 15 kDa in the presence of 2-mercaptoethanol. The molar ratios of neurotoxins, nontoxic non-HAs, and each HA subcomponent of the 19 S and 16 S toxins showed that only HA-35 of the 19 S toxin was approximately twice the size of that of the 16 S toxin, suggesting that the 19 S toxin is a dimer of the 16 S toxin cross-linked by the 35-kDa subcomponent. The nontoxic non-HA of the 12 S toxin, but not those of the 19 S and 16 S toxins, demonstrated two bands with molecular masses of 106 and 13 kDa on SDS-PAGE with or without 2-mercaptoethanol. It was concluded from the N-terminal amino acid sequences that 106- and 13-kDa proteins were generated by a cleavage of whole nontoxic non-HA. This may explain why the 12 S and 16 S (and 19 S) toxins exist in the same culture. We also found that the HA and its 35-kDa subcomponent exist in a free state in the culture fluid along with three types of progenitor toxins. PMID:8613365

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

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

    2009-10-06

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

  9. hCINAP is an atypical mammalian nuclear adenylate kinase with an ATPase motif: structural and functional studies.

    Drakou, Christina E; Malekkou, Anna; Hayes, Joseph M; Lederer, Carsten W; Leonidas, Demetres D; Oikonomakos, Nikos G; Lamond, Angus I; Santama, Niovi; Zographos, Spyros E

    2012-01-01

    Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. Despite its intriguing structure, unique properties and cellular localization, the enzymatic mechanism and biological function of hCINAP have remained poorly characterized. Here, we offer the first high-resolution structure of hCINAP in complex with the substrate ADP (and dADP), the structure of hCINAP with a sulfate ion bound at the AMP binding site, and the structure of the ternary complex hCINAP-Mg(2+) ADP-Pi. Induced fit docking calculations are used to predict the structure of the hCINAP-Mg(2+) ATP-AMP ternary complex. Structural analysis suggested a functional role for His79 in the Walker B motif. Kinetic analysis of mutant hCINAP-H79G indicates that His79 affects both AK and ATPase catalytic efficiency and induces homodimer formation. Finally, we show that in vivo expression of hCINAP-H79G in human cells is toxic and drastically deregulates the number and appearance of CBs in the cell nucleus. Our findings suggest that hCINAP may not simply regulate nucleotide homeostasis, but may have broader functionality, including control of CB assembly and disassembly in the nucleus of human cells. PMID:22038794

  10. Mechanism of adenylate kinase. Is there a relationship between local substrate dynamics, local binding energy, and the catalytic mechanism?

    Adenylyl (β,γ-methylene)diphosphonic acid (AMPPCP) labeled with deuterium at the adenine ring ([8-2H]AMPPCP) and at the β,γ-methylene group (AMPPCD2P), as well as adenosine 5'-monophosphate labeled at the adenine ring ([8-2H]AMP), was synthesized and used for deuterium nuclear magnetic resonance (NMR) determination of effective correlation times (τc) of the free nucleotide and the complexes with adenylate kinase (AK). Extensive and rigorous control experiments and theoretical analysis were performed to justify the validity of the experimental approaches, particularly the fast exchange condition, and the reliability of the τc values obtained. For the free nucleotide, the results suggest that the phosphonate group of free AMPPCP possesses appreciable local mobility relative to the adenine ring and that complexation with Mg2+ greatly reduced such a local mobility. These results suggest that the adenine ring of substrates is rigidly bound in all cases, that the phosphonate chain of AMPPCP possesses considerable local mobility, and that Mg2+ reduces such local mobility but does not totally immobilize it. The results suggest that no general correlation exists between the local rigidity of portions of a bound substrate and the corresponding (ground state) local binding energy contributed by these portions. The authors have found that the Ki values for the mixture, the Δ isomer, and the Λ isomer of CrATP are 16, 11, and 20 μM, respectively, which suggest that ground-state binding by AK is stereochemically permissive. The results of both problems fully support the conclusion that the phosphonate chain of AK-MgAMPPCP possesses considerable local mobility and illuminate the relationship between the dynamics of bound substrates and the catalytic mechanism

  11. Shiga Toxin Interaction with Human Intestinal Epithelium

    Stephanie Schüller

    2011-06-01

    Full Text Available After ingestion via contaminated food or water, enterohaemorrhagic E. coli colonises the intestinal mucosa and produces Shiga toxins (Stx. No Stx-specific secretion system has been described so far, and it is assumed that Stx are released into the gut lumen after bacterial lysis. Human intestinal epithelium does not express the Stx receptor Gb3 or other Stx binding sites, and it remains unknown how Stx cross the intestinal epithelial barrier and gain access to the systemic circulation. This review summarises current knowledge about the influence of the intestinal environment on Stx production and release, Stx interaction with intestinal epithelial cells and intracellular uptake, and toxin translocation into underlying tissues. Furthermore, it highlights gaps in understanding that need to be addressed by future research.

  12. Clostridium perfringens epsilon toxin: the third most potent bacterial toxin known.

    Alves, Guilherme Guerra; Machado de Ávila, Ricardo Andrez; Chávez-Olórtegui, Carlos Delfin; Lobato, Francisco Carlos Faria

    2014-12-01

    Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains and causes enterotoxemia, a highly lethal disease with major impacts on the farming of domestic ruminants, particularly sheep. ETX belongs to the aerolysin-like pore-forming toxin family. Although ETX has striking similarities to other toxins in this family, ETX is often more potent, with an LD50 of 100 ng/kg in mice. Due to this high potency, ETX is considered as a potential bioterrorism agent and has been classified as a category B biological agent by the Centers for Disease Control and Prevention (CDC) of the United States. The protoxin is converted to an active toxin through proteolytic cleavage performed by specific proteases. ETX is absorbed and acts locally in the intestines then subsequently binds to and causes lesions in other organs, including the kidneys, lungs and brain. The importance of this toxin for veterinary medicine and its possible use as a biological weapon have drawn the attention of researchers and have led to a large number of studies investigating ETX. The aim of the present work is to review the existing knowledge on ETX from C. perfringens type B and D. PMID:25234332

  13. Frey syndrome treatment with botulinum toxin

    Dulguerov, Pavel; Quinodoz, Didier François; Cosendai, Grégoire; Piletta Zanin, Pierre; Lehmann, Willy

    2000-01-01

    The goal of this work is to present our results of the intradermic infiltration with botulinum toxin in patients with Frey syndrome. Sixteen hemifaces in 15 patients were studied. Gustatory stimulation was evoked by sucking on a slice of lemon while measurements were done on both hemifaces, with the normal side being used as a control. Skin temperature and color (erythema) were measured with a digital surface thermometer and a skin chromameter, respectively. Sweat quantity and surface were me...

  14. Abstract Action Potential Models for Toxin Recognition

    Peterson, James; Khan, Taufiquar

    2005-01-01

    In this paper, we present a robust methodology using mathematical pattern recognition schemes to detect and classify events in action potentials for recognizing toxins in biological cells. We focus on event detection in action potential via abstraction of information content into a low dimensional feature vector within the constrained computational environment of a biosensor. We use generated families of action potentials from a classic Hodgkin–Huxley model to verify our methodology and build...

  15. Natural Toxins for Use in Pest Management

    Schrader, Kevin K.; Nurhayat Tabanca; Wedge, David E; Meepagala, Kumudini M.; Cantrell, Charles L.; Duke, Stephen O.

    2010-01-01

    Natural toxins are a source of new chemical classes of pesticides, as well as environmentally and toxicologically safer molecules than many of the currently used pesticides. Furthermore, they often have molecular target sites that are not exploited by currently marketed pesticides. There are highly successful products based on natural compounds in the major pesticide classes. These include the herbicide glufosinate (synthetic phosphinothricin), the spinosad insecticides, and the strobilurin f...

  16. Botulinum Toxin in the Treatment of Cystitis

    Lucia Lucan; EnachDan Enache; Bodo Ors Zsombor

    2014-01-01

    Objective. This study will follow the effectiveness of treatment of chronic cystitis in menopausal women, associated with overactive bladder syndrome, performed by endoscopic injection of botulinum toxin type A to patients who previously attended drug treatment and bladder instilational treatment but with persistence of clinical manifestations.Materials and methods. We studied 43 patients, in menopause with chronic recurrent cystitis and overactive bladder. The data were statistically analyze...

  17. Why do we study animal toxins?

    Zhang, Yun

    2015-01-01

    Venom (toxins) is an important trait evolved along the evolutionary tree of animals. Our knowledges on venoms, such as their origins and loss, the biological relevance and the coevolutionary patterns with other organisms are greatly helpful in understanding many fundamental biological questions, i.e., the environmental adaptation and survival competition, the evolution shaped development and balance of venoms, and the sophisticated correlations among venom, immunity, body power, intelligence,...

  18. Toxins for Transgenic Resistance to Hemipteran Pests

    Bryony C. Bonning; Chougule, Nanasaheb P.

    2012-01-01

    The sap sucking insects (Hemiptera), which include aphids, whiteflies, plant bugs and stink bugs, have emerged as major agricultural pests. The Hemiptera cause direct damage by feeding on crops, and in some cases indirect damage by transmission of plant viruses. Current management relies almost exclusively on application of classical chemical insecticides. While the development of transgenic crops expressing toxins derived from the bacterium Bacillus thuringiensis (Bt) ha...

  19. Shiga Toxin Interaction with Human Intestinal Epithelium

    Stephanie Schüller

    2011-01-01

    After ingestion via contaminated food or water, enterohaemorrhagic E. coli colonises the intestinal mucosa and produces Shiga toxins (Stx). No Stx-specific secretion system has been described so far, and it is assumed that Stx are released into the gut lumen after bacterial lysis. Human intestinal epithelium does not express the Stx receptor Gb3 or other Stx binding sites, and it remains unknown how Stx cross the intestinal epithelial barrier and gain access to the systemic circulation. This ...

  20. Emerging food pathogens and bacterial toxins.

    Bielecki, Jacek

    2003-01-01

    Many different foodborne diseases have been described. For example, Shigella bacteria, hepatitis A virus and Norwalk virus were shown as a unwashed hands microorganisms, but pathogen Campylobacter and Escherichia coli were named as raw and undercooked meat and poultry or raw milk and untreated water born bacteria. However, two of them: Listeria monocytogenes and Yersinia enterocolitica are known as growing at refrigerator temperatures. Essential virulence determinants of Listeria monocytogenes pathogenicity are well known as a bacterial toxins. Basic molecular mechanisms of pathogenicity depending from these toxins were presented. It was shown that other bacterial toxins may act as very danger food poisoning substances. This is why elimination of pathogenic microorganisms from foods is an obvious solution in some food processes, however this approach is not practical or even desirable in many processes. Thus, risk assessment and microbial monitoring will continue to play important roles in ensuring food safety. Some technological advances have the capability of delivering detection systems that can not only monitor pathogenic microorganisms, but also entire microbial populations in the food matrix. PMID:15058810

  1. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    Stefanie Verherstraeten

    2015-05-01

    Full Text Available The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin, a pore-forming cholesterol-dependent cytolysin (CDC. PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis.

  2. Bioengineered kidney tubules efficiently excrete uremic toxins

    Jansen, J.; Fedecostante, M.; Wilmer, M. J.; Peters, J. G.; Kreuser, U. M.; van den Broek, P. H.; Mensink, R. A.; Boltje, T. J.; Stamatialis, D.; Wetzels, J. F.; van den Heuvel, L. P.; Hoenderop, J. G.; Masereeuw, R.

    2016-01-01

    The development of a biotechnological platform for the removal of waste products (e.g. uremic toxins), often bound to proteins in plasma, is a prerequisite to improve current treatment modalities for patients suffering from end stage renal disease (ESRD). Here, we present a newly designed bioengineered renal tubule capable of active uremic toxin secretion through the concerted action of essential renal transporters, viz. organic anion transporter-1 (OAT1), breast cancer resistance protein (BCRP) and multidrug resistance protein-4 (MRP4). Three-dimensional cell monolayer formation of human conditionally immortalized proximal tubule epithelial cells (ciPTEC) on biofunctionalized hollow fibers with maintained barrier function was demonstrated. Using a tailor made flow system, the secretory clearance of human serum albumin-bound uremic toxins, indoxyl sulfate and kynurenic acid, as well as albumin reabsorption across the renal tubule was confirmed. These functional bioengineered renal tubules are promising entities in renal replacement therapies and regenerative medicine, as well as in drug development programs. PMID:27242131

  3. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

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

    Waldman, Scott A

    2010-01-01

    Colon cancer, the second leading cause of cancer-related mortality worldwide, originates from the malignant transformation of intestinal epithelial cells. The intestinal epithelium undergoes a highly organized process of rapid regeneration along the crypt-villus axis, characterized by proliferation, migration, differentiation and apoptosis, whose coordination is essential to maintaining the mucosal barrier. Disruption of these homeostatic processes predisposes cells to mutations in tumor suppressors or oncogenes, whose dysfunction provides transformed cells an evolutionary growth advantage. While sequences of genetic mutations at different stages along the neoplastic continuum have been established, little is known of the events initiating tumorigenesis prior to adenomatous polyposis coli (APC) mutations. Here, we examine a role for the corruption of homeostasis induced by silencing novel tumor suppressors, including the intestine-specific transcription factor CDX2 and its gene target guanylyl cyclase C (GCC), as early events predisposing cells to mutations in APC and other sequential genes that initiate colorectal cancer. CDX2 and GCC maintain homeostatic regeneration in the intestine by restricting cell proliferation, promoting cell maturation and adhesion, regulating cell migration and defending the intestinal barrier and genomic integrity. Elimination of CDX2 or GCC promotes intestinal tumor initiation and growth in aged mice, mice carrying APC mutations or mice exposed to carcinogens. The roles of CDX2 and GCC in suppressing intestinal tumorigenesis, universal disruption in their signaling through silencing of hormones driving GCC, and the uniform overexpression of GCC by tumors underscore the potential value of oral replacement with GCC ligands as targeted prevention and therapy for colorectal cancer. PMID:20592492

  5. A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B☆

    Robinson, Jerid W.; Dickey, Deborah M.; Miura, Kohji; Michigami, Toshimi; Ozono, Keiichi; Potter, Lincoln R.

    2015-01-01

    C-type natriuretic peptide (CNP) increases long bone growth by stimulating guanylyl cyclase (GC)-B/NPR-B/NPR2. Recently, a Val to Met missense mutation at position 883 in the catalytic domain of GC-B was identified in humans with increased blood cGMP levels that cause abnormally long bones. Here, we determined how this mutation activates GC-B. In the absence of CNP, cGMP levels in cells expressing V883M-GC-B were increased more than 20 fold compared to cells expressing wild-type (WT)-GC-B, and the addition of CNP only further increased cGMP levels 2-fold. In the absence of CNP, maximal enzymatic activity (Vmax) of V883M-GC-B was increased 15-fold compared to WT-GC-B but the affinity of the enzymes for substrate as revealed by the Michaelis constant (Km) was unaffected. Surprisingly, CNP decreased the Km of V883M-GC-B 10-fold in a concentration dependent manner without increasing Vmax. Unlike the WT enzyme the Km reduction of V883M-GC-B did not require ATP. Unexpectedly, V883M-GC-B, but not WT-GC-B, failed to inactivate with time. Phosphorylation elevated but was not required for the activity increase associated with the mutation because the Val to Met substitution also activated a GC-B mutant lacking all known phosphorylation sites. We conclude that the V883M mutation increases maximal velocity in the absence of CNP, eliminates the requirement for ATP in the CNP-dependent Km reduction, and disrupts the normal inactivation process. PMID:23827346

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

    Turek, Ilona; Gehring, Chris

    2016-06-01

    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. PMID:26945740

  7. Diversity of squalene-hopene cyclases in a tropical carbonate-rich environment

    Leavitt, W. D.; Pearson, A.

    2007-12-01

    Hopanoids are isoprenoid lipids which derive primarily from bacteria and are ubiquitous in contemporary Earth surface environments. In the geologic record, hopanes found in sedimentary rocks are used as proxies to help decipher ancient biological communities. However, in contrast to the ubiquity of these lipid products, biosynthesis of hopanoids appears to be a relatively rare physiological trait among bacteria in complex environmental communities. We have recently estimated that fewer than one in ten bacterial cells in soils and fewer than one in twenty bacterial cells in the ocean contains the gene squalene-hopene cyclase (sqhC) [1]. Biosynthesis of hopanoids is rarer in natural communities than it is among species that have been propagated in pure culture [2]. Here we continue our previous work to survey the phylogeny and diversity of hopanoid producers using culture-independent methods. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analyzed previously [1]. One possible explanation is that hopanoid-producing strains of cyanobacteria are regionally localized. It has been suggested that throughout the long-term sedimentary record there is a correlation between 2-methylhopanoid index (a putative indicator of cyanobacterial biomass) and the global prevalence of shallow carbonate platform environments [3], and in previous work we did not analyze any such environments. To address this question we surveyed a land-sea gradient across the Bahamian island of San Salvador. Samples were taken from upland soil, a hypersaline lake, a tidal creek, and the shallow open ocean. The data are remarkably similar to our previous results: environmental sqhCs average Gen. Microbiol. 130, 1137-1150. [3] Summons, RE (personal communication).

  8. Adenylyl cyclase regulation in heart failure due to myocardial infarction in rats.

    Bräunig, Jörg H; Albrecht-Küpper, Barbara; Seifert, Roland

    2014-04-01

    Cardiac adenylyl cyclase (AC) activity was described to be differentially regulated in left and right ventricles (LVs and RVs) of rats with heart failure (HF) due to LV myocardial infarction (MI) (Sethi et al. Am J Physiol 272:H884-H893, 1997). AC activities in LVs and RVs were increased and decreased respectively in rats 8 and 16 weeks post MI under basal and stimulatory conditions including AC activation via β-adrenergic receptors (β-ARs), stimulatory G protein (Gs), and direct AC activation with forskolin (FS). The current study aimed to detect alterations in rat heart AC activities in a comparable model of HF 9 weeks post LV MI. Therefore, cardiac AC activities were measured under basal and β-AR-, Gs-, or FS-stimulated conditions as well as under inhibition with various MANT [2'(3')-O-(N-methylanthraniloyl)]-nucleotide AC inhibitors and the P-site AC inhibitors NKY80 [2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone] and vidarabine (9-β-D-arabinosyladenine, AraAde). Basal and stimulated AC activities along with AC inhibition experiments did not reveal evidence for changes in AC activity in LVs and RVs from MI group animals despite the presence of congestive HF. However, our study is indeterminate. Further studies are required to identify the factors responsible for previously described changes in cardiac AC activity in MI induced HF and to elucidate the role of altered AC regulation in the pathophysiology of HF. In order to detect small changes in AC regulation, larger group sizes than the ones used in our present study are required. PMID:24276219

  9. Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque

    Titti Fausto

    2004-09-01

    Full Text Available Abstract Background The CD38 transmembrane glycoprotein is an ADP-ribosyl cyclase that moonlights as a receptor in cells of the immune system. Both functions are independently implicated in numerous areas related to human health. This study originated from an inherent interest in studying CD38 in the cynomolgus monkey (Macaca fascicularis, a species closely related to humans that also represents a cogent animal model for the biomedical analysis of CD38. Results A cDNA was isolated from cynomolgus macaque peripheral blood leukocytes and is predicted to encode a type II membrane protein of 301 amino acids with 92% identity to human CD38. Both RT-PCR-mediated cDNA cloning and genomic DNA PCR surveying were possible with heterologous human CD38 primers, demonstrating the striking conservation of CD38 in these primates. Transfection of the cDNA coincided with: (i surface expression of cynomolgus macaque CD38 by immunofluorescence; (ii detection of ~42 and 84 kDa proteins by Western blot and (iii the appearance of ecto-enzymatic activity. Monoclonal antibodies were raised against the cynomolgus CD38 ectodomain and were either species-specific or cross-reactive with human CD38, in which case they were directed against a common disulfide-requiring conformational epitope that was mapped to the C-terminal disulfide loop. Conclusion This multi-faceted characterization of CD38 from cynomolgus macaque demonstrates its high genetic and biochemical similarities with human CD38 while the immunological comparison adds new insights into the dominant epitopes of the primate CD38 ectodomain. These results open new prospects for the biomedical and pharmacological investigations of this receptor-enzyme.

  10. A peptide against soluble guanylyl cyclase α1: a new approach to treating prostate cancer.

    Shuai Gao

    Full Text Available Among the many identified androgen-regulated genes, sGCα1 (soluble guanylyl cyclase α1 appears to play a pivotal role in mediating the pro-cancer effects of androgens and androgen receptor. The classical role for sGCα1 is to heterodimerize with the sGCβ1 subunit, forming sGC, the enzyme that mediates nitric oxide signaling by catalyzing the synthesis of cyclic guanosine monophosphate. Our published data show that sGCα1 can drive prostate cancer cell proliferation independent of hormone and provide cancer cells a pro-survival function, via a novel mechanism for p53 inhibition, both of which are independent of sGCβ1, NO, and cGMP. All of these properties make sGCα1 an important novel target for prostate cancer therapy. Thus, peptides were designed targeting sGCα1 with the aim of disrupting this protein's pro-cancer activities. One peptide (A-8R was determined to be strongly cytotoxic to prostate cancer cells, rapidly inducing apoptosis. Cytotoxicity was observed in both hormone-dependent and, significantly, hormone-refractory prostate cancer cells, opening the possibility that this peptide can be used to treat the usually lethal castration-resistant prostate cancer. In mouse xenograft studies, Peptide A-8R was able to stop tumor growth of not only hormone-dependent cells, but most importantly from hormone-independent cells. In addition, the mechanism of Peptide A cytotoxicity is generation of reactive oxygen species, which recently have been recognized as a major mode of action of important cancer drugs. Thus, this paper provides strong evidence that targeting an important AR-regulated gene is a new paradigm for effective prostate cancer therapy.

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

    Eliane Q Lin

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

  12. Recent insights into Clostridium perfringens beta-toxin.

    Nagahama, Masahiro; Ochi, Sadayuki; Oda, Masataka; Miyamoto, Kazuaki; Takehara, Masaya; Kobayashi, Keiko

    2015-02-01

    Clostridium perfringens beta-toxin is a key mediator of necrotizing enterocolitis and enterotoxemia. It is a pore-forming toxin (PFT) that exerts cytotoxic effect. Experimental investigation using piglet and rabbit intestinal loop models and a mouse infection model apparently showed that beta-toxin is the important pathogenic factor of the organisms. The toxin caused the swelling and disruption of HL-60 cells and formed a functional pore in the lipid raft microdomains of sensitive cells. These findings represent significant progress in the characterization of the toxin with knowledge on its biological features, mechanism of action and structure-function having been accumulated. Our aims here are to review the current progresses in our comprehension of the virulence of C. perfringens type C and the character, biological feature and structure-function of beta-toxin. PMID:25654787

  13. Fold modulating function: Bacterial toxins to functional amyloids

    AdnanKhawajaSyed

    2014-08-01

    Full Text Available Many bacteria produce cytolytic toxins that target host cells or other competing microbes. It is well known that environmental factors control toxin expression, however recent work suggests that some bacteria manipulate the fold of these protein toxins to control their function. The β-sheet rich amyloid fold is a highly stable ordered aggregate that many toxins form in response to specific environmental conditions. When in the amyloid state, toxins become inert, losing the cytolytic activity they display in the soluble form. Emerging evidence suggest that some amyloids function as toxin storage systems until they are again needed, while other bacteria utilize amyloids as a structural matrix component of biofilms. This amyloid matrix component facilitates resistance to biofilm disruptive challenges. The bacterial amyloids discussed in this review reveal an elegant system where changes in protein fold and solubility dictate the function of proteins in response to the environment.

  14. Cytoskeleton as an Emerging Target of Anthrax Toxins

    Jean-Nicolas Tournier

    2012-02-01

    Full Text Available Bacillus anthracis, the agent of anthrax, has gained virulence through its exotoxins produced by vegetative bacilli and is composed of three components forming lethal toxin (LT and edema toxin (ET. So far, little is known about the effects of these toxins on the eukaryotic cytoskeleton. Here, we provide an overview on the general effects of toxin upon the cytoskeleton architecture. Thus, we shall discuss how anthrax toxins interact with their receptors and may disrupt the interface between extracellular matrix and the cytoskeleton. We then analyze what toxin molecular effects on cytoskeleton have been described, before discussing how the cytoskeleton may help the pathogen to corrupt general cell processes such as phagocytosis or vascular integrity.

  15. Recent Insights into Clostridium perfringens Beta-Toxin

    Masahiro Nagahama

    2015-02-01

    Full Text Available Clostridium perfringens beta-toxin is a key mediator of necrotizing enterocolitis and enterotoxemia. It is a pore-forming toxin (PFT that exerts cytotoxic effect. Experimental investigation using piglet and rabbit intestinal loop models and a mouse infection model apparently showed that beta-toxin is the important pathogenic factor of the organisms. The toxin caused the swelling and disruption of HL-60 cells and formed a functional pore in the lipid raft microdomains of sensitive cells. These findings represent significant progress in the characterization of the toxin with knowledge on its biological features, mechanism of action and structure-function having been accumulated. Our aims here are to review the current progresses in our comprehension of the virulence of C. perfringens type C and the character, biological feature and structure-function of beta-toxin.

  16. Fusarial toxins: secondary metabolites of Fusarium fungi.

    Nesic, Ksenija; Ivanovic, Snezana; Nesic, Vladimir

    2014-01-01

    Exposure to mycotoxins occurs worldwide, even though there are geographic and climatic differences in the amounts produced and occurrence of these substances.Mycotoxins are secondary chemical metabolites of different fungi. They are natural contaminants of cereals, so their presence is often inevitable. Among many genera that produce mycotoxins, Fusarium fungi are the most widespread in cereal-growing areas of the planet. Fusarium fungi produce a diversity of mycotoxin types, whose distributions are also diverse. What is produced and where it is produced is influenced primarily by environmental conditions, and crop production and storage methods. The amount of toxin produced depends on physical (viz., moisture, relative humidity, temperature, and mechanical damage), chemical (viz., carbon dioxide,oxygen, composition of substrate, insecticides and fungicides), and biological factors (viz., plant variety, stress, insects, spore load, etc.). Moisture and temperature have a major influence on mold growth rate and mycotoxin production.Among the most toxic and prevalent fusaria) toxins are the following: zearalenone,fumonisins, moniliformin and trichothecenes (T-2/HT-2 toxin, deoxynivalenol,diacetoxyscirpenol, nivalenol). Zearalenone (ZEA; ZON, F-2 toxin) isaphy to estrogenic compound, primarily a field contaminant, which exhibits estrogenic activity and has been implicated in numerous mycotoxicoses of farm animals,especially pigs. Recently, evidence suggests that ZEA has potential to stimulate the growth of human breast cancer cells. Fumonisins are also cancer-promoting metabolites,of which Fumonisin 8 I (FBI) is the most important. Moniliformin (MON) isalso highly toxic to both animals and humans. Trichothecenes are classified as gastrointestinal toxins, dermatotoxins, immunotoxins, hematotoxins, and gene toxins.T-2 and HT-2 toxin, and diacetoxyscirpenol (DAS, anguidine) are the most toxic mycotoxins among the trichothecene group. Deoxynivalenol (DON, vomitoxin) and

  17. Staphylococcus aureus α-Toxin: Nearly a Century of Intrigue

    Bryan J. Berube

    2013-06-01

    Full Text Available Staphylococcus aureus secretes a number of host-injurious toxins, among the most prominent of which is the small β-barrel pore-forming toxin α-hemolysin. Initially named based on its properties as a red blood cell lytic toxin, early studies suggested a far greater complexity of α-hemolysin action as nucleated cells also exhibited distinct responses to intoxication. The hemolysin, most aptly referred to as α-toxin based on its broad range of cellular specificity, has long been recognized as an important cause of injury in the context of both skin necrosis and lethal infection. The recent identification of ADAM10 as a cellular receptor for α-toxin has provided keen insight on the biology of toxin action during disease pathogenesis, demonstrating the molecular mechanisms by which the toxin causes tissue barrier disruption at host interfaces lined by epithelial or endothelial cells. This review highlights both the historical studies that laid the groundwork for nearly a century of research on α-toxin and key findings on the structural and functional biology of the toxin, in addition to discussing emerging observations that have significantly expanded our understanding of this toxin in S. aureus disease. The identification of ADAM10 as a proteinaceous receptor for the toxin not only provides a greater appreciation of truths uncovered by many historic studies, but now affords the opportunity to more extensively probe and understand the role of α-toxin in modulation of the complex interaction of S. aureus with its human host.

  18. Military Importance of Natural Toxins and Their Analogs.

    Pitschmann, Vladimír; Hon, Zdeněk

    2016-01-01

    Toxin weapon research, development, production and the ban on its uses is an integral part of international law, with particular attention paid to the protection against these weapons. In spite of this, hazards associated with toxins cannot be completely excluded. Some of these hazards are also pointed out in the present review. The article deals with the characteristics and properties of natural toxins and synthetic analogs potentially constituting the basis of toxin weapons. It briefly describes the history of military research and the use of toxins from distant history up to the present age. With respect to effective disarmament conventions, it mentions certain contemporary concepts of possible toxin applications for military purposes and the protection of public order (suppression of riots); it also briefly refers to the question of terrorism. In addition, it deals with certain traditional as well as modern technologies of the research, synthesis, and use of toxins, which can affect the continuing development of toxin weapons. These are, for example, cases of new toxins from natural sources, their chemical synthesis, production of synthetic analogs, the possibility of using methods of genetic engineering and modern biotechnologies or the possible applications of nanotechnology and certain pharmaceutical methods for the effective transfer of toxins into the organism. The authors evaluate the military importance of toxins based on their comparison with traditional chemical warfare agents. They appeal to the ethics of the scientific work as a principal condition for the prevention of toxin abuse in wars, military conflicts, as well as in non-military attacks. PMID:27136512

  19. Military Importance of Natural Toxins and Their Analogs

    Vladimír Pitschmann

    2016-04-01

    Full Text Available Toxin weapon research, development, production and the ban on its uses is an integral part of international law, with particular attention paid to the protection against these weapons. In spite of this, hazards associated with toxins cannot be completely excluded. Some of these hazards are also pointed out in the present review. The article deals with the characteristics and properties of natural toxins and synthetic analogs potentially constituting the basis of toxin weapons. It briefly describes the history of military research and the use of toxins from distant history up to the present age. With respect to effective disarmament conventions, it mentions certain contemporary concepts of possible toxin applications for military purposes and the protection of public order (suppression of riots; it also briefly refers to the question of terrorism. In addition, it deals with certain traditional as well as modern technologies of the research, synthesis, and use of toxins, which can affect the continuing development of toxin weapons. These are, for example, cases of new toxins from natural sources, their chemical synthesis, production of synthetic analogs, the possibility of using methods of genetic engineering and modern biotechnologies or the possible applications of nanotechnology and certain pharmaceutical methods for the effective transfer of toxins into the organism. The authors evaluate the military importance of toxins based on their comparison with traditional chemical warfare agents. They appeal to the ethics of the scientific work as a principal condition for the prevention of toxin abuse in wars, military conflicts, as well as in non-military attacks.

  20. ArachnoServer: a database of protein toxins from spiders

    Kaas Quentin; Raven Robert J; Cai Shuzhi; Miljenović Tomas; Wood David LA; Escoubas Pierre; Herzig Volker; Wilson David; King Glenn F

    2009-01-01

    Abstract Background Venomous animals incapacitate their prey using complex venoms that can contain hundreds of unique protein toxins. The realisation that many of these toxins may have pharmaceutical and insecticidal potential due to their remarkable potency and selectivity against target receptors has led to an explosion in the number of new toxins being discovered and characterised. From an evolutionary perspective, spiders are the most successful venomous animals and they maintain by far t...

  1. ATDB: a uni-database platform for animal toxins

    He, Quan-Yuan; He, Quan-Ze; Deng, Xing-Can; Yao, Lei; Meng, Er; Liu, Zhong-Hua; Liang, Song-Ping

    2007-01-01

    Venomous animals possess an arsenal of toxins for predation and defense. These toxins have great diversity in function and structure as well as evolution and therefore are of value in both basic and applied research. Recently, toxinomics researches using cDNA library sequencing and proteomics profiling have revealed a large number of new toxins. Although several previous groups have attempted to manage these data, most of them are restricted to certain taxonomic groups and/or lack effective s...

  2. ClanTox: a classifier of short animal toxins

    Naamati, Guy; Askenazi, Manor; Linial, Michal

    2009-01-01

    Toxins are detected in sporadic species along the evolutionary tree of the animal kingdom. Venomous animals include scorpions, snakes, bees, wasps, frogs and numerous animals living in the sea such as the stonefish, snail, jellyfish, hydra and more. Interestingly, proteins that share a common scaffold with animal toxins also exist in non-venomous species. However, due to their short length and primary sequence diversity, these, toxin-like proteins remain undetected by classical search engines...

  3. Transcriptional Stimulation of Anthrax Toxin Receptors by Anthrax Edema Toxin and Bacillus anthracis Sterne Spore

    Xu, Qingfu; Hesek, Eric D.; Zeng, Mingtao

    2007-01-01

    We used quantitative real-time RT-PCR to not only investigate the mRNA levels of anthrax toxin receptor 1 (ANTXR1) and 2 (ANTXR2) in the murine J774A.1 macrophage cells and different tissues of mice, but also evaluate the effect of anthrax edema toxin and Bacillus anthracis Sterne spores on the expression of mRNA of these receptors. The mRNA transcripts of both receptors was detected in J774A.1 cells and mouse tissues such as the lung, heart, kidney, spleen, stomach, jejunum, brain, skeleton ...

  4. Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals.

    Tresguerres, Martin; Barott, Katie L; Barron, Megan E; Roa, Jinae N

    2014-03-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3(-), and sAC has been confirmed to be a HCO3(-) sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3(-)-regulated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H(+) absorption. In the intestine of bony fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved. PMID:24574382

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

    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.

  6. Natriuretic peptide receptor-B (guanylyl cyclase-B) mediates C-type natriuretic peptide relaxation of precontracted rat aorta.

    Drewett, J G; Fendly, B M; Garbers, D L; Lowe, D G

    1995-03-01

    The most potent known agonist for the natriuretic peptide receptor-B (NPR-B)/guanylyl cyclase-B is C-type natriuretic peptide (CNP). A homologous ligand-receptor system consists of atrial natriuretic peptide (ANP) and NPR-A/guanylyl cyclase-A. A third member of this family is NPR-C, a non-guanylyl cyclase receptor. Monoclonal antibodies were raised against NPR-B by immunizing mice with a purified receptor-IgG fusion protein consisting of the extracellular domain of NPR-B and the Fc portion of human IgG-gamma 1. One monoclonal antibody, 3G12, did not recognize NPR-A or NPR-C and bound to human and rat NPR-B. CNP binding to NPR-B and stimulation of cGMP synthesis were inhibited by 3G12. With cells isolated from either the media or adventitia layers of rat thoracic aorta, 3G12 did not interfere with ANP-stimulated cGMP synthesis, but it inhibited CNP-stimulated cGMP levels in cells from both layers. CNP (IC50 = 10 nM) and ANP (IC50 = 1 nM) caused relaxation of phenylephrine-contracted rat aortic rings. 3G12 caused a marked increase in the IC50 for CNP, from 10 nM to 140 nM, but failed to affect ANP-mediated relaxation. Therefore, our results for the first time demonstrate that CNP relaxes vascular smooth muscle by virtue of its binding to NPR-B. PMID:7876238

  7. Role of receptors in Bacillus thuringiensis crystal toxin activity.

    Pigott, Craig R; Ellar, David J

    2007-06-01

    Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

  8. Animal Toxins: How is Complexity Represented in Databases?

    Jungo F.; Estreicher A.; Bairoch A.; Bougueleret L.; Xenarios I.

    2010-01-01

    Peptide toxins synthesized by venomous animals have been extensively studied in the last decades. To be useful to the scientific community, this knowledge has been stored, annotated and made easy to retrieve by several databases. The aim of this article is to present what type of information users can access from each database. ArachnoServer and ConoServer focus on spider toxins and cone snail toxins, respectively. UniProtKB, a generalist protein knowledgebase, has an animal toxin-dedicated a...

  9. Gene therapy for carcinoma of the breast: Genetic toxins

    Gene therapy was initially envisaged as a potential treatment for genetically inherited, monogenic disorders. The applications of gene therapy have now become wider, however, and include cardiovascular diseases, vaccination and cancers in which conventional therapies have failed. With regard to oncology, various gene therapy approaches have been developed. Among them, the use of genetic toxins to kill cancer cells selectively is emerging. Two different types of genetic toxins have been developed so far: the metabolic toxins and the dominant-negative class of toxins. This review describes these two different approaches, and discusses their potential applications in cancer gene therapy

  10. Tumor Targeting and Drug Delivery by Anthrax Toxin.

    Bachran, Christopher; Leppla, Stephen H

    2016-01-01

    Anthrax toxin is a potent tripartite protein toxin from Bacillus anthracis. It is one of the two virulence factors and causes the disease anthrax. The receptor-binding component of the toxin, protective antigen, needs to be cleaved by furin-like proteases to be activated and to deliver the enzymatic moieties lethal factor and edema factor to the cytosol of cells. Alteration of the protease cleavage site allows the activation of the toxin selectively in response to the presence of tumor-associated proteases. This initial idea of re-targeting anthrax toxin to tumor cells was further elaborated in recent years and resulted in the design of many modifications of anthrax toxin, which resulted in successful tumor therapy in animal models. These modifications include the combination of different toxin variants that require activation by two different tumor-associated proteases for increased specificity of toxin activation. The anthrax toxin system has proved to be a versatile system for drug delivery of several enzymatic moieties into cells. This highly efficient delivery system has recently been further modified by introducing ubiquitin as a cytosolic cleavage site into lethal factor fusion proteins. This review article describes the latest developments in this field of tumor targeting and drug delivery. PMID:27376328

  11. Botulinum toxin for treatment of glandular hypersecretory disorders.

    Laing, T A

    2012-02-03

    SUMMARY: The use of botulinum toxin to treat disorders of the salivary glands is increasing in popularity in recent years. Recent reports of the use of botulinum toxin in glandular hypersecretion suggest overall favourable results with minimal side-effects. However, few randomised clinical trials means that data are limited with respect to candidate suitability, treatment dosages, frequency and duration of treatment. We report a selection of such cases from our own department managed with botulinum toxin and review the current data on use of the toxin to treat salivary gland disorders such as Frey\\'s syndrome, excessive salivation (sialorrhoea), focal and general hyperhidrosis, excessive lacrimation and chronic rhinitis.

  12. Entry of diphtheria toxin into cells: possible existence of cellular factor(s) for entry of diphtheria toxin into cells was studied in somatic cell hybrids and hybrid toxins

    1984-01-01

    Ehrlich ascites tumor cells were found to be very insensitive to diphtheria toxin. We formed 37 hybrids from Ehrlich tumor cells and diphtheria toxin-sensitive human fibroblasts. The effects of diphtheria toxin on protein synthesis in those hybrids were examined. The hybrids were divided into three groups on the basis of toxin sensitivity. Group A hybrids were as sensitive to diphtheria toxin as human fibroblasts, Group C were as resistant as Ehrlich tumor cells, and Group B had intermediate ...

  13. Molecular Cloning,Expression,and Characterization of an Adenylyl Cyclase-associated Protein from Gossypium arboreum Fuzzless Mutant

    WANG Sheng; ZHAO Guo-hong; JIA Yin-hua; DU Xiong-ming

    2008-01-01

    @@ CAP,an adenylyl cyclase-associated protein,is predicted to be involved in cytoskeletal organization and signal transduction.Recently,we found that CAP may play an important role in fuzz-like fiber cell initiation in cotton.For the further research,we isolated two CAP homologues from wild type cotton Gossypium arboreum L.(DPL971) and its natural fuzzless mutant (DPL972).The gene consisted of an open reading frame of 1,416 nucleotides encoding a protein of 471 amino acid residues with a calculated molecular weight of 50.6 kDa.

  14. Tyrosine Phosphatase TpbA Controls Rugose Colony Formation in Pseudomonas aeruginosa by Dephosphorylating Diguanylate Cyclase TpbB

    Pu, Mingming; Wood, Thomas K.

    2010-01-01

    Tyrosine phosphatase TpbA in Pseudomonas aeruginosa PA14 is a negative regulator of the diguanylate cyclase TpbB. Inactivation of TpbA caused rugose colony morphology which is related to cell persistence in clinical infections. We show here that TpbA is a dual specific tyrosine phosphatase, that TpbB is phosphorylated, and that TpbA controls phosphorylation of TpbB at both Tyr and Ser/Thr residues in vivo as detected by Western blot analysis. In addition, TpbB is demonstrated to be a substrat...

  15. Temporal and Regional Regulation of Gene Expression by Calcium-Stimulated Adenylyl Cyclase Activity during Fear Memory

    Wieczorek, Lindsay; Maas, James W.; Muglia, Lisa M.; Vogt, Sherri K.; Muglia, Louis J.

    2010-01-01

    Background The Ca2+-stimulated adenylyl cyclases (ACs), AC1 and AC8, are key components of long-term memory processing. AC1 and AC8 double knockout mice (Adcy1−/−Adcy8−/−; DKO) display impaired fear memory processing; the mechanism of this impairment is largely unknown. Methodology/Principal Findings We hypothesize that the Ca2+-stimulated ACs modulate long-lasting transcriptional changes essential for fear memory consolidation and maintenance. Here, we report a genome-wide study of gene expr...

  16. Isolation and Characterization of toxin A-negative, toxin B-positive Clostridium difficile in Dublin, Ireland

    Drudy, Denise; Harnedy, N.; Fanning, S; O’Mahony, R.; Kyne, L.

    2006-01-01

    Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients. Most pathogenic C. difficile strains produce two toxins, A and B; however, clinically relevant toxin A-negative, toxin Bpositive (A– B+ ) strains of C. difficile that cause diarrhoea and colitis in humans have been isolated worldwide. The aims of this study were to isolate and characterise A– B+ strains from two university hospitals in Dublin, Ireland. Samples positive for C. difficile were identified dai...

  17. SVM-Based Prediction of Propeptide Cleavage Sites in Spider Toxins Identifies Toxin Innovation in an Australian Tarantula

    Wong, Emily S. W.; Hardy, Margaret C.; David Wood; Timothy Bailey; Glenn F. King

    2013-01-01

    Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin tran...

  18. Fast collapse but slow formation of secondary structure elements in the refolding transition of E. coli adenylate kinase.

    Ratner, V; Amir, D; Kahana, E; Haas, E

    2005-09-23

    The various models proposed for protein folding transition differ in their order of appearance of the basic steps during this process. In this study, steady state and time-resolved dynamic non-radiative excitation energy transfer (FRET and trFRET) combined with site specific labeling experiments were applied in order to characterize the initial transient ensemble of Escherichia coli adenylate kinase (AK) molecules upon shifting conditions from those favoring denaturation to refolding and from folding to denaturing. Three sets of labeled AK mutants were prepared, which were designed to probe the equilibrium and transient distributions of intramolecular segmental end-to-end distances. A 176 residue section (residues 28-203), which spans most of the 214 residue molecule, and two short secondary structure chain segments including an alpha-helix (residues 169-188) and a predominantly beta-strand region (residues 188-203), were labeled. Upon fast change of conditions from denaturing to folding, the end-to-end distance of the 176 residue chain section showed an immediate collapse to a mean value of 26 A. Under the same conditions, the two short secondary structure elements did not respond to this shift within the first ten milliseconds, and retained the characteristics of a fully unfolded state. Within the first 10 ms after changes of the solvent from folding to denaturing, only minor changes were observed at the local environments of residues 203 and 169. The response of these same local environments to the shift of conditions from denaturing to folding occurred within the dead time of the mixing device. Thus, the response of the CORE domain of AK to fast transfer from folding to unfolding conditions is slow at all three conformational levels that were probed, and for at least a few milliseconds the ensemble of folded molecules is maintained under unfolding conditions. A different order of the changes was observed upon initiation of refolding. The AK molecules undergo

  19. Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis

    Adenylate kinase (AK) from D. gigas was purified and crystallized in three different metal-bound forms: Zn2+–AK, Co2+–AK and Fe2+–AK. Adenylate kinase (AK; ATP:AMP phosphotransferase; EC 2.7.4.3) is involved in the reversible transfer of the terminal phosphate group from ATP to AMP. AKs contribute to the maintenance of a constant level of cellular adenine nucleotides, which is necessary for the energetic metabolism of the cell. Three metal ions, cobalt, zinc and iron(II), have been reported to be present in AKs from some Gram-negative bacteria. Native zinc-containing AK from Desulfovibrio gigas was purified to homogeneity and crystallized. The crystals diffracted to beyond 1.8 Å resolution. Furthermore, cobalt- and iron-containing crystal forms of recombinant AK were also obtained and diffracted to 2.0 and 3.0 Å resolution, respectively. Zn2+–AK and Fe2+–AK crystallized in space group I222 with similar unit-cell parameters, whereas Co2+–AK crystallized in space group C2; a monomer was present in the asymmetric unit for both the Zn2+–AK and Fe2+–AK forms and a dimer was present for the Co2+–AK form. The structures of the three metal-bound forms of AK will provide new insights into the role and selectivity of the metal in these enzymes

  20. Structure of the D-alanylgriseoluteic acid biosynthetic protein EhpF, an atypical member of the ANL superfamily of adenylating enzymes

    Bera, A.K.; Robinson, H.; Atanasova, V.; Gamage, S.; Parsons, J. F.

    2010-06-01

    The structure of EhpF, a 41 kDa protein that functions in the biosynthetic pathway leading to the broad-spectrum antimicrobial compound D-alanylgriseoluteic acid (AGA), is reported. A cluster of approximately 16 genes, including ehpF, located on a 200 kbp plasmid native to certain strains of Pantoea agglomerans encodes the proteins that are required for the conversion of chorismic acid to AGA. Phenazine-1,6-dicarboxylate has been identified as an intermediate in AGA biosynthesis and deletion of ehpF results in accumulation of this compound in vivo. The crystallographic data presented here reveal that EhpF is an atypical member of the acyl-CoA synthase or ANL superfamily of adenylating enzymes. These enzymes typically catalyze two-step reactions involving adenylation of a carboxylate substrate followed by transfer of the substrate from AMP to coenzyme A or another phosphopantetheine. EhpF is distinguished by the absence of the C-terminal domain that is characteristic of enzymes from this family and is involved in phosphopantetheine binding and in the second half of the canonical two-step reaction that is typically observed. Based on the structure of EhpF and a bioinformatic analysis, it is proposed that EhpF and EhpG convert phenazine-1,6-dicarboxylate to 6-formylphenazine-1-carboxylate via an adenylyl intermediate.

  1. A common origin for the bacterial toxin-antitoxin systems parD and ccd, suggested by analyses of toxin/target and toxin/antitoxin interactions.

    Andrew B Smith

    Full Text Available Bacterial toxin-antitoxin (TA systems encode two proteins, a potent inhibitor of cell proliferation (toxin and its specific antidote (antitoxin. Structural data has revealed striking similarities between the two model TA toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid toxin, two activities that are specifically counteracted by its cognate Kis antitoxin, are altered, but not inhibited, by the CcdA antitoxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the toxins and antitoxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these toxins evolved to reach different, rather than common, cellular targets.

  2. Shared Binding Sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A Toxins

    Herrero, Salvador; González-Cabrera, Joel; Tabashnik, Bruce E; Ferré, Juan

    2001-01-01

    Bacillus thuringiensis toxins act by binding to specific target sites in the insect midgut epithelial membrane. The best-known mechanism of resistance to B. thuringiensis toxins is reduced binding to target sites. Because alteration of a binding site shared by several toxins may cause resistance to all of them, knowledge of which toxins share binding sites is useful for predicting cross-resistance. Conversely, cross-resistance among toxins suggests that the toxins share a binding site. At lea...

  3. Toxin production in Dinophysis and the fate of these toxins in marine mussels

    Nielsen, Lasse Tor

    Diarrhetic shellfish poisoning (DSP) poses a considerable threat to food safety and to the economy of shellfish fishers and farmers in many parts of the world. Thousands of DSP intoxications have been reported, and bivalve harvesting can sometimes be closed down several months in a row. The toxins...

  4. Tetanus toxin : primary structure, expression in E. coli, and homology with botulinum toxins

    Eisel, Ulrich; Jarausch, Wolfgang; Goretzki, Karin; Henschen, Agnes; Engels, Joachim; Weller, Ulrich; Hudel, Martina; Habermann, Ernst; Niemann, Heiner; Rott, R.

    1986-01-01

    A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75-kbp plasmid from a toxigenic non-sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a

  5. Binding of Diphtheria Toxin to Phospholipids in Liposomes

    Alving, Carl R.; Iglewski, Barbara H.; Urban, Katharine A.; Moss, Joel; Richards, Roberta L.; Sadoff, Jerald C.

    1980-04-01

    Diphtheria toxin bound to the phosphate portion of some, but not all, phospholipids in liposomes. Liposomes consisting of dimyristoyl phosphatidylcholine and cholesterol did not bind toxin. Addition of 20 mol% (compared to dimyristoyl phosphatidylcholine) of dipalmitoyl phosphatidic acid, dicetyl phosphate, phosphatidylinositol phosphate, cardiolipin, or phosphatidylserine in the liposomes resulted in substantial binding of toxin. Inclusion of phosphatidylinositol in dimyristol phosphatidylcholine / cholesterol liposomes did not result in toxin binding. The calcium salt of dipalmitoyl phosphatidic acid was more effective than the sodium salt, and the highest level of binding occurred with liposomes consisting only of dipalmitoyl phosphatidic acid (calcium salt) and cholesterol. Binding of toxin to liposomes was dependent on pH, and the pattern of pH dependence varied with liposomes having different compositions. Incubation of diphtheria toxin with liposomes containing dicetyl phosphate resulted in maximal binding at pH 3.6, whereas binding to liposomes containing phosphatidylinositol phosphate was maximal above pH 7. Toxin did not bind to liposomes containing 20 mol% of a free fatty acid (palmitic acid) or a sulfated lipid (3-sulfogalactosylceramide). Toxin binding to dicetyl phosphate or phosphatidylinositol phosphate was inhibited by UTP, ATP, phosphocholine, or p-nitrophenyl phosphate, but not by uracil. We conclude that (a) diphtheria toxin binds specifically to the phosphate portion of certain phospholipids, (b) binding to phospholipids in liposomes is dependent on pH, but is not due only to electrostatic interaction, and (c) binding may be strongly influenced by the composition of adjacent phospholipids that do not bind toxin. We propose that a minor membrane phospholipid (such as phosphatidylinositol phosphate or phosphatidic acid), or that some other phosphorylated membrane molecule (such as a phosphoprotein) may be important in the initial binding of

  6. Prevention, control and detection of Fusarial toxins

    Nešić Ksenija D.

    2013-01-01

    Full Text Available The past couple of decades have provided considerable details on fungi and the toxins that they produce, as well on the mechanism of toxin action, toxicity and effects on animal and human health. But, since they are natural contaminants, their presence is often inevitable. Fusaria are widespread in all cereal-growing territories of the world, but they are especially common in our geographic area. Therefore, special attention is paid to the prevention and control, and also to the improvement of methods for their detection. Although all collected data were critical for understanding this worldwide problem, managing the impact of these toxins on the feed and food safety is still great practical challenge. There are a number of approaches that can be taken to minimize mycotoxin contamination in this chain: prevention of fungal growth and thus mycotoxin formation, strategies to reduce or eliminate mycotoxins from contaminated feedstuffs or diverting the contaminated products to low risk uses. A control program for mycotoxins from field to table should in­volve the criteria of an HACCP (Hazard Analysis Critical Control Points approach. It requires an understanding of the important aspects of the interactions of the toxigenic fungi with crop plants, the on-farm production and harvest methods for crops, the production of livestock using grains and processed feeds, including diagnostic capabilities for mycotoxicoses, and all the way to the development of processed foods for human consumption, as well as understanding the marketing and trade channels including storage and delivery of foods to the consumer’s table. A good testing protocol for mycotoxins is necessary to manage all of the control points and in order to be able to ensure a food supply free of toxic levels of mycotoxins for the consumer. [Projekat Ministarstva nauke Republike Srbije, br. III 46009

  7. Glycolipid binding preferences of Shiga toxin variants.

    Sayali S Karve

    Full Text Available The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx, an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3 and globotetraosylceramide (Gb4 in the presence of cell membrane components such as phosphatidylcholine (PC, cholesterol (Ch and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.

  8. Overview of Botulinum Toxins for Aesthetic Uses.

    Gart, Michael S; Gutowski, Karol A

    2016-07-01

    Botulinum toxin type A (BTA) can be used for facial aesthetics. The 3 currently available BTA types include onabotulinumtoxinA (Botox; Botox Cosmetic, Allergan, Irvine, CA), abobotulinumtoxinA (Dysport; Ipsen, Ltd, Berkshire, UK), and incobotulinumtoxinA (Xeomin; Merz Pharmaceuticals, Frankfurt, Germany). The mechanism of action and clinical uses for treatment of dynamic lines of the forehead, brow, glabella, lateral orbit, nose, and lips are presented, as well as treatment of masseter hypertrophy, platysmal bands, and improvements of the perioral region. Specific BTA injection sites and suggested doses are presented. PMID:27363760

  9. Molecular Cloning and Characterization of an Allene Oxide Cyclase Gene Associated with Fiber Strength in Cotton

    WANG Li-man; ZHU You-min; TONG Xiang-chao; HU Wen-jing; CAI Cai-ping; GUO Wang-zhen

    2014-01-01

    Allene oxide cyclase (AOC) is one of the most important enzymes in the biosynthetic pathway of the plant hormone jasmonic acid (JA). AOC catalyzes the conversion of allene oxide into 12-oxo-phytodienoic acid (OPDA), a precursor of JA. Using 28K cotton genome array hybridization, an expressed sequence tag (EST;GenBank accession no. ES792958) was investigated that exhibited signiifcant expression differences between lintless-fuzzless XinWX and linted-fuzzless XinFLM isogenic lines during ifber initiation stages. The EST was used to search the Gossypium EST database (http://www.ncbi.nlm.nih.gov/) for corresponding cDNA sequences encoding full-length open reading frames (ORFs). Identiifed ORFs were conifrmed using transcriptional and genomic data. As a result, a novel gene encoding AOC in cotton (Gossypium hirsutum AOC;GenBank accession no. KF383427) was cloned and characterized. The 741-bp GhAOC gene comprises three exons and two introns and encodes a polypeptide of 246 amino acids. Two homologous copies were identiifed in the tetraploid cotton species G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124, and one copy in the diploid cotton species G. herbaceum and G. raimondii. qRT-PCR showed that the GhAOC transcript was abundant in cotton ifber tissues from 8 to 23 days post anthesis (DPA), and the expression proifles were similar in the two cultivated tetraploid cotton species G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124, with a higher level of transcription in the former. One copy of GhAOC in tetraploid cotton was localized to chromosome 24 (Chr. D8) using the subgenome-speciifc single nucleotide polymorphism (SNP) marker analysis, which co-localized GhAOC to within 10 cM of a ifber strength quantitative trait locus (QTL) reported previously. GhAOC was highly correlated with ifber quality and strength (P=0.014) in an association analysis, suggesting a possible role in cotton ifber development, especially in secondary cell wall thickening.

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

    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

  11. Guanylate cyclase C deficiency causes severe inflammation in a murine model of spontaneous colitis.

    Eleana Harmel-Laws

    Full Text Available BACKGROUND: Guanylate Cyclase C (GC-C; Gucy2c is a transmembrane receptor expressed in intestinal epithelial cells. Activation of GC-C by its secreted ligand guanylin stimulates intestinal fluid secretion. Familial mutations in GC-C cause chronic diarrheal disease or constipation and are associated with intestinal inflammation and infection. Here, we investigated the impact of GC-C activity on mucosal immune responses. METHODS: We utilized intraperitoneal injection of lipopolysaccharide to elicit a systemic cytokine challenge and then measured pro-inflammatory gene expression in colonic mucosa. GC-C(+/+ and GC-C(-/- mice were bred with interleukin (IL-10 deficient animals and colonic inflammation were assessed. Immune cell influx and cytokine/chemokine expression was measured in the colon of wildtype, IL-10(-/-, GC-C(+/+IL-10(-/- and GC-C(-/-IL-10(-/- mice. GC-C and guanylin production were examined in the colon of these animals and in a cytokine-treated colon epithelial cell line. RESULTS: Relative to GC-C(+/+ animals, intraperitoneal lipopolysaccharide injection into GC-C(-/- mice increased proinflammatory gene expression in both whole colon tissue and in partially purified colonocyte isolations. Spontaneous colitis in GC-C(-/-IL-10(-/- animals was significantly more severe relative to GC-C(+/+IL-10(-/- mice. Unlike GC-C(+/+IL-10(-/- controls, colon pathology in GC-C(-/-IL-10(-/- animals was apparent at an early age and was characterized by severely altered mucosal architecture, crypt abscesses, and hyperplastic subepithelial lesions. F4/80 and myeloperoxidase positive cells as well as proinflammatory gene expression were elevated in GC-C(-/-IL-10(-/- mucosa relative to control animals. Guanylin was diminished early in colitis in vivo and tumor necrosis factor α suppressed guanylin mRNA and protein in intestinal goblet cell-like HT29-18-N2 cells. CONCLUSIONS: The GC-C signaling pathway blunts colonic mucosal inflammation that is initiated by

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

    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

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

    Rameshwar K Sharma

    2014-07-01

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

  14. Elevation of soluble guanylate cyclase suppresses proliferation and survival of human breast cancer cells.

    Hui-Chin Wen

    Full Text Available Nitric oxide (NO is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC, composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells.

  15. Metal Ion Activation of Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B

    Genth, Harald; Schelle, Ilona; Just, Ingo

    2016-01-01

    Lethal Toxin from Clostridium sordellii (TcsL) and Toxin B from Clostridium difficile (TcdB) belong to the family of the “Large clostridial glycosylating toxins.” These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. TcsL is casually involved in the toxic shock syndrome and the gas gangrene. TcdB—together with Toxin A (TcdA)—is causative for the pseudomembranous colitis (PMC). Here, we present evidence for the in vitro metal ion activation of the glucosyltransferase and the UDP-glucose hydrolysis activity of TcsL and TcdB. The following rating is found for activation by divalent metal ions: Mn2+ > Co2+ > Mg2+ >> Ca2+, Cu2+, Zn2+. TcsL and TcdB thus require divalent metal ions providing an octahedral coordination sphere. The EC50 values for TcsL were estimated at about 28 µM for Mn2+ and 180 µM for Mg2+. TcsL and TcdB further require co-stimulation by monovalent K+ (not by Na+). Finally, prebound divalent metal ions were dispensible for the cytopathic effects of TcsL and TcdB, leading to the conclusion that TcsL and TcdB recruit intracellular metal ions for activation of the glucosyltransferase activity. With regard to the intracellular metal ion concentrations, TcsL and TcdB are most likely activated by K+ and Mg2+ (rather than Mn2+) in mammalian target cells. PMID:27089365

  16. Metal Ion Activation of Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B

    Harald Genth

    2016-04-01

    Full Text Available Lethal Toxin from Clostridium sordellii (TcsL and Toxin B from Clostridium difficile (TcdB belong to the family of the “Large clostridial glycosylating toxins.” These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. TcsL is casually involved in the toxic shock syndrome and the gas gangrene. TcdB—together with Toxin A (TcdA—is causative for the pseudomembranous colitis (PMC. Here, we present evidence for the in vitro metal ion activation of the glucosyltransferase and the UDP-glucose hydrolysis activity of TcsL and TcdB. The following rating is found for activation by divalent metal ions: Mn2+ > Co2+ > Mg2+ >> Ca2+, Cu2+, Zn2+. TcsL and TcdB thus require divalent metal ions providing an octahedral coordination sphere. The EC50 values for TcsL were estimated at about 28 µM for Mn2+ and 180 µM for Mg2+. TcsL and TcdB further require co-stimulation by monovalent K+ (not by Na+. Finally, prebound divalent metal ions were dispensible for the cytopathic effects of TcsL and TcdB, leading to the conclusion that TcsL and TcdB recruit intracellular metal ions for activation of the glucosyltransferase activity. With regard to the intracellular metal ion concentrations, TcsL and TcdB are most likely activated by K+ and Mg2+ (rather than Mn2+ in mammalian target cells.

  17. Distribution of the Toxin following Medial Rectus Muscular Injection of Botulinum Toxin Gel in Rabbits

    Jingchang Chen; Guanghuan Mai; Xinping Yu; Huanyuan Yu; Heping Wu; Xiaoming Lin; Daming Deng; Ying Kang

    2005-01-01

    Purpose: To determine the distribution of the toxin among individual muscles after intramuscular injection of botulinum toxin gel.Methods: One eye of 7 New Zealand white rabbits was randomized into group A, and the contralateral eye was into group B. Eyes in group A received medial rectus intramuscular injection of 2.5 IU of 125I-BTX-A gel in 0.1 ml, and those in group B received equivalent amount of 125I-BTX-A solution by medial rectus intramuscular injection. Four rectus muscles and the levator palpebrae superioris were harvested and the radioactivity of muscles was measured 16 hours after the injection.Results: In group A, the radioactivity of per gram medial rectus was significantly higher than that of other muscles (P < 0.01), and there was no statistically significant difference in the radioactivity of per gram muscles among other muscles (P > 0.05). In group B, the radioactivity of per gram medial rectus and levator palpebrae superioris was significantly higher than that of other muscles respectively(P < 0.05), and the difference in the radioactivity of per gram muscles between medial rectus and levator palpebrae superioris was not statistically significant (P > 0.05). The radioactivity of per gram medial rectus in group A was higher than that in group B (t=3.731 ,P=0.01), and there was no significant difference in the radioactivity of per gram muscles among other homonymous muscles (P > 0.05).Conclusion: The toxin dispersed principally in the injected muscle and the local concentration of the toxin was much high following intramuscular injection of botulinum toxin gel.

  18. Diarrhetic Shellfish Toxins and Other Lipophilic Toxins of Human Health Concern in Washington State

    Bich-Thuy L. Eberhart

    2013-05-01

    Full Text Available The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 μg okadaic acid (OA + dinophysistoxins (DTXs/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1 with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2 and yessotoxin (YTX with azaspiracid-2 (AZA-2 also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1 and azaspiracid-3 (AZA-3 were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS. A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.

  19. Cloning and Functional Analysis of Lycopeneε-Cyclase (IbLCYe) Gene from Sweetpotato, Ipomoea batatas (L.) Lam

    YU Ling; ZHAI Hong; CHEN Wei; HE Shao-zhen; LIU Qing-chang

    2013-01-01

    This paper reported firstly successful cloning of lycopeneε-cyclase (IbLCYe) gene from sweetpotato, Ipomoea batatas (L.) Lam. Using rapid amplification of cDNA ends (RACE), IbLCYe gene was cloned from sweetpotato cv. Nongdafu 14 with high carotenoid content. The 1 805 bp cDNA sequence of IbLCYe gene contained a 1 236 bp open reading frame (ORF) encoding a 411 amino acids polypeptide with a molecular weight of 47 kDa and an isoelectric point (pI) of 6.95. IbLCYe protein contained one potential lycopeneε-cyclase domain and one potential FAD (flavinadenine dinucleotide)/NAD(P) (nicotinamide adenine dinucleotide phosphate)-binding domain, indicating that this protein shares the typical characteristics of LCYe proteins. The gDNA of IbLCYe gene was 4 029 bp and deduced to contain 5 introns and 6 exons. Real-time quantitative PCR analysis revealed that the expression level of IbLCYe gene was significantly higher in the storage roots of Nongdafu 14 than those in the leaves and stems. Transgenic tobacco (cv. Wisconsin 38) expressing IbLCYe gene accumulated significantly moreβ-carotene compared to the untransformed control plants. These results showed that IbLCYe gene has an important function for the accumulation of carotenoids of sweetpotato.

  20. Adenylyl cyclase types I and VI but not II and V are selectively inhibited by nitric oxide

    J. Goldstein

    2002-02-01

    Full Text Available Adenylyl cyclase (AC isoforms catalyze the synthesis of 3',5'-cyclic AMP from ATP. These isoforms are critically involved in the regulation of gene transcription, metabolism, and ion channel activity among others. Nitric oxide (NO is a gaseous product whose synthesis from L-arginine is catalyzed by the enzyme NO synthase. It has been well established that NO activates the enzyme guanylyl cyclase, but little has been reported on the effects of NO on other important second messengers, such as AC. In the present study, the effects of sodium nitroprusside (SNP, a nitric oxide-releasing compound, on COS-7 cells transfected with plasmids containing AC types I, II, V and VI were evaluated. Total inhibition (~98.5% of cAMP production was observed in COS-7 cells transfected with the AC I isoform and previously treated with SNP (10 mM for 30 min, when stimulated with ionomycin. A high inhibition (~76% of cAMP production was also observed in COS-7 cells transfected with the AC VI isoform and previously treated with SNP (10 mM for 30 min, when stimulated with forskolin. No effect on cAMP production was observed in cells transfected with AC isoforms II and V.