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Sample records for adenylate cyclase toxin

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-08-01

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

  2. Adenylate cyclase toxin promotes internalisation of integrins and raft components and decreases macrophage adhesion capacity.

    Directory of Open Access Journals (Sweden)

    César Martín

    Full Text Available Bordetella pertussis, the bacterium that causes whooping cough, secretes an adenylate cyclase toxin (ACT that must be post-translationally palmitoylated in the bacterium cytosol to be active. The toxin targets phagocytes expressing the CD11b/CD18 integrin receptor. It delivers a catalytic adenylate cyclase domain into the target cell cytosol producing a rapid increase of intracellular cAMP concentration that suppresses bactericidal functions of the phagocyte. ACT also induces calcium fluxes into target cells. Biochemical, biophysical and cell biology approaches have been applied here to show evidence that ACT and integrin molecules, along with other raft components, are rapidly internalized by the macrophages in a toxin-induced calcium rise-dependent process. The toxin-triggered internalisation events occur through two different routes of entry, chlorpromazine-sensitive receptor-mediated endocytosis and clathrin-independent internalisation, maybe acting in parallel. ACT locates into raft-like domains, and is internalised, also in cells devoid of receptor. Altogether our results suggest that adenylate cyclase toxin, and maybe other homologous pathogenic toxins from the RTX (Repeats in Toxin family to which ACT belongs, may be endowed with an intrinsic capacity to, directly and efficiently, insert into raft-like domains, promoting there its multiple activities. One direct consequence of the integrin removal from the cell surface of the macrophages is the hampering of their adhesion ability, a fundamental property in the immune response of the leukocytes that could be instrumental in the pathogenesis of Bordetella pertussis.

  3. Pertussis toxin inhibits cAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

    NARCIS (Netherlands)

    Snaar-Jagalska, B. Ewa; Haastert, Peter J.M. van

    1990-01-01

    cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two component

  4. Adenylate cyclase toxin-mediated delivery of the S1 subunit of pertussis toxin into mammalian cells.

    Science.gov (United States)

    Iwaki, Masaaki; Konda, Toshifumi

    2016-02-01

    The adenylate cyclase toxin (ACT) of Bordetella pertussis internalizes its catalytic domain into target cells. ACT can function as a tool for delivering foreign protein antigen moieties into immune effector cells to induce a cytotoxic T lymphocyte response. In this study, we replaced the catalytic domain of ACT with an enzymatically active protein moiety, the S1 (ADP-ribosyltransferase) subunit of pertussis toxin (PT). The S1 moiety was successfully internalized independent of endocytosis into sheep erythrocytes. The introduced polypeptide exhibited ADP-ribosyltransferase activity in CHO cells and induced clustering typical to PT. The results indicate that ACT can act as a vehicle for not only epitopes but also enzymatically active peptides to mammalian cells.

  5. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain.

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    Kepa B Uribe

    Full Text Available Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC". The calpain-mediated ACT processing allows trafficking of the "soluble AC" domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools", which would play different roles in the cell pathophysiology.

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

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    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

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

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    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.

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

    Science.gov (United States)

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

    2014-10-10

    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 (Rh) and reduced thermal stability in the mutant complex. Taken together, our data provide new structural insights into the β-hairpin's role in stabilizing interactions between CyaA-ACD and N-CaM.

  9. Amidate prodrugs of 9-[2-(phosphonomethoxy)ethyl]adenine as inhibitors of adenylate cyclase toxin from Bordetella pertussis.

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    Šmídková, Markéta; Dvoráková, Alexandra; Tloust'ová, Eva; Česnek, Michal; Janeba, Zlatko; Mertlíková-Kaiserová, Helena

    2014-01-01

    Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.

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

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    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.

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

    OpenAIRE

    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...

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

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    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

  13. Pituitary adenylate cyclase activating polypeptide and migraine

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  14. Adenylate cyclases involvement in pathogenicity, a minireview.

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    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

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

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

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    Česnek, Michal; Jansa, Petr; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Brust, Tarsis F; Pávek, Petr; Trejtnar, František; Watts, Val J; Janeba, Zlatko

    2015-08-01

    Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 =0.145 μM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.

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

    OpenAIRE

    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 ...

  17. Calcium, acylation, and molecular confinement favor folding of Bordetella pertussis adenylate cyclase CyaA toxin into a monomeric and cytotoxic form.

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    Karst, Johanna C; Ntsogo Enguéné, V Yvette; Cannella, Sara E; Subrini, Orso; Hessel, Audrey; Debard, Sylvain; Ladant, Daniel; Chenal, Alexandre

    2014-10-31

    The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.

  18. Bordetella adenylate cyclase toxin differentially modulates toll-like receptor-stimulated activation, migration and T cell stimulatory capacity of dendritic cells.

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    Irena Adkins

    Full Text Available Adenylate cyclase toxin (CyaA is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR-activated murine and human dendritic cells (DCs. cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4(+ and CD8(+ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4+ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4(+CD25(+Foxp3(+ T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8(+ T cell proliferation and limited the induction of IFN-γ producing CD8(+ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.

  19. Guanylate cyclase in Dictyostelium discoideum with the topology of mammalian adenylate cyclase

    NARCIS (Netherlands)

    Roelofs, J; Snippe, H; Kleineidam, RG; Van Haastert, PJM

    2001-01-01

    The core of adenylate and guanylate cyclases is formed by an intramolecular ol intermolecular dimer of two cyclase domains arranged in an antiparallel fashion. Metazoan membrane-bound adenylate cyclases are composed of 12 transmembrane spanning regions, and two cyclase domains which function as a he

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

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    Giorgio Fedele

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

  1. Adenyl cyclase in the human placenta.

    Science.gov (United States)

    Sato, K; Ryan, K J

    1971-09-21

    This study demonstrated that the human placenta possesses an adenyl cyclase system responsive to catecholamines and sodium flouride (NaF). 2.5 gm human term placentas were homogenized, centrifuged, washed, resuspended, and used as the enzyme system when placed with various agents. Incubations and the determination of adenosine 3', 5' monophosphate (cyclic AMP) formed were performed. Samples stimulated by .0001 M catecholamines (L-epinephrine or L-norepinephrine) or .01 M NaF had higher levels of cyclic AMP than the controls (p. 005 for catecholamine-treated samples and p. 001 for NaF-treated samples). A concentration of .0001 M L-epinephrine or L-norepinephrine appeared to be a maximum effective dose and .0000001 M a minimum. L=epinephrine was 10 times as effective in the stimulation as L-norepinephrine. With .0001 M, 499 and 439 pmoles/10 minutes per 25 mg of tissue was formed, whereas in the control (no added hormones) 256 pmoles/10 minutes were formed. 3.2% ethanol activated the system by a small amount (p.02). Propranolol alone did not appear to have any effect; however, the effect of .0001 M L-epinephrine was reduced by 95% in the presence of .00001 M propranolol. Propranolol had no effect on NaF-stimulated activity.

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

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    Yasmeen Faiz Kazi

    2012-06-01

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

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

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    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.

    Directory of Open Access Journals (Sweden)

    Kepa B Uribe

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

  5. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    International Nuclear Information System (INIS)

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

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    Tomoya eNakamachi

    2012-11-01

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

  9. Adenylate cyclase regulates elongation of mammalian primary cilia

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-10-01

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

  10. Adenylate cyclase regulates elongation of mammalian primary cilia

    International Nuclear Information System (INIS)

    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.

  11. 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)

    OpenAIRE

    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...

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

    Science.gov (United States)

    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

  13. Pituitary adenylate cyclase-activating polypeptide stimulates renin secretion via activation of PAC1 receptors

    DEFF Research Database (Denmark)

    Hautmann, Matthias; Friis, Ulla G; Desch, Michael;

    2007-01-01

    Besides of its functional role in the nervous system, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is involved in the regulation of cardiovascular function. Therefore, PACAP is a potent vasodilator in several vascular beds, including the renal vasculature. Because t...

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-04-08

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

  16. Age-associated alterations in hepatic. beta. -adrenergic receptor/adenylate cyclase complex

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-09-01

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

  17. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) in the circulation after sumatriptan

    DEFF Research Database (Denmark)

    Hansen, Jakob Møller; Fahrenkrug, Jan; Petersen, Jesper Troensegaard;

    2013-01-01

    The origin of migraine pain is still elusive, but increasingly researchers focus on the neuropeptides in the perivascular space of cranial vessels as important mediators of nociceptive input during migraine attacks. The parasympathetic neurotransmitters, pituitary adenylate cyclase activating...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    Directory of Open Access Journals (Sweden)

    Sarah J. Casey

    2014-03-01

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

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

    OpenAIRE

    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...

  2. Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments.

    Science.gov (United States)

    Imbert, M; Chabardès, D; Montégut, M; Clique, A; Morel, F

    1975-01-01

    A method is described, which allows adenylate cyclase activity measurement in single pieces of various nephron segments. Tubular samples of 0.5 to 2 mm length were isolated by microdissection from collagenase treated slices of rabbit kidney. A photograph of each piece was taken in order to measure its length. After a permeabilisation treatment involving preincubation in a hypoosmotic medium and a freezing step, each sample was incubated for 30 mm at 30 degrees C in a medium containing high specific (alpha-32-P)-ATP 3-10-4 M, final volume 2.5 mu 1. The (32P)-cAMP formed was separated from the other labelled nucleotides by filtering the incubate on a dry aluminium oxide microcolumn, 3H cAMP was added as a tracer for measuring cAMP recovery. The sensitivity of the method was found to be a few fentomoles (10-15 M) cAMP. cAMP generation increased linearly as a function of the incubation time up to more than 30 min, and as a function of the length of the segment used. Control and fluoride (5 mM) stimulated adenvlate cyclase activities were measured in the following segments of the nephron: early proximal convoluted tubule (PCT), pars recta of the proximal tubule (PR), thin descending limb of the loop (TDL), cortical portion of the thick ascending limb (CAL), distal convoluted tubule (dct), first branched portion of the collecting tubule (BCT), further cortical (CCT) and medullary (MCT) portions of the collecting tubule. Mean control adenylate cyclase activity varied from 7 (PR) to 75 (BCT) fmoles/mm/30 min. Flouride addition resulted in a 10 (BCT) to 50 (PR) fold increase in enzyme activity. Series of replicates gave a scatter equal to plus or minus 20% (S.D. as a per cent of the mean). The method described appears to be suitable to determine which nephron segments contain hormone-dependent adenylate cyclase.

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

    KAUST Repository

    Hussein, Rana

    2012-11-01

    Cyclic nucleotide, cAMP, is an important signaling molecule in animals and plants. However, in plants the enzymes that synthesize this second messenger, adenyl cyclases (ACs), remain elusive. Given the physiological importance of cAMP in signaling, particularly in response to biotic and abiotic stresses, it is thus important to identify and characterize ACs in higher plants. Using computational approaches, a disease resistance protein from Arabidopsis thaliana, At3g04220 was found to have an AC catalytic center motif. In an attempt to prove that this candidate has adenyl cyclases activity in vitro, the coding sequence of the putative AC catalytic domain of this protein was cloned and expressed in E. coli and the recombinant protein was purified. The nucleotide cyclase activity of the recombinant protein was examined using cyclic nucleotide enzyme immunoassays. In parallel, the expression of At3g04220 was measured in leaves under three different stress conditions in order to determine under which conditions the disease resistance protein could function. Results show that the purified recombinant protein has Mn2+ dependent AC activity in vitro, and the expression analysis supports a role for At3g04220 and cAMP in plant defense.

  4. The effect of adrenergic receptor—adenyl cyclase system on myocardial ischemic preconditioning in rats

    Institute of Scientific and Technical Information of China (English)

    LANXiao-Li; LANJi-Cheng; 等

    2002-01-01

    In order to study the effects of every part of adrenergic receptor-adenyl cyclase system on ischemic preconditioning of myocardium in rats in vivo,SD rats were divided into three groups:IP group,I/R group and CON group.Rate were received surgical procedure and undergone left coronary artery occlusion and reperfusion.Hearts were extracted to analyze the infarct size by TTC staining,to measure serum myocardial enzymes,to study β-AR Bamx and Kd by radioligand binding assay of receptors(RAB),and to check the activity of AC and the content of cAMP by radioimmunoassay(RIA).The infarct area was found much smaller in IP group than I/R group(P<0.001);CK,CK-MB and LDH were found significantly higher in I/R group (P<0.001),The Bmax of β-AR in IP group were higher than in I/R group (P<0.001), No difference of Kd could be seen between IP and I/R group,In IP group,the activity of Ac and the content of cAMP were higher than I/R group(P<0.05 and 0.001,respectively).It is concluded that ischemic preconditioning can protect the hearts from necrosis and reduce endo-enzyme leakage.The system of adrenergic receptor-adenyl cyclase system probably takes part in the protection of the IP.

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Adenylate cyclase 5 is required for melanophore and male pattern development in the guppy (Poecilia reticulata).

    Science.gov (United States)

    Kottler, Verena A; Künstner, Axel; Koch, Iris; Flötenmeyer, Matthias; Langenecker, Tobias; Hoffmann, Margarete; Sharma, Eshita; Weigel, Detlef; Dreyer, Christine

    2015-09-01

    Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects life span and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments.

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

    KAUST Repository

    Gehring, Christoph A

    2010-06-25

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Conner, D.A.

    1988-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Mounira Tlili

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tatjana Arsenijevic

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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Satoko eHattori

    2012-10-01

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

  13. Pertussis toxin

    Energy Technology Data Exchange (ETDEWEB)

    Sekura, R.D.; Moss, J.; Vaughan, M.

    1985-01-01

    This book contains 13 selections. Some of the titles are: Genetic and Functional Studies of Pertussis Toxin Substrates; Effect of Pertussis Toxin on the Hormonal Responsiveness of Different Tissues; Extracellular Adenylate Cyclase of Bordetella pertussis; and GTP-Regulatory Proteins are Introcellular Messagers: A Model for Hormone Action.

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  16. Effect of Cardiopulmonary Bypass on Beta Adrenergic ReceptorAdenylate Cyclase System on Surfaces of Peripheral Lymphocytes

    Institute of Scientific and Technical Information of China (English)

    LUO Ailin; TIAN Yuke; JIN Shiao

    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.

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

    KAUST Repository

    Al-Younis, Inas

    2015-11-27

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

  18. The Arabidopsis thaliana K(+)-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre.

    Science.gov (United States)

    Al-Younis, Inas; Wong, Aloysius; Gehring, Chris

    2015-12-21

    Adenylate cyclases (ACs) catalyse the formation of the second messenger cyclic adenosine 3',5'-monophosphate (cAMP) from adenosine 5'-triphosphate (ATP). Although cAMP is increasingly recognised as an important signalling molecule in higher plants, ACs have remained somewhat elusive. Here we used a search motif derived from experimentally tested guanylyl cyclases (GCs), substituted the residues essential for substrate specificity and identified the Arabidopsis thaliana K(+)-uptake permease 7 (AtKUP7) as one of several candidate ACs. Firstly, we show that a recombinant N-terminal, cytosolic domain of AtKUP7(1-100) is able to complement the AC-deficient mutant cyaA in Escherichia coli and thus restoring the fermentation of lactose, and secondly, we demonstrate with both enzyme immunoassays and mass spectrometry that a recombinant AtKUP7(1-100) generates cAMP in vitro. PMID:26638082

  19. Studies on cell migration, adenylate cyclase and membrane-coating granules in the buccal epithelium of the zinc-deficient rabbit, including the influence of isoproterenol.

    Science.gov (United States)

    Chen, S Y

    1988-01-01

    Cell migration was slightly increased; cytochemical reaction deposits of adenylate cyclase and the area density of membrane-coating granules (MCG) were significantly increased. Upon isoproterenol stimulation, the MCG area density was significantly increased, whereas the cell migration rate was unchanged. Thus in zinc deficiency, there may be a simultaneous increase in the production and secretion of MCGs, in adenylate cyclase activity, and in cell migration. The non-significantly increased cell migration rate may not keep pace with the significantly increased cell-production rate, resulting in thickening of the epithelium.

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

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A;

    2012-01-01

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

  1. Effect of the pituitary adenylate cyclase-activating polypeptide on the autophagic activation observed in in vitro and in vivo models of Parkinson's disease.

    Science.gov (United States)

    Lamine-Ajili, Asma; Fahmy, Ahmed M; Létourneau, Myriam; Chatenet, David; Labonté, Patrick; Vaudry, David; Fournier, Alain

    2016-04-01

    Parkinson's disease (PD) is a neurodegenerative disorder that leads to destruction of the midbrain dopaminergic (DA) neurons. This phenomenon is related to apoptosis and its activation can be blocked by the pituitary adenylate cyclase-activating polypeptide (PACAP). Growing evidence indicates that autophagy, a self-degradation activity that cleans up the cell, is induced during the course of neurodegenerative diseases. However, the role of autophagy in the pathogenesis of neuronal disorders is yet poorly understood and the potential ability of PACAP to modulate the related autophagic activation has never been significantly investigated. Hence, we explored the putative autophagy-modulating properties of PACAP in in vitro and in vivo models of PD, using the neurotoxic agents 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), respectively, to trigger alterations of DA neurons. In both models, following the toxin exposure, PACAP reduced the autophagic activity as evaluated by the production of LC3 II, the modulation of the p62 protein levels, and the formation of autophagic vacuoles. The ability of PACAP to inhibit autophagy was also observed in an in vitro cell assay by the blocking of the p62-sequestration activity produced with the autophagy inducer rapamycin. Thus, the results demonstrated that autophagy is induced in PD experimental models and that PACAP exhibits not only anti-apoptotic but also anti-autophagic properties.

  2. Effects of cimetidine on adenylate cyclase activity of guinea pig gastric mucosa stimulated by histamine, sodium fluoride and 5'-guanylylimidodiphosphate.

    Science.gov (United States)

    Anttila, P; Westermann, E

    1976-08-01

    Cimetidine, a recently developed histamine H2-receptor blocking agent has been shown to be a potent inhibitor of histamine-stimulated gastric acid secretion in rat, cat, dog and man. To study the mode of action of cimetidine the modification of stimulatory effects of histamine, sodium flouride and 5'-guanylylimidodiphosphate by cimetidine on the adenylate cyclase activity of guinea pig gastric mucosa was studied. The effect of cimetidine was also compared to that of metiamide, an older histamine H2-receptor antagonist. The effect of cimetidine was qualitatively similar to that of metiamide, i.e. a selective blockade of histamine H2-receptors. Quantitatively cimetidine was about 10-fold more potent than metiamide.

  3. Subtyping of Salmonella enterica subspecies I using single nucleotide polymorphisms in adenylate cyclase (cyaA)

    Science.gov (United States)

    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single nucleotide polymorphisms (SNPs) were characterized within adenylate cyclas...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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 ...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  7. Linalool from rosewood (Aniba rosaeodora Ducke) oil inhibits adenylate cyclase in the retina, contributing to understanding its biological activity.

    Science.gov (United States)

    Sampaio, Lucia de Fatima S; Maia, José Guilherme S; de Parijós, Amanda M; de Souza, Rita Z; Barata, Lauro Euclides S

    2012-01-01

    Rosewood oil (RO) (Aniba rosaeodora Ducke) is rich in linalool, a monoterpene alcohol, which has well studied anxiolytic, sedative and anticonvulsant effects. The inhibition of the increases in cAMP protects against seizures in a diversity of models of epilepsy. In this paper, the principal aim was to investigate the effects of RO, (±)-linalool and (-)-linalool) on adenylate cyclase. They were tested in chick retinas and forskolin was used to stimulate the enzyme target. The phosphodiesterase inhibitor, 4-(3-butoxy-4-methoxybenzyl)-imidazolidin-2-one, and the non-selective adenosine receptor antagonist 3-isobutyl-methyl-xanthine (IBMX), were used to control the participation of phosphodiesterase and adenosine receptors in the resulting effects, respectively. The cAMP accumulation was measured by enzyme immune assay (EIA). Rosewood oil, (-)-linalool and (±)-linalool inhibited exclusively the cAMP accumulation stimulated by forskolin, even when adenosine receptors were blocked with IBMX. The IC(50) values (in μ m concentration range) calculated from their concentration response-curves were not statistically different, however, the compounds presented a different relative efficacy. These results extend the range of subcellular mechanisms underlying the relaxant action of linalool on the central nervous system.

  8. PPARgamma-dependent regulation of adenylate cyclase 6 amplifies the stimulatory effect of cAMP on renin gene expression.

    Science.gov (United States)

    Desch, Michael; Schubert, Thomas; Schreiber, Andrea; Mayer, Sandra; Friedrich, Björn; Artunc, Ferruh; Todorov, Vladimir T

    2010-11-01

    The second messenger cAMP plays an important role in the regulation of renin gene expression. Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is known to stimulate renin gene transcription acting through PPARγ-binding sequences in renin promoter. We show now that activation of PPARγ by unsaturated fatty acids or thiazolidinediones drastically augments the cAMP-dependent increase of renin mRNA in the human renin-producing cell line Calu-6. The underlying mechanism involves potentiation of agonist-induced cAMP increase and up-regulation of adenylate cyclase 6 (AC6) gene expression. We identified a palindromic element with a 3-bp spacer (Pal3) in AC6 intron 1 (AC6Pal3). AC6Pal3 bound PPARγ and mediated trans-activation by PPARγ agonist. AC6 knockdown decreased basal renin mRNA level and attenuated the maximal PPARγ-dependent stimulation of the cAMP-induced renin gene expression. AC6Pal3 decoy oligonucleotide abrogated the PPARγ-dependent potentiation of cAMP-induced renin gene expression. Treatment of mice with PPARγ agonist increased AC6 mRNA kidney levels. Our data suggest that in addition to its direct effect on renin gene transcription, PPARγ "sensitizes" renin gene to cAMP via trans-activation of AC6 gene. AC6 has been identified as PPARγ target gene with a functional Pal3 sequence.

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

    Directory of Open Access Journals (Sweden)

    Tóth Gábor

    2002-02-01

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

  10. Regulation by the quorum sensor from Vibrio indicates a receptor function for the membrane anchors of adenylate cyclases.

    Science.gov (United States)

    Beltz, Stephanie; Bassler, Jens; Schultz, Joachim E

    2016-02-27

    Adenylate cyclases convert intra- and extracellular stimuli into a second messenger cAMP signal. Many bacterial and most eukaryotic ACs possess membrane anchors with six transmembrane spans. We replaced the anchor of the AC Rv1625c by the quorum-sensing receptor from Vibrio harveyi which has an identical 6TM design and obtained an active, membrane-anchored AC. We show that a canonical class III AC is ligand-regulated in vitro and in vivo. At 10 µM, the cholera-autoinducer CAI-1 stimulates activity 4.8-fold. A sequence based clustering of membrane domains of class III ACs and quorum-sensing receptors established six groups of potential structural and functional similarities. The data support the notion that 6TM AC membrane domains may operate as receptors which directly regulate AC activity as opposed and in addition to the indirect regulation by GPCRs in eukaryotic congeners. This adds a completely novel dimension of potential AC regulation in bacteria and vertebrates.

  11. Stress tolerance of the Saccharomyces cerevisiae adenylate cyclase fil1 (CYR1) mutant depends on Hsp26.

    Science.gov (United States)

    Vianna, Cristina R; Ferreira, Mariana C; Silva, Carol L C; Tanghe, An; Neves, Maria J; Thevelein, Johan M; Rosa, Carlos A; Van Dijck, Patrick

    2010-01-01

    Fermentation-induced loss of stress resistance in yeast is an important phenotype from an industrial point of view. It hampers optimal use of frozen dough applications as well as high gravity brewing fermentations because these applications require stress-tolerant yeast strains during active fermentation. Different mutants (e.g. fil1, an adenylate cyclase mutant CYR1(lys1682)) that are affected in this loss of stress resistance have been isolated, but so far the identification of the target genes important for the increased tolerance has failed. Previously we have shown that neither trehalose nor Hsp104 nor STRE-controlled genes are involved in the higher stress tolerance of the fil1 mutant. The contribution of other putative downstream factors of the PKA pathway was investigated and here we show that the small heat-shock protein Hsp26 is required for the high heat stress tolerance of the fil1 mutant, both in stationary phase cells as well as during active fermentation. PMID:20924200

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

    Science.gov (United States)

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

    2015-03-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2013-09-01

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

  15. Pituitary adenylate cyclase activating-peptide and its receptor antagonists in development of acute pancreatitis in rats

    Institute of Scientific and Technical Information of China (English)

    You-Dai Chen; Zong-Guang Zhou; Zhao Wang; Hong-Kai Gao; Wen-Wei Yan; Cun Wang; Gao-Ping Zhao; Xiao-Hui Peng

    2005-01-01

    AIM: Pituitary adenylate cyclase activating-peptide (PACAP) is a late member of the secretin/glucagon/vasoactive intestinal peptide (VIP) family of brain-gut peptides. It is unknown whether PACAP takes part in the development of acute pancreatitis and whether PACAP or its antagonists can be used to suppress the progression of acute pancreatitis.We investigated the actions of PACAP and its receptor antagonists in acute pancreatitis on rats.METHODS: Acute pancreatitis was induced in rats with caerulein or 3.5% sodium taurocholate. The rats were continuously infused with 5-30 μg/kg PACAP via jugular vein within the first 90 min, while 10-100 μg/kg PACAP6-27 and (4-Cl-D-Phe6, Leu17) VIP (PACAP receptor antagonists) were intravenously infused for 1 h. Biochemical and histopathological assessments were made at 4 h after infusion. Pancreatic and duodenal PACAP concentrations were determined by enzyme-linked immunosorbent assay (ELISA). Chinese ink-perfused pancreas was fixed, sectioned and cleared for counting the functional capillary density.RESULTS: PACAP augmented caerulein-induced pancreatitis and failed to ameliorate sodium taurocholate-induced pancreatitis. ELISA revealed that relative concentrations of PACAP in pancreas and duodenum were significantly increased in both sodium taurocholate- and caeruleininduced pancreatitis compared with those in normal controls.Unexpectedly, PACAP6-27 and (4-Cl-DPhe6, Leu17) VIP could induce mild acute pancreatitis and aggravate caeruleininduced pancreatitis with characteristic manifestations of acute hemorrhagic/necrotizing pancreatitis. Functional capillary density of pancreas was interpreted in the context of pancreatic edema, and calibrated functional capillary density (calibrated FCD), which combined measurement of functional capillary density with dry weight/wet weight ratio, was introduced. Hyperemia or congestion, rather than ischemia, characterized pancreatic microcirculatory changes in acute pancreatitis

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

    Science.gov (United States)

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

    2010-10-13

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-09-23

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

  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

    Institute of Scientific and Technical Information of China (English)

    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.

    Science.gov (United States)

    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☆

    OpenAIRE

    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. First report of the pituitary adenylate cyclase activating polypeptide (PACAP) in crustaceans: conservation of its functions as growth promoting factor and immunomodulator in the white shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Lugo, Juana María; Carpio, Yamila; Morales, Reynold; Rodríguez-Ramos, Tania; Ramos, Laida; Estrada, Mario Pablo

    2013-12-01

    The high conservation of the pituitary adenylate cyclase activating polypeptide (PACAP) sequence indicates that this peptide fulfills important biological functions in a broad spectrum of organisms. However, in invertebrates, little is known about its presence and its functions remain unclear. Up to now, in non-mammalian vertebrates, the majority of studies on PACAP have focused mainly on the localization, cloning and structural evolution of this peptide. As yet, little is known about its biological functions as growth factor and immunomodulator in lower vertebrates. Recently, we have shown that PACAP, apart from its neuroendocrine role, influences immune functions in larval and juvenile fish. In this work, we isolated for the first time the cDNA encoding the mature PACAP from a crustacean species, the white shrimp Litopenaeus vannamei, corroborating its high degree of sequence conservation, when compared to sequences reported from tunicates to mammalian vertebrates. Based on this, we have evaluated the effects of purified recombinant Clarias gariepinus PACAP administrated by immersion baths on white shrimp growth and immunity. We demonstrated that PACAP improves hemocyte count, superoxide dismutase, lectins and nitric oxide synthase derived metabolites in treated shrimp related with an increase in total protein concentration and growth performance. From our results, PACAP acts as a regulator of shrimp growth and immunity, suggesting that in crustaceans, as in vertebrate organisms, PACAP is an important molecule shared by both the endocrine and the immune systems.

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

    Science.gov (United States)

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

    2016-01-01

    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

  3. Testosterone regulates levels of cystic fibrosis transmembrane regulator, adenylate cyclase, and cAMP in the seminal vesicles of orchidectomized rats.

    Science.gov (United States)

    Ramli, Nur Siti Khadijah; Giribabu, Nelli; Muniandy, Sekaran; Salleh, Naguib

    2016-01-15

    Secretions of chloride (Cl(-))- and bicarbonate (HCO3(-))-rich fluid by the seminal vesicles could involve cystic fibrosis transmembrane regulator (CFTR), which activity can be stimulated by cAMP generated from the reaction involving adenylate cyclase (AC). In this study, we investigated levels of CFTR, AC, and cAMP in the seminal vesicles under testosterone influence. Orchidectomized adult male rats received 7-day treatment with 125 or 250 μg/kg/day of testosterone with or without flutamide or finasteride. At the end of the treatment, animals were sacrificed and seminal vesicles were harvested for analyses of CFTR and AC protein expression level by Western blotting. Distribution of CFTR and AC in seminal vesicles was observed by immunohistochemistry. Levels of cAMP and dihydrotestosterone in seminal vesicle homogenates were measured by ELISA. Cystic fibrosis transmembrane regulator, AC, and cAMP levels increased with increasing doses of testosterone (P seminal vesicle lumen with higher expression levels observed in testosterone-treated rats than in non-treated orchidectomized rats (P seminal vesicle homogenates after treatment with 250 μg/kg/day than with 125 μg/kg/day of testosterone (P seminal vesicles might contribute toward an increase in Cl(-) and HCO3(-) concentrations in the seminal fluid as reported under testosterone influence.

  4. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) regulate murine neural progenitor cell survival, proliferation, and differentiation.

    Science.gov (United States)

    Scharf, Eugene; May, Victor; Braas, Karen M; Shutz, Kristin C; Mao-Draayer, Yang

    2008-11-01

    Neural stem/progenitor cells (NPC) have gained wide interest over the last decade from their therapeutic potential, either through transplantation or endogenous replacement, after central nervous system (CNS) disease and damage. Whereas several growth factors and cytokines have been shown to promote NPC survival, proliferation, or differentiation, the identification of other regulators will provide much needed options for NPC self-renewal or lineage development. Although previous studies have shown that pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal peptide (VIP) can regulate stem/progenitor cells, the responses appeared variable. To examine the direct roles of these peptides in NPCs, postnatal mouse NPC cultures were withdrawn from epidermal growth factor (EGF) and fibroblastic growth factor (FGF) and maintained under serum-free conditions in the presence or absence of PACAP27, PACAP38, or VIP. The NPCs expressed the PAC1(short)null receptor isoform, and the activation of these receptors decreased progenitor cell apoptosis more than 80% from TUNEL assays and facilitated proliferation more than fivefold from bromodeoxyuridine (BrdU) analyses. To evaluate cellular differentiation, replicate control and peptide-treated cultures were examined for cell fate marker protein and transcript expression. In contrast with previous work, PACAP peptides downregulated NPC differentiation, which appeared consistent with the proliferation status of the treated cells. Accordingly, these results demonstrate that PACAP signaling is trophic and can maintain NPCs in a multipotent state. With these attributes, PACAP may be able to promote endogenous NPC self-renewal in the adult CNS, which may be important for endogenous self-repair in disease and ageing processes.

  5. Optogenetic Modulation of an Adenylate Cyclase in Toxoplasma gondii Demonstrates a Requirement of the Parasite cAMP for Host-Cell Invasion and Stage Differentiation*

    Science.gov (United States)

    Hartmann, Anne; Arroyo-Olarte, Ruben Dario; Imkeller, Katharina; Hegemann, Peter; Lucius, Richard; Gupta, Nishith

    2013-01-01

    Successful infection and transmission of the obligate intracellular parasite Toxoplasma gondii depends on its ability to switch between fast-replicating tachyzoite (acute) and quiescent bradyzoite (chronic) stages. Induction of cAMP in the parasitized host cells has been proposed to influence parasite differentiation. It is not known whether the parasite or host cAMP is required to drive this phenomenon. Other putative roles of cAMP for the parasite biology also remain to be identified. Unequivocal research on cAMP-mediated signaling in such intertwined systems also requires a method for an efficient and spatial control of the cAMP pool in the pathogen or in the enclosing host cell. We have resolved these critical concerns by expressing a photoactivated adenylate cyclase that allows light-sensitive control of the parasite or host-cell cAMP. Using this method, we reveal multiple roles of the parasite-derived cAMP in host-cell invasion, stage-specific expression, and asexual differentiation. An optogenetic method provides many desired advantages such as: (i) rapid, transient, and efficient cAMP induction in extracellular/intracellular and acute/chronic stages; (ii) circumvention of the difficulties often faced in cultures, i.e. poor diffusion, premature degradation, steady activation, and/or pleiotropic effects of cAMP agonists and antagonists; (iii) genetically encoded enzyme expression, thus inheritable to the cell progeny; and (iv) conditional and spatiotemporal control of cAMP levels. Importantly, a successful optogenetic application in Toxoplasma also illustrates its wider utility to study cAMP-mediated signaling in other genetically amenable two-organism systems such as in symbiotic and pathogen-host models. PMID:23525100

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Institute of Scientific and Technical Information of China (English)

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

    Science.gov (United States)

    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

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

    OpenAIRE

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

    2015-01-01

    BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. METHODS: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing...

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

    NARCIS (Netherlands)

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

    2015-01-01

    BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectio

  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.

    Science.gov (United States)

    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. Changes in vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide and neuropeptide Y-ergic structures of the enteric nervous system in the carcinoma of the human large intestine.

    Directory of Open Access Journals (Sweden)

    Ireneusz Mirosław Łakomy

    2010-08-01

    Full Text Available This investigation was aimed at immunohistochemical analysis of potential changes in the enteric nervous system caused by cancer of the large intestine. In this purpose, neurons and nerve fibers of intestinal plexuses containing neuropeptides: vasoactive intestinal peptide (VIP, pituitary adenylate cyclase-activating polypeptide (PACAP and neuropeptide Y (NPY, in pathologically changed part of the large intestine were microscpically observed and compared. Samples were taken from patients operated due to cancer of the sigmoid colon and rectum. The number of neurons and density of nerve fibres containing neuropeptides found in sections with cancer tissues were compared to those observed in sections from the uninvolved intestinal wall. Changes relating to reductions in the number of NPY-ergic neurons and density of nerve fibres in submucous and myenteric plexuses in the sections with cancer tissues (pathological sections were statistically significant. A statistically similar presence of VIP-ergic and PACAP-ergic neurons in the submucosal and myenteric plexuses was observed in both the pathological and control sections. On the other hand, in the pathological sections, VIP-ergic nerve fibres in the myenteric plexuses and PACAP-ergic nerve fibres in the submucosal and myenteric plexuses were found to be less dense. Analysis revealed changes in pathologically affected part of the large intestine may caused disruption of proper intestinal function. Observed changes in the neural elements which are responsible for relaxation of the intestine may suggest dysfunction in the innervation of this part of the colon.

  15. 阿尔茨海默病G蛋白-腺苷酸环化酶系统信号途经的机理、防治综述%Review of Mechanism Prevention and Treatment of G Protein-Adenylate Cyclase Signal Pathway in Alzheimer' s Disease

    Institute of Scientific and Technical Information of China (English)

    罗本华; 张雪竹; 赵岚; 成海燕; 韩景献; 于建春

    2012-01-01

    在AD的发病过程中,多种信号分子的含量异常及分布改变,以及信息传递通路异常,在AD病理改变改变中发挥重要作用;信息传递与信号通路异常是AD发病机制的一个共同途径.第一,AD中,Gia、Gsa蛋白的表达差异是引起腺苷酸环化酶信号系统功能失调;第二,G蛋白的偶联活性的差异是引起腺苷酸环化酶系统信号功能失调的原因:(1)AC与Gia、Gsa蛋白偶联环节出现障碍是AD中AC功能失调的重要原因.(2)G蛋白及其GTP酶活性失常是AD信号转导功能障碍的原因;第三,AD中,G蛋白与受体偶联障碍及受体脱敏是影响G蛋白活性的重要方面;第四,配体激活G蛋白偶联受体的能力损害也是AD中AC功能失调的重要原因;最后,腺苷酸环化酶信号系统功能失调是导致AD病理改变的原因.AD中,G蛋白介导腺苷酸环化酶信号系统功能失调对AD的防治策略有重要启示作用.%In AD, both the abnormal content and the change of distribution of many kinds of signaling molecules and the abnormal signal pathways play very important roles in Alzheimer' s disease pathological change, and the abnormal information transfer and signal pathways are common channel in Alzheimer' s disease pathogenesis. Firstly,the difference of expressed content in Gsot and Gia protein is the important cause of functional disorder of adenylate cyclase signal system. Secondly, the difference of G protein coupled activity can bring about the functional disorder of adenylate cyclase signal system. (1) The coupled disorder between Gsα,Giα protein and adenylate cyclase can cause functional disorder of adenylate cyclase in AD. (2)The disorder of GT-Pase activity which can hydrolyses Gα-GTP into Gα-GDP is the important cause of signal transduction dysfunction. Thirdly, the receptors coupled G protein activity disorder and receptor desensitization are the important aspects of affecting G protein activity. Fourthly

  16. 斜带石斑鱼PACAP的原核表达及活性分析%The prokaryotic expression and biological activity of the pituitary adenylate cyclase activating polypeptide in groupers Epinephelus coioides

    Institute of Scientific and Technical Information of China (English)

    江湧; 李文笙; 林浩然

    2005-01-01

    自1989年从绵羊下丘脑提取物发现垂体腺苷酸环化酶激活多肽(Pituitary adenylate cyclase activating polypeptide,PACAP)以来(Miyata et al.,1989),已证明它能促进垂体激素释放,同时还具有神经递质、神经调质和神经营养等作用,使对PACAP的研究成为十分活跃的领域。PACAP属于血管活性肠肽(VIP)-胰高血糖素-生长激素释放因子-分泌素家族(Campbell and Scanes,1992)成员,已鉴别出包含27和38个氨基酸两种类型。对原索动物(McRory et al.,1997)、两栖类(蛙)(Alexandre et al.,2000)、爬行类(蜥蜴)(Pohland Wank,1998)、鸟类(鸡)(McRory et al.,1997),啮齿类(鼠)(Ghatei et al.,1993)等脊椎动物PACAP的研究多集中在结构与进化方面,对功能了解甚少。

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

    OpenAIRE

    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...

  18. Interactions between Autophagy and Bacterial Toxins: Targets for Therapy?

    Science.gov (United States)

    Mathieu, Jacques

    2015-08-04

    Autophagy is a physiological process involved in defense mechanisms for clearing intracellular bacteria. The autophagic pathway is finely regulated and bacterial toxins interact with this process in a complex manner. Bacterial toxins also interact significantly with many biochemical processes. Evaluations of the effects of bacterial toxins, such as endotoxins, pore-forming toxins and adenylate cyclases, on autophagy could support the development of new strategies for counteracting bacterial pathogenicity. Treatment strategies could focus on drugs that enhance autophagic processes to improve the clearance of intracellular bacteria. However, further in vivo studies are required to decipher the upregulation of autophagy and potential side effects limiting such approaches. The capacity of autophagy activation strategies to improve the outcome of antibiotic treatment should be investigated in the future.

  19. Interactions between Autophagy and Bacterial Toxins: Targets for Therapy?

    Directory of Open Access Journals (Sweden)

    Jacques Mathieu

    2015-08-01

    Full Text Available Autophagy is a physiological process involved in defense mechanisms for clearing intracellular bacteria. The autophagic pathway is finely regulated and bacterial toxins interact with this process in a complex manner. Bacterial toxins also interact significantly with many biochemical processes. Evaluations of the effects of bacterial toxins, such as endotoxins, pore-forming toxins and adenylate cyclases, on autophagy could support the development of new strategies for counteracting bacterial pathogenicity. Treatment strategies could focus on drugs that enhance autophagic processes to improve the clearance of intracellular bacteria. However, further in vivo studies are required to decipher the upregulation of autophagy and potential side effects limiting such approaches. The capacity of autophagy activation strategies to improve the outcome of antibiotic treatment should be investigated in the future.

  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

    Science.gov (United States)

    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

    Science.gov (United States)

    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 cause problems ...

  2. Synthetic peptides with antigenic specificity for bacterial toxins.

    Science.gov (United States)

    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

    International Nuclear Information System (INIS)

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Allosteric activation of Bordetella pertussis adenylyl cyclase by calmodulin: molecular dynamics and mutagenesis studies.

    Science.gov (United States)

    Selwa, Edithe; Davi, Marilyne; Chenal, Alexandre; Sotomayor-Pérez, Ana-Cristina; Ladant, Daniel; Malliavin, Thérèse E

    2014-07-25

    Adenylyl cyclase (AC) toxin is an essential toxin that allows Bordetella pertussis to invade eukaryotic cells, where it is activated after binding to calmodulin (CaM). Based on the crystal structure of the AC catalytic domain in complex with the C-terminal half of CaM (C-CaM), our previous molecular dynamics simulations (Selwa, E., Laine, E., and Malliavin, T. (2012) Differential role of calmodulin and calcium ions in the stabilization of the catalytic domain of adenyl cyclase CyaA from Bordetella pertussis. Proteins 80, 1028–1040) suggested that three residues (i.e. Arg(338), Asn(347), and Asp(360)) might be important for stabilizing the AC/CaM interaction. These residues belong to a loop-helix-loop motif at the C-terminal end of AC, which is located at the interface between CaM and the AC catalytic loop. In the present study, we conducted the in silico and in vitro characterization of three AC variants, where one (Asn(347); ACm1A), two (Arg(338) and Asp(360); ACm2A), or three residues (Arg(338), Asn(347), and Asp(360); ACm3A) were substituted with Ala. Biochemical studies showed that the affinities of ACm1A and ACm2A for CaM were not affected significantly, whereas that of ACm3A was reduced dramatically. To understand the effects of these modifications, molecular dynamics simulations were performed based on the modified proteins. The molecular dynamics trajectories recorded for the ACm3AC-CaM complex showed that the calcium-binding loops of C-CaM exhibited large fluctuations, which could be related to the weakened interaction between ACm3A and its activator. Overall, our results suggest that the loop-helix-loop motif at the C-terminal end of AC is crucial during CaM binding for stabilizing the AC catalytic loop in an active configuration.

  6. 百日咳杆菌腺苷酸环化酶毒素基因的克隆及原核表达%Cloning and Prokaryotic Expression of Bordetella pertussis Adenylate Cyclase Toxin Gene

    Institute of Scientific and Technical Information of China (English)

    石碧珠; 张华捷; 孟民杰; 张庶民

    2010-01-01

    目的 克隆百日咳杆菌腺苷酸环化酶毒素(CyaA,ACT)基因,表达并纯化重组CyaA蛋白.方法 从百日咳杆菌CS株的基因组DNA中PCR扩增CyaA编码基因,克隆人载体pET30a,构建重组原核表达质粒pET30a/cyaA,转化感受态大肠杆菌BL21(DE3),IPTG诱导表达.表达的重组蛋白经8 mol/L尿素变性、透析复性、DEAE阴离子交换柱纯化后,采用Western blot法鉴定其反应原性.结果 重组原核表达质粒pET30a/cyaA经PCR、双酶切及测序证明构建正确;表达的重组蛋白主要以包涵体形式存在,表达量约占菌体总蛋白的20%;纯化的重组蛋白纯度达90%左右,可与全细胞百日咳疫苗和无细胞百日咳疫苗免疫血清结合.结论 已成功克隆了百日咳杆菌cyaA基因,并在大肠杆菌中表达了重组CyaA蛋白,为进一步开展CyaA的应用研究奠定了基础.

  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

    Institute of Scientific and Technical Information of China (English)

    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. Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

    Directory of Open Access Journals (Sweden)

    Laakso Kati

    2007-10-01

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

  9. Disorder-to-Order Transition in the CyaA Toxin RTX Domain: Implications for Toxin Secretion

    Directory of Open Access Journals (Sweden)

    Ana-Cristina Sotomayor-Pérez

    2014-12-01

    Full Text Available The past decade has seen a fundamental reappraisal of the protein structure-to-function paradigm because it became evident that a significant fraction of polypeptides are lacking ordered structures under physiological conditions. Ligand-induced disorder-to-order transition plays a key role in the biological functions of many proteins that contain intrinsically disordered regions. This trait is exhibited by RTX (Repeat in ToXin motifs found in more than 250 virulence factors secreted by Gram-negative pathogenic bacteria. We have investigated several RTX-containing polypeptides of different lengths, all derived from the Bordetella pertussis adenylate cyclase toxin, CyaA. Using a combination of experimental approaches, we showed that the RTX proteins exhibit the hallmarks of intrinsically disordered proteins in the absence of calcium. This intrinsic disorder mainly results from internal electrostatic repulsions between negatively charged residues of the RTX motifs. Calcium binding triggers a strong reduction of the mean net charge, dehydration and compaction, folding and stabilization of secondary and tertiary structures of the RTX proteins. We propose that the intrinsically disordered character of the RTX proteins may facilitate the uptake and secretion of virulence factors through the bacterial secretion machinery. These results support the hypothesis that the folding reaction is achieved upon protein secretion and, in the case of proteins containing RTX motifs, could be finely regulated by the calcium gradient across bacterial cell wall.

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

    Directory of Open Access Journals (Sweden)

    Nguyen Chinh

    2012-01-01

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

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

    OpenAIRE

    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...

  12. Targeted silencing of anthrax toxin receptors protects against anthrax toxins.

    Science.gov (United States)

    Arévalo, Maria T; Navarro, Ashley; Arico, Chenoa D; Li, Junwei; Alkhatib, Omar; Chen, Shan; Diaz-Arévalo, Diana; Zeng, Mingtao

    2014-05-30

    Anthrax spores can be aerosolized and dispersed as a bioweapon. Current postexposure treatments are inadequate at later stages of infection, when high levels of anthrax toxins are present. Anthrax toxins enter cells via two identified anthrax toxin receptors: tumor endothelial marker 8 (TEM8) and capillary morphogenesis protein 2 (CMG2). We hypothesized that host cells would be protected from anthrax toxins if anthrax toxin receptor expression was effectively silenced using RNA interference (RNAi) technology. Thus, anthrax toxin receptors in mouse and human macrophages were silenced using targeted siRNAs or blocked with specific antibody prior to challenge with anthrax lethal toxin. Viability assays were used to assess protection in macrophages treated with specific siRNA or antibody as compared with untreated cells. Silencing CMG2 using targeted siRNAs provided almost complete protection against anthrax lethal toxin-induced cytotoxicity and death in murine and human macrophages. The same results were obtained by prebinding cells with specific antibody prior to treatment with anthrax lethal toxin. In addition, TEM8-targeted siRNAs also offered significant protection against lethal toxin in human macrophage-like cells. Furthermore, silencing CMG2, TEM8, or both receptors in combination was also protective against MEK2 cleavage by lethal toxin or adenylyl cyclase activity by edema toxin in human kidney cells. Thus, anthrax toxin receptor-targeted RNAi has the potential to be developed as a life-saving, postexposure therapy against anthrax.

  13. Uridylation and adenylation of RNAs.

    Science.gov (United States)

    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. Role of Toxin Functional Domains in Anthrax Pathogenesis

    OpenAIRE

    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...

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    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...

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    OpenAIRE

    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) ...

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    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...... a 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 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...

  20. Integrative Signaling Networks of Membrane Guanylate Cyclases: Biochemistry and Physiology

    Science.gov (United States)

    Sharma, Rameshwar K.; Duda, Teresa; Makino, Clint L.

    2016-01-01

    This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs), highlighting contributions made by the authors and their collaborators. Upon resolution of early contentious studies, cyclic GMP emerged alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca2+-sensing subunits that either increase or decrease cyclic GMP synthesis, depending on subunit identity. In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and Transient Receptor Potential (TRP) ion channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory.

  1. Cytosolic adenylate changes during exercise in prawn muscle

    Energy Technology Data Exchange (ETDEWEB)

    Thebault, M.T. [College de France, 29 - Concarneau (France); Raffin, J.P.; Pichon, R. [Brest Univ., 29 (France)

    1994-11-01

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

  2. Structural studies of Schistosoma mansoni adenylate kinases

    Energy Technology Data Exchange (ETDEWEB)

    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)

  3. Polyamine toxins

    DEFF Research Database (Denmark)

    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 have...

  4. Bicarbonate-Regulated Soluble Adenylyl Cyclase

    Directory of Open Access Journals (Sweden)

    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.

  5. Botulinum toxin.

    OpenAIRE

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

    Directory of Open Access Journals (Sweden)

    Janna eBlechman

    2013-05-01

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

  7. Adenylate cyclase and the cyclic AMP receptor protein modulate stress resistance and virulence capacity of uropathogenic Escherichia coli.

    Science.gov (United States)

    Donovan, Grant T; Norton, J Paul; Bower, Jean M; Mulvey, Matthew A

    2013-01-01

    In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, the cyaA and crp deletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H(2)O(2)) and acid stress. In the mutant strains, H(2)O(2) resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    OpenAIRE

    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...

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

    DEFF Research Database (Denmark)

    Aakerlund, L; Gether, U; Fuhlendorff, J;

    1990-01-01

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

  11. Botulinum toxin

    Directory of Open Access Journals (Sweden)

    Nigam P

    2010-01-01

    Full Text Available 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 three months. Botulinum toxins now play a very significant role in the management of a wide variety of medical conditions, especially strabismus and focal dystonias, hemifacial spasm, and various spastic movement disorders, headaches, hypersalivation, hyperhidrosis, and some chronic conditions that respond only partially to medical treatment. The list of possible new indications is rapidly expanding. The cosmetological applications include correction of lines, creases and wrinkling all over the face, chin, neck, and chest to dermatological applications such as hyperhidrosis. Injections with botulinum toxin are generally well tolerated and side effects are few. A precise knowledge and understanding of the functional anatomy of the mimetic muscles is absolutely necessary to correctly use botulinum toxins in clinical practice.

  12. Diterpene Cyclases and the Nature of the Isoprene Fold

    Science.gov (United States)

    Cao, Rong; Zhang, Yonghui; Mann, Francis M.; Huang, Cancan; Mukkamala, Dushyant; Hudock, Michael P.; Mead, Matthew; Prisic, Sladjana; Wang, Ke; Lin, Fu-Yang; Chang, Ting-Kai; Peters, Reuben; Oldfield, Eric

    2013-01-01

    The structures and mechanism of action of many terpene cyclases are known, but there are no structures of diterpene cyclases. Here, we propose structural models based on bioinformatics, site-directed mutagenesis, domain swapping, enzyme inhibition and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain ∼20 α-helices and the same conserved “QW” and DxDD motifs as in triterpene cyclases, indicating the presence of a βγ barrel structure. Plant diterpene cyclases have a similar catalytic motif and βγ-domain structure together with a third, α-domain, forming an αβγ structure, and in H+-initiated cyclases, there is an EDxxD-like Mg2+/diphosphate binding motif located in the γ-domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (βγ) bacterial triterpene cyclases to (βγ) bacterial and thence to (αβγ) plant diterpene cyclases. PMID:20602361

  13. Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

    Directory of Open Access Journals (Sweden)

    Hildebrand Dagmar

    2012-08-01

    Full Text Available Abstract Background Lipopolysaccharide (LPS-triggered Toll-like receptor (TLR 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement. Results Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gβγ-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gβγ-mediated activation of PI3kinase and JNK activation. Conclusions On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host’s immune

  14. Correlated inter-domain motions in adenylate kinase.

    Directory of Open Access Journals (Sweden)

    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.

  15. Botox (Botulinum Toxin)

    Science.gov (United States)

    ... 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 ...

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

    Directory of Open Access Journals (Sweden)

    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.

  17. An aberrant adenylate kinase isoenzyme from the serum of patients with Duchenne muscular dystrophy.

    Science.gov (United States)

    Hamada, M; Okuda, H; Oka, K; Watanabe, T; Ueda, K; Nojima, M; Kuby, S A; Manship, M; Tyler, F H; Ziter, F A

    1981-08-13

    The sera from patients with human Duchenne (X-linked) progressive muscular dystrophy contain elevated adenylate kinase (ATP: AMP phosphotransferase, EC 2.7.4.3) activities, in addition to their characteristically high creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) activities. By agarose gel electrophoresis of human Duchenne dystrophic serum, the presence of an apparently normal human serum adenylate kinase together with a variant species of adenylate kinase was detected. The latter enzyme species appeared, in its mobility, to be similar to that of the normal human liver-type adenylate kinase. The presence of this aberrant liver-type adenylate kinase could also be demonstrated by characteristic (for the liver type) inhibition patterns with P1,P5-di-(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate. On the other hand, by inhibition titrations with an anti-muscle-type adenylate kinase, hemolysates from the erythrocytes of several Duchenne and Becker's dystrophics were found to contain approx. 96% muscle-type adenylate kinase and their serum approx. 97% muscle-type adenylate kinase. These same patients contained approx. 89% M-M type creatine kinase in their serum (by inhibition against anti-human muscle-type creatine kinase) indicative of the presence also of M-B plus B-B type active isoenzymes. All of these data can best be explained by the presence of a variant or mutant adenylate kinase isoenzyme in the dystrophic serum. This isoenzyme appears to resemble the liver type in its inhibition patterns with P1,P5-di(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate, and in its heat stability (compare also the agarose gel electrophoresis pattern); but structurally, it is a muscle type, or derived from a muscle type, as shown immunologically by inhibition reactions with anti-muscle-type adenylate kinase. Whether this is a fetal-type isoenzyme of adenylate kinase will require further

  18. Characterization of cereulide synthetase, a toxin-producing macromolecular machine.

    Directory of Open Access Journals (Sweden)

    Diego A Alonzo

    Full Text Available Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride.

  19. Bacterial glycosyltransferase toxins.

    Science.gov (United States)

    Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-12-01

    Mono-glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide-binding proteins of the Rho family. However, toxin-induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin-catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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...

  2. Botulinum toxin injection - larynx

    Science.gov (United States)

    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; ...

  3. *CYANOBACTERIA AND THEIR TOXINS

    Science.gov (United States)

    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...

  4. Stool C. difficile toxin

    Science.gov (United States)

    ... 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 ...

  5. Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

    Science.gov (United States)

    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

  6. 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.

    Directory of Open Access Journals (Sweden)

    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.

  7. Computational identification of candidate nucleotide cyclases in higher plants

    KAUST Repository

    Wong, Aloysius Tze

    2013-09-03

    In higher plants guanylyl cyclases (GCs) and adenylyl cyclases (ACs) cannot be identified using BLAST homology searches based on annotated cyclic nucleotide cyclases (CNCs) of prokaryotes, lower eukaryotes, or animals. The reason is that CNCs are often part of complex multifunctional proteins with different domain organizations and biological functions that are not conserved in higher plants. For this reason, we have developed CNC search strategies based on functionally conserved amino acids in the catalytic center of annotated and/or experimentally confirmed CNCs. Here we detail this method which has led to the identification of >25 novel candidate CNCs in Arabidopsis thaliana, several of which have been experimentally confirmed in vitro and in vivo. We foresee that the application of this method can be used to identify many more members of the growing family of CNCs in higher plants. © Springer Science+Business Media New York 2013.

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

    Directory of Open Access Journals (Sweden)

    Abir U Igamberdiev

    2015-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-12-22

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

  10. Bioterrorism: toxins as weapons.

    Science.gov (United States)

    Anderson, Peter D

    2012-04-01

    The potential for biological weapons to be used in terrorism is a real possibility. Biological weapons include infectious agents and toxins. Toxins are poisons produced by living organisms. Toxins relevant to bioterrorism include ricin, botulinum, Clostridium perfrigens epsilson toxin, conotoxins, shigatoxins, saxitoxins, tetrodotoxins, mycotoxins, and nicotine. Toxins have properties of biological and chemical weapons. Unlike pathogens, toxins do not produce an infection. Ricin causes multiorgan toxicity by blocking protein synthesis. Botulinum blocks acetylcholine in the peripheral nervous system leading to muscle paralysis. Epsilon toxin damages cell membranes. Conotoxins block potassium and sodium channels in neurons. Shigatoxins inhibit protein synthesis and induce apoptosis. Saxitoxin and tetrodotoxin inhibit sodium channels in neurons. Mycotoxins include aflatoxins and trichothecenes. Aflatoxins are carcinogens. Trichothecenes inhibit protein and nucleic acid synthesis. Nicotine produces numerous nicotinic effects in the nervous system.

  11. Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.

    Science.gov (United States)

    Siedenburg, Gabriele; Breuer, Michael; Jendrossek, Dieter

    2013-02-01

    Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst. PMID:22526778

  12. AKAPs and Adenylyl Cyclase in Cardiovascular Physiology and Pathology

    Science.gov (United States)

    Efendiev, Riad; Dessauer, Carmen W.

    2011-01-01

    Cyclic AMP, generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins (AKAPs) localize the effect of cAMP in space and time by organizing receptors, adenylyl cyclase, protein kinase A and other components of the cAMP cascade into multiprotein complexes. In this review we discuss how interaction of AKAPs with distinct AC isoforms affects cardiovascular physiology. PMID:21978991

  13. Tyrosine phosphorylation of the human guanylyl cyclase C receptor

    Indian Academy of Sciences (India)

    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.

  14. Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis.

    Science.gov (United States)

    Tükenmez, Hasan; Magnussen, Helge Magnus; Kovermann, Michael; Byström, Anders; Wolf-Watz, Magnus

    2016-01-01

    Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies. PMID:27642758

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  16. General base-general acid catalysis by terpenoid cyclases.

    Science.gov (United States)

    Pemberton, Travis A; Christianson, David W

    2016-07-01

    Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains.

  17. Evidences for involvement of endogenous cAMP in Arabidopsis defense responses to Verticillium toxins

    Institute of Scientific and Technical Information of China (English)

    Jing JIANG; Ling Wen FAN; Wei Hua WU

    2005-01-01

    Although there were reports suggesting the involvement of endogenous cAMP in plant defense signaling cascades,there is no direct evidence supporting this notion yet and the detailed mechanism is unclear. In the present study, we have used pathogenic fungi Verticillium dahliae and Arabidopsis plants as a model system of plant-microb interaction to demonstrate the function of endogenous cAMP in Arabidopsis defense responses. Both V. dahliae inoculation and Verticillium toxins injection induced typical "wilt" symptoms in Arabidopsis seedlings. When either 8-Br-AMP (a membrane permeable cAMP analogue) or salicylic acid (SA) was applied to Arabidopsis, the plants became resistant to V. dahliae toxins. However, addition of 8-Br-AMP did not increase the resistance of Arabidopsis transgenic plants deficient in SA to the toxins, suggesting that cAMP might act upstream of SA in plant defense signaling pathway.Indeed, 8-Br-cAMP and forskolin, an activator of adenylyl cyclase, significantly stimulated the endogenous SA level in plants, whereas DDA, an inhibitor of adenylyl cyclase dramatically reduced toxin-induced SA increase. Both the endogenous cAMP and SA increased significantly in Arabidopsis seedlings treated with toxins. Furthermore, transcription level of pathogenesis-related protein 1 gene (PR1) was strongly induced by both 8-Br-cAMP and the toxin treatment. Taken together, our data demonstrate that endogenous cAMP is involved in plant defense responses against Verticilliumsecreted toxins by regulating the production of the known signal SA in plant defense pathway.

  18. [Intoxication of botulinum toxin].

    Science.gov (United States)

    Chudzicka, Aleksandra

    2015-09-01

    Botulinum toxin is an egzotoxin produced by Gram positive bacteria Clostridium botulinum. It is among the most potent toxins known. The 3 main clinical presentations of botulism are as follows: foodborne botulism, infant botulism and wound botulism. The main symptom of intoxication is flat muscles paralysis. The treatment is supportive care and administration of antitoxin. In prevention the correct preparing of canned food is most important. Botulinum toxin is accepted as a biological weapon.

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

    Directory of Open Access Journals (Sweden)

    Andre Terzic

    2009-04-01

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

  20. Stimulation of adenylate cyclase in relation to dopamine-induced long-term enhancement (LTE) of muscarinic depolarization in the rabbit superior cervical ganglion.

    Science.gov (United States)

    Mochida, S; Kobayashi, H; Libet, B

    1987-02-01

    Dopamine (DA) induction of the long-term enhancement (LTE) of the slow muscarinic depolarizing response to methacholine (MCh), equivalent to the slow EPSP (S-EPSP), was previously found to be mimicked by exogenous cyclic AMP (cAMP) in the rabbit superior cervical ganglion (SCG). DA-induced LTE of the S-EPSP was shown to be depressed by some DA antagonists. We now show that DA (15 microM), its analog, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (ADTN), and a D2 receptor antagonist, metoclopramide, each can induce both LTE of MCh depolarization and an increase in ganglionic cAMP. Conversely, antagonists of DA-induced LTE also depress DA-induced rises in cAMP; these antagonists include haloperidol (1 microM), both (+) and (-) enantiomers of butaclamol (0.7-7 microM), flupenthixol (1 microM), and (+)-R-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-o l (SCH-23390) (7 microM). The selective D2 antagonists sulpiride (10 microM) and domperidone (10 microM) affect neither DA action. Alpha-2 adrenergic agonists (alpha-methyl-norepinephrine and clonidine) produce no LTE; alpha-antagonist dihydroergotamine (35 microM) does not affect either DA action, although it can completely block the hyperpolarizing response to DA or other catecholamines. Beta-antagonist propranolol (5 microM) partially depresses DA-induced rises in cAMP but has no effect on the DA-induced LTE. (Butaclamol and propranolol in combination can completely block the cAMP rise induced by DA.) Beta-agonist isoproterenol can induce appreciable LTE of MCh depolarization, but this LTE is not depressed by propranolol (10 microM). Isoproterenol can elicit a substantial rise in cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. 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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    DEFF Research Database (Denmark)

    Frödin, M; Hannibal, J; Wulff, B S;

    1995-01-01

    medulla showed PACAP38 immunoreactivity in a widely distributed network of delicate nerve fibers surrounding the chromaffin cells. In a primary culture system, PACAP38 inhibited growth factor-stimulated DNA synthesis by 90% in neonatal and adult rat chromaffin cells with half-maximal inhibition at 4 and 0...... cells, 100 nM PACAP38 and 1 microM corticosterone added together abolished proliferation completely (99.8% inhibition). Finally, PACAP38 increased cell survival but showed little neurite-promoting activity in the chromaffin cells. Our data suggest that neurally derived PACAP38, in conjunction...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    OpenAIRE

    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. Understanding malarial toxins.

    Science.gov (United States)

    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.

  6. Understanding malarial toxins.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  8. Receptor number and caveolar co-localization determine receptor coupling efficiency to adenylyl cyclase.

    Science.gov (United States)

    Ostrom, R S; Gregorian, C; Drenan, R M; Xiang, Y; Regan, J W; Insel, P A

    2001-11-01

    Recent evidence suggests that many signaling molecules localize in microdomains of the plasma membrane, particularly caveolae. In this study, overexpression of adenylyl cyclase was used as a functional probe of G protein-coupled receptor (GPCR) compartmentation. We found that three endogenous receptors in neonatal rat cardiomyocytes couple with different levels of efficiency to the activation of adenylyl cyclase type 6 (AC6), which localizes to caveolin-rich membrane fractions. Overexpression of AC6 enhanced the maximal cAMP response to beta(1)-adrenergic receptor (beta(1)AR)-selective activation 3.7-fold, to beta(2)AR-selective activation only 1.6-fold and to prostaglandin E(2) (PGE(2)) not at all. Therefore, the rank order of efficacy in coupling to AC6 is beta(1)AR > beta(2)AR > prostaglandin E(2) receptor (EP(2)R). beta(2)AR coupling efficiency was greater when we overexpressed the receptor or blocked its desensitization by expressing betaARKct, an inhibitor of G protein-coupled receptor kinase activation, but was not significantly greater when cells were treated with pertussis toxin. Assessment of receptor and AC expression indicated co-localization of AC5/6, beta(1)AR, and beta(2)AR, but not EP(2)R, in caveolin-rich membranes and caveolin-3 immunoprecipitates, likely explaining the observed activation of AC6 by betaAR subtypes but lack thereof by PGE(2). When cardiomyocytes were stimulated with a betaAR agonist, beta(2)AR were no longer found in caveolin-3 immunoprecipitates; an effect that was blocked by expression of betaARKct. Thus, agonist-induced translocation of beta(2)AR out of caveolae causes a sequestration of receptor from effector and likely contributes to the lower efficacy of beta(2)AR coupling to AC6 as compared with beta(1)AR, which do not similarly translocate. Therefore, spatial co-localization is a key determinant of efficiency of coupling by particular extracellular signals to activation of GPCR-linked effectors. PMID:11533056

  9. Staphylococcus aureus toxins.

    Science.gov (United States)

    Otto, Michael

    2014-02-01

    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 against bacterial elimination by innate host defense. Furthermore, S. aureus secretes many factors that inhibit the complement cascade or prevent recognition by host defenses. Several further toxins add to this multi-faceted program of S. aureus to evade elimination in the host. This review will give an overview over S. aureus toxins focusing on recent advances in our understanding of how leukotoxins work in receptor-mediated or receptor-independent fashions.

  10. Skeletal muscle contractile performance and ADP accumulation in adenylate kinase-deficient mice

    NARCIS (Netherlands)

    Hancock, C.R.; Janssen, E.E.W.; Terjung, R.L.

    2005-01-01

    The production of AMP by adenylate kinase (AK) and subsequent deamination by AMP deaminase limits ADP accumulation during conditions of high-energy demand in skeletal muscle. The goal of this study was to investigate the consequences of AK deficiency (-/-) on adenine nucleotide management and whole

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

    Energy Technology Data Exchange (ETDEWEB)

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

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

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

    International Nuclear Information System (INIS)

    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.)

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

    OpenAIRE

    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...

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

    Science.gov (United States)

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

    2015-05-29

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

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

    OpenAIRE

    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....

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  19. Toxins Best Paper Award 2015

    OpenAIRE

    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...

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

    Directory of Open Access Journals (Sweden)

    Rouhiainen Leo

    2008-09-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  2. Multiforms of mammalian adenylate kinase and its monoclonal antibody against AK1.

    Science.gov (United States)

    Kurokawa, Y; Takenaka, H; Sumida, M; Oka, K; Hamada, M; Kuby, S A

    1990-01-01

    An attempt has been made to determine the intracellular distribution of the multiforms of the adenylate kinase (AK) isoenzymes in mammalian tissues, to shed some light on their physiological roles, especially in energy metabolism. The adenylate kinase zymograms obtained from isoelectric focusing yielded two typical isoform patterns: (1) with a pI greater than or equal to 9 and 8.6, specific for bovine skeletal muscle, heart, aorta and brain, and (2) with a pI = 7.9 and 7.1, specific for liver and kidney. Pattern (1) was attributed to the cytosolic isoenzyme (AK1) as demonstrated by immunostaining with anti-AK1. Pattern (2) was attributed to the mitochondrial isoenzyme (AK2). These results were largely confirmed by chromatofocusing experiments. The AK1 isoenzyme was partially purified from the cytosol fraction of bovine aortic smooth muscle and had an apparent Mr of 23.5 kilodaltons. Its kinetic features are discussed from a comparative standpoint. Finally, the human serum AK1 isoform was also detected by Western blotting with a monoclonal antibody directed against crystalline porcine muscle AK1. These results are to form the basis of further studies on the 'aberrant' adenylate kinase isoenzyme from the serum of Duchenne muscular dystrophics.

  3. NMR studies of the AMP-binding site and mechanism of adenylate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Fry, D.C.; Kuby, S.A.; Mildvan, A.S.

    1987-03-24

    NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr/sup 3 +/AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T/sub 1/ method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine.

  4. Botulinum Toxin Therapy

    Science.gov (United States)

    ... 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 ...

  5. [Protein toxins of Staphylococcus aureus].

    Science.gov (United States)

    Shamsutdinov, A F; Tiurin, Iu A

    2014-01-01

    Main scientific-research studies regarding protein bacterial toxins of the most widespread bacteria that belong to Staphylococcus spp. genus and in particular the most pathogenic species for humans--Staphylococcus aureus, are analyzed. Structural and biological properties of protein toxins that have received the name of staphylococcus pyrogenic toxins (PTSAg) are presented. Data regarding genetic regulation of secretion and synthesis of these toxins and 3 main regulatory genetic systems (agr--accessory gene regulator, xpr--extracellular protein regulator, sar--staphylococcal accessory regulator) that coordinate synthesis of the most important protein toxins and enzymes for virulence of S. aureus, are presented.

  6. Toxins Best Paper Award 2015

    Directory of Open Access Journals (Sweden)

    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:[...

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

    Science.gov (United States)

    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

  8. Autoproteolytic Activation of Bacterial Toxins

    Directory of Open Access Journals (Sweden)

    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.

  9. Inferring biological functions of guanylyl cyclases with computational methods

    KAUST Repository

    Alquraishi, May Majed

    2013-09-03

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

  10. Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum.

    Science.gov (United States)

    Witters, Erwin; Quanten, Lieve; Bloemen, Jo; Valcke, Roland; Van Onckelen, Harry

    2004-01-01

    In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled (15)N-ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) method was developed to measure de novo formed (15)N-adenosine 3',5'-cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv. Petit Havana and the kinetic parameters and influence of various metabolic effectors are discussed in their context.

  11. Toxins and drug discovery.

    Science.gov (United States)

    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

  12. Method for detecting biological toxins

    Energy Technology Data Exchange (ETDEWEB)

    Ligler, F.S.; Campbell, J.R.

    1992-01-01

    Biological toxins are indirectly detected by using polymerase chain reaction to amplify unique nucleic acid sequences coding for the toxins or enzymes unique to toxin synthesis. Buffer, primers coding for the unique nucleic acid sequences and an amplifying enzyme are added to a sample suspected of containing the toxin. The mixture is then cycled thermally to exponentially amplify any of these unique nucleic acid sequences present in the sample. The amplified sequences can be detected by various means, including fluorescence. Detection of the amplified sequences is indicative of the presence of toxin in the original sample. By using more than one set of labeled primers, the method can be used to simultaneously detect several toxins in a sample.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-20

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

  15. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    Science.gov (United States)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α 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. PMID:21602811

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

    Science.gov (United States)

    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

  17. Clostridium difficile binary toxin CDT

    OpenAIRE

    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...

  18. Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface.

    Science.gov (United States)

    Peshenko, Igor V; Olshevskaya, Elena V; Dizhoor, Alexander M

    2015-08-01

    The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met(823) was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg(822). The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met(823) or Arg(822) was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg(822) and Met(823).

  19. NMR studies of the AMP-binding site and mechanism of adenylate kinase.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1987-03-24

    NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y

  20. Requirements for anthrax toxin entry into cells

    OpenAIRE

    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...

  1. Shiga Toxin Detection Methods : A Short Review

    OpenAIRE

    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...

  2. Guanylyl cyclase C signaling axis and colon cancer prevention

    Science.gov (United States)

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-01-01

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C’s role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention. PMID:27688649

  3. Guanylyl cyclase C signaling axis and colon cancer prevention

    Science.gov (United States)

    Pattison, Amanda M; Merlino, Dante J; Blomain, Erik S; Waldman, Scott A

    2016-01-01

    Colorectal cancer (CRC) is a major cause of cancer-related mortality and morbidity worldwide. While improved treatments have enhanced overall patient outcome, disease burden encompassing quality of life, cost of care, and patient survival has seen little benefit. Consequently, additional advances in CRC treatments remain important, with an emphasis on preventative measures. Guanylyl cyclase C (GUCY2C), a transmembrane receptor expressed on intestinal epithelial cells, plays an important role in orchestrating intestinal homeostatic mechanisms. These effects are mediated by the endogenous hormones guanylin (GUCA2A) and uroguanylin (GUCA2B), which bind and activate GUCY2C to regulate proliferation, metabolism and barrier function in intestine. Recent studies have demonstrated a link between GUCY2C silencing and intestinal dysfunction, including tumorigenesis. Indeed, GUCY2C silencing by the near universal loss of its paracrine hormone ligands increases colon cancer susceptibility in animals and humans. GUCY2C’s role as a tumor suppressor has opened the door to a new paradigm for CRC prevention by hormone replacement therapy using synthetic hormone analogs, such as the FDA-approved oral GUCY2C ligand linaclotide (Linzess™). Here we review the known contributions of the GUCY2C signaling axis to CRC, and relate them to a novel clinical strategy targeting tumor chemoprevention.

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

    OpenAIRE

    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...

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

    CERN Document Server

    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...

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

    Science.gov (United States)

    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

  7. 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.

    Science.gov (United States)

    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

  8. 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.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  11. Cloning and Characterization of Oxidosqualene Cyclases from Kalanchoe daigremontiana

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2012-06-01

    The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. PMID:22426196

  13. Food toxin detection with atomic force microscope

    Science.gov (United States)

    Externally introduced toxins or internal spoilage correlated pathogens and their metabolites are all potential sources of food toxins. To prevent and protect unsafe food, many food toxin detection techniques have been developed to detect various toxins for quality control. Although several routine m...

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

    Directory of Open Access Journals (Sweden)

    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

  15. Shiga Toxin Producing Escherichia coli.

    Science.gov (United States)

    Bryan, Allen; Youngster, Ilan; McAdam, Alexander J

    2015-06-01

    Shiga toxin-producing Escherichia coli (STEC) is among the common causes of foodborne gastroenteritis. STEC is defined by the production of specific toxins, but within this pathotype there is a diverse group of organisms. This diversity has important consequences for understanding the pathogenesis of the organism, as well as for selecting the optimum strategy for diagnostic testing in the clinical laboratory. This review includes discussions of the mechanisms of pathogenesis, the range of manifestations of infection, and the several different methods of laboratory detection of Shiga toxin-producing E coli.

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

    OpenAIRE

    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...

  17. Indirect activation of neuronal noncapacitative Ca2+ entry is the final step involved in the neurotoxic effect of Tityus serrulatus scorpion beta-toxin.

    Science.gov (United States)

    Grolleau, Françoise; Stankiewicz, Maria; Kielbasiewicz, Ewa; Martin-Eauclaire, Marie-France; Lavialle, Céline; De Vente, Jan; Lapied, Bruno

    2006-03-01

    Interweaving strategies of electrophysiology, calcium imaging and immunocytochemistry bring new insights into the mode of action of the Brazilian scorpion Tityus serrulatusbeta-toxin VII. Pacemaker dorsal unpaired median neurons isolated from the cockroach central nervous system were used to study the effects of toxin VII. In current-clamp, 50 nm toxin VII produced a membrane depolarization and reduced spiking. At 200 nM, depolarization associated with multiphasic effects was seen. After artificial hyperpolarization, plateau potentials on which spontaneous electrical activity appeared were observed. In voltage clamp, toxin VII induced a negative shift of the voltage dependence of sodium current activation without significant effect on steady-state inactivation. In addition, toxin VII produced a permanent TTX-sensitive holding inward current, indicating that background sodium channels were targeted by beta-toxin. Cell-attached patch recordings indicated that these channels were switched from unclustered single openings to current fluctuating between distinct subconductance levels exhibiting increased open probability and open-time distribution. Toxin VII also produced a TTX-sensitive [Ca2+]i rise. Immunostaining with Cav2.2(alpha1b) antibodies and calcium imaging data obtained with omega-CgTx GVIA indicated that N-type high-voltage-activated calcium channels initiated calcium influx and were an essential intermediate in the pathway linking toxin VII-modified sodium channels to the activation of an additional route for calcium entry. By using inhibitors of (i) noncapacitative calcium entry (inhibitor LOE-908), (ii) NO-sensitive guanylyl cyclase (ODQ) and (iii) phosphodiesterase 2 (EHNA), together with cGMP antibodies, we demonstrated that noncapacitative calcium entry was the final step in a complex combination of events that was initiated by toxin VII-alteration of sodium channels and then involved successive activation of other membrane ion channels. PMID:16553610

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

    International Nuclear Information System (INIS)

    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

  19. VIP/PACAP receptors in cerebral arteries of rat

    DEFF Research Database (Denmark)

    Erdling, André; Sheykhzade, Majid; Maddahi, Aida;

    2013-01-01

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

  20. NCBI nr-aa BLAST: CBRC-ACAR-01-0801 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-0801 ref|NP_001092076.1| adenylate cyclase activating polypeptide 1 (pituitary...) receptor type I [Gallus gallus] gb|ABQ63080.1| pituitary adenylate cyclase-activating polypeptide

  1. NCBI nr-aa BLAST: CBRC-XTRO-01-3244 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-3244 ref|NP_001092076.1| adenylate cyclase activating polypeptide 1 (pituitary...) receptor type I [Gallus gallus] gb|ABQ63080.1| pituitary adenylate cyclase-activating polypeptide

  2. NCBI nr-aa BLAST: CBRC-TNIG-22-0273 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TNIG-22-0273 ref|NP_001092076.1| adenylate cyclase activating polypeptide 1 (pituitary...) receptor type I [Gallus gallus] gb|ABQ63080.1| pituitary adenylate cyclase-activating polypeptide

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Synthetic genes for human muscle-type adenylate kinase in Escherichia coli.

    Science.gov (United States)

    Kim, H J; Nishikawa, S; Tanaka, T; Uesugi, S; Takenaka, H; Hamada, M; Kuby, S A

    1989-01-01

    An artificial gene coding for the human muscle-type cytosolic adenylate kinase (hAK1) was chemically synthesized and directly expressed in Escherichia coli under the control of trp promoter. The DNA duplex of 596 bp was designed and constructed from 40 oligonucleotide fragments of typically 30 nucleotides in length. Twelve unique restriction sites were fairly evenly spaced in the synthetic gene to facilitate site-specific mutagenesis at any part of this recombinant protein. The genes for mutant hAK1 (Tyr 95----Phe 95, Y95F hAK1; Arg 97----Ala 97, R97A hAK1) were constructed by cassette mutagenesis and utilized restriction sites incorporated in the hAK1 gene. The recombinant hAK1 was purified to homogeneity by a two-step chromatographic procedure with a good yield, and showed the same adenylate kinase activity as that of authentic hAK1. Preliminary kinetic studies show that the enzymatic activity (Vmax app,cor/Et) of Y95F hAK1 was slightly greater than that of recombinant hAK1, whereas R97A hAK1 still possessed approximately 4% of recombinant hAK1 activity. These results suggest that the Arg-97 residue is important but not essential for catalytic activity, and that Tyr-95 can be replaced by phenylalanine without substantial effects on the enzymatic activity. Moreover, preliminary estimates of the apparent kinetic parameters suggest that these residues are not required for MgATP binding, and therefore they do not appear to be part of the MgATP binding site.

  6. NMr studies of the AMP binding site and mechanism of adenylate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Kuby, S.A.; Fry, D.C.; Mildvan, A.S.

    1986-05-01

    The authors recently located by NMR the MgATP binding site on adenylate kinase correcting the proposed location for this site based on X-ray studies of the binding of salicylate. To determine the conformation and location of the other substrate, they have determined distances from Cr/sup 3 +/ AMPPCP to 6 protons and to the phosphorus atom of AMP on adenylate kinase using the paramagnetic-probe-T/sub 1/ method. They have also used time-dependent NOEs to measure five interproton distances on AMP, permitting evaluation of the conformation of enzyme-bound AMP and its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high-anti glycosyl torsional angle (X = 110/sup 0/), a 3'-endo sugar pucker (delta = 105/sup 0/), and a gauche-trans orientation about the C/sub 4/'-C/sub 5/' bond (..gamma.. = 180/sup 0/). The distance from Cr/sup 3 +/ to the phosphorus of AMP is 6.4 +/- 0.3 A, indicating a reaction coordinate distance of greater than or equal to A which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP were detected. These constraints, together with the conformation of AMP and the X-ray structure of the enzyme, suggest proximity (less than or equal to A) of AMP to leu 116, arg 171, val 173, gln 185, thr 188, and asp 191.

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

    OpenAIRE

    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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Masure, H.R.; Donovan, M.G.; Storm, D.R.

    1991-01-01

    An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.

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

    International Nuclear Information System (INIS)

    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

  11. Ectopic expression of cyclase associated protein CAP restores the streaming and aggregation defects of adenylyl cyclase a deficient Dictyostelium discoideum cells

    Directory of Open Access Journals (Sweden)

    Sultana Hameeda

    2012-01-01

    Full Text Available Abstract Background Cell adhesion, an integral part of D. discoideum development, is important for morphogenesis and regulated gene expression in the multicellular context and is required to trigger cell-differentiation. G-protein linked adenylyl cyclase pathways are crucially involved and a mutant lacking the aggregation specific adenylyl cyclase ACA does not undergo multicellular development. Results Here, we have investigated the role of cyclase-associated protein (CAP, an important regulator of cell polarity and F-actin/G-actin ratio in the aca- mutant. We show that ectopic expression of GFP-CAP improves cell polarization, streaming and aggregation in aca- cells, but it fails to completely restore development. Our studies indicate a requirement of CAP in the ACA dependent signal transduction for progression of the development of unicellular amoebae into multicellular structures. The reduced expression of the cell adhesion molecule DdCAD1 together with csA is responsible for the defects in aca- cells to initiate multicellular development. Early development was restored by the expression of GFP-CAP that enhanced the DdCAD1 transcript levels and to a lesser extent the csA mRNA levels. Conclusions Collectively, our data shows a novel role of CAP in regulating cell adhesion mechanisms during development that might be envisioned to unravel the functions of mammalian CAP during animal embryogenesis.

  12. Antibody-based biological toxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Menking, D.E.; Goode, M.T. [Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD (United States)

    1995-12-01

    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a direct competition assay, antibodies against Cholera toxin, Staphylococcus Enterotoxin B or ricin were noncovalently immobilized on quartz fibers and probed with fluorescein isothiocyanate (FITC) - labeled toxins. In the indirect competition assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified anti-toxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or anti-toxin IgG in a dose dependent manner and the detection of the toxins was in the nanomolar range.

  13. Targeted Toxins in Brain Tumor Therapy

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

    Chen, Yicai; Yuan, Mingjun; Mohanty, Anee;

    2015-01-01

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

  15. Unusual guanylyl cyclases and cGMP signaling in Dictyostelium discoideum

    NARCIS (Netherlands)

    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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Chen Yun-Ru

    2012-09-01

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

  18. Botulinum Toxin Injections: A Treatment for Muscle Spasms

    Science.gov (United States)

    ... 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 ...

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

    OpenAIRE

    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...

  20. Sodium Channel Inhibiting Marine Toxins

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2012-07-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

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

    Science.gov (United States)

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

    2012-01-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  3. Clostridium difficile and C. difficile Toxin Testing

    Science.gov (United States)

    ... 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. ...

  4. Toxin synergism in snake venoms

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard

    2016-01-01

    Synergism between venom toxins exists for a range of snake species. Synergism can be derived from both intermolecular interactions and supramolecular interactions between venom components, and can be the result of toxins targeting the same protein, biochemical pathway or physiological process. Few...... simple systematic tools and methods for determining the presence of synergism exist, but include co-administration of venom components and assessment of Accumulated Toxicity Scores. A better understanding of how to investigate synergism in snake venoms may help unravel strategies for developing novel...

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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...

  7. Adenylate kinase 2 (AK2 promotes cell proliferation in insect development

    Directory of Open Access Journals (Sweden)

    Chen Ru-Ping

    2012-09-01

    Full Text Available Abstract Background Adenylate kinase 2 (AK2 is a phosphotransferase that catalyzes the reversible reaction 2ADP(GDP ↔ ATP(GTP + AMP and influences cellular energy homeostasis. However, the role of AK2 in regulating cell proliferation remains unclear because AK2 has been reported to be involved in either cell proliferation or cell apoptosis in different cell types of various organisms. Results This study reports AK2 promotion of cell proliferation using the lepidopteran insect Helicoverpa armigera and its epidermal cell line HaEpi as models. Western blot analysis indicates that AK2 constitutively expresses in various tissues during larval development. Immunocytochemistry analysis indicates that AK2 localizes in the mitochondria. The recombinant expressed AK2 in E. coli promotes cell growth and viability of HaEpi cell line by 3-(4, 5-Dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide (MTT assay. AK2 knockdown in larvae by RNA interference causes larval growth defects, including body weight decrease and development delay. AK2 knockdown in larvae also decreases the number of circulating haemocytes. The mechanism for such effects might be the suppression of gene transcription involved in insect development caused by AK2 knockdown. Conclusion These results show that AK2 regulates cell growth, viability, and proliferation in insect growth and development.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  9. Adenylate nucleotide levels and energy charge in Arthrobacter crystallopoietes during growth and starvation.

    Science.gov (United States)

    Leps, W T; Ensign, J C

    1979-07-01

    The adenylate nucleotide concentrations, based on internal water space, were determined in cells of Arthrobacter crystallopoietes during growth and starvation and the energy charge of the cells was calculated. The energy charge of spherical cells rose during the first 10 h of growth, then remained nearly constant for as long as 20 h into the stationary phase. The energy charge of rod-shaped cells rose during the first 4 h of growth, then remained constant during subsequent growth and decreased in the stationary growth phase. Both spherical and rod-shaped cells excreted adenosine monophosphate but not adenosine triphosphate or adenosine diphosphate during starvation. The intracellular energy charge of spherical cells declined during the initial 10 h and then remained constant for 1 week of starvation at a value of 0.78. The intracellular energy charge of rod-shaped cells declined during the first 24 h of starvation, remained constant for the next 80 h, then decreased to a value of 0.73 after a total of 168 h starvation. Both cell forms remained more than 90% viable during this time. Addition of a carbon and energy source to starving cells resulted in an increase in the ATP concentration and as a result the energy charge increased to the smae levels as found during growth.

  10. Structure and function of adenylate kinase isozymes in normal humans and muscular dystrophy patients.

    Science.gov (United States)

    Hamada, M; Takenaka, H; Fukumoto, K; Fukamachi, S; Yamaguchi, T; Sumida, M; Shiosaka, T; Kurokawa, Y; Okuda, H; Kuby, S A

    1987-01-01

    Two isozymes of adenylate kinase from human Duchenne muscular dystrophy serum, one of which was an aberrant form specific to DMD patients, were separated by Blue Sepharose CL-6B affinity chromatography. The separated aberrant form possessed a molecular weight of 98,000 +/- 1,500, whereas the normal serum isozyme had a weight of 87,000 +/- 1,600, as determined by SDS-polyacrylamide gel electrophoresis, gel filtration, and sedimentation equilibrium. The sedimentation coefficients were 5.8 S and 5.6 S for the aberrant form and the normal form, respectively. Both serum isozymes are tetramers. The subunit size of the aberrant isozyme (Mr = 24,700) was very similar to that of the normal human liver isozyme, and the subunit size of the normal isozyme (Mr = 21,700) was very similar to that of the normal human muscle enzyme. The amino acid composition of the normal serum isozyme was similar to that of the muscle-type enzyme, and that of the aberrant isozyme was similar to that of the liver enzyme, with some exceptions in both cases.

  11. Risk Assessment of Shellfish Toxins

    Directory of Open Access Journals (Sweden)

    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.

  12. Shigella Sonnei and Shiga Toxin

    Centers for Disease Control (CDC) Podcasts

    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.

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

    OpenAIRE

    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...

  14. Identification of the chlE gene encoding oxygen-independent Mg-protoporphyrin IX monomethyl ester cyclase in cyanobacteria.

    Science.gov (United States)

    Yamanashi, Kaori; Minamizaki, Kei; Fujita, Yuichi

    2015-08-01

    The fifth ring (E-ring) of chlorophyll (Chl) a is produced by Mg-protoporphyrin IX monomethyl ester (MPE) cyclase. There are two evolutionarily unrelated MPE cyclases: oxygen-independent (BchE) and oxygen-dependent (ChlA/AcsF) MPE cyclases. Although ChlA is the sole MPE cyclase in Synechocystis PCC 6803, it is yet unclear whether BchE exists in cyanobacteria. A BLAST search suggests that only few cyanobacteria possess bchE. Here, we report that two bchE candidate genes from Cyanothece strains PCC 7425 and PCC 7822 restore the photosynthetic growth and bacteriochlorophyll production in a bchE-lacking mutant of Rhodobacter capsulatus. We termed these cyanobacterial bchE orthologs "chlE."

  15. Novel receptors for bacterial protein toxins.

    Science.gov (United States)

    Schmidt, Gudula; Papatheodorou, Panagiotis; Aktories, Klaus

    2015-02-01

    While bacterial effectors are often directly introduced into eukaryotic target cells by various types of injection machines, toxins enter the cytosol of host cells from endosomal compartments or after retrograde transport via Golgi from the ER. A first crucial step of toxin-host interaction is receptor binding. Using optimized protocols and new methods novel toxin receptors have been identified, including metalloprotease ADAM 10 for Staphylococcus aureus α-toxin, laminin receptor Lu/BCAM for Escherichia coli cytotoxic necrotizing factor CNF1, lipolysis stimulated lipoprotein receptor (LSR) for Clostridium difficile transferase CDT and low-density lipoprotein receptor-related protein (LRP) 1 for Clostridium perfringens TpeL toxin.

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

    Science.gov (United States)

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

    2015-12-01

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

  17. 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

    OpenAIRE

    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...

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

    OpenAIRE

    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 ...

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

    OpenAIRE

    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 ...

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

    Science.gov (United States)

    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

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

    OpenAIRE

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    Science.gov (United States)

    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

  5. Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver.

    Science.gov (United States)

    Kuby, S A; Fleming, G; Frischat, A; Cress, M C; Hamada, M

    1983-02-10

    Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.

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

    Science.gov (United States)

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

    2013-01-01

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

  7. H2S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway.

    Science.gov (United States)

    Li, Sen; Ping, Na-Na; Cao, Lei; Mi, Yan-Ni; Cao, Yong-Xiao

    2015-12-15

    Hydrogen sulfide (H2S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H2S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H2S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-type Ca(2+) channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H2S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway.

  8. The first structure of a bacterial diterpene cyclase: CotB2.

    Science.gov (United States)

    Janke, Ronja; Görner, Christian; Hirte, Max; Brück, Thomas; Loll, Bernhard

    2014-06-01

    Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacterium Streptomyces melanosporofaciens, at 1.64 Å resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclooctatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2(W288G) produced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistant Staphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-01-01

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

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

    Directory of Open Access Journals (Sweden)

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    OpenAIRE

    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.

  14. Why do we study animal toxins?

    Science.gov (United States)

    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

    Directory of Open Access Journals (Sweden)

    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 in Pediatric Neurology

    OpenAIRE

    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...

  17. Bacterial protein toxins in human cancers.

    Science.gov (United States)

    Rosadi, Francesca; Fiorentini, Carla; Fabbri, Alessia

    2016-02-01

    Many bacteria causing persistent infections produce toxins whose mechanisms of action indicate that they could have a role in carcinogenesis. Some toxins, like CDT and colibactin, directly attack the genome by damaging DNA whereas others, as for example CNF1, CagA and BFT, impinge on key eukaryotic processes, such as cellular signalling and cell death. These bacterial toxins, together with other less known toxins, mimic carcinogens and tumour promoters. The aim of this review is to fulfil an up-to-date analysis of toxins with carcinogenic potential that have been already correlated to human cancers. Bacterial toxins-induced carcinogenesis represents an emerging aspect in bacteriology, and its significance is increasingly recognized.

  18. Bt Toxin Modification for Enhanced Efficacy

    Directory of Open Access Journals (Sweden)

    Benjamin R. Deist

    2014-10-01

    Full Text Available Insect-specific toxins derived from Bacillus thuringiensis (Bt provide a valuable resource for pest suppression. Here we review the different strategies that have been employed to enhance toxicity against specific target species including those that have evolved resistance to Bt, or to modify the host range of Bt crystal (Cry and cytolytic (Cyt toxins. These strategies include toxin truncation, modification of protease cleavage sites, domain swapping, site-directed mutagenesis, peptide addition, and phage display screens for mutated toxins with enhanced activity. Toxin optimization provides a useful approach to extend the utility of these proteins for suppression of pests that exhibit low susceptibility to native Bt toxins, and to overcome field resistance.

  19. Toxin production in Dinophysis and the fate of these toxins in marine mussels

    DEFF Research Database (Denmark)

    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....... acuta. I grew the two species in laboratory cultures at different irradiances (7-130 μmol photons m-2 s-1) and with different food availability. The results showed that irradiance had no effects on toxin profiles, and only limited effects of the cellular toxin contents. Rather, toxin production rates...... followed growth rates, thus giving stable toxin contents. Food availability also did not change the toxin profiles of either species, but starvation did increase the cellular contents of each of the toxins present. The observation that toxin production continued for several weeks after the ciliate food...

  20. Rho-modifying bacterial protein toxins.

    Science.gov (United States)

    Aktories, Klaus

    2015-12-01

    Rho proteins are targets of numerous bacterial protein toxins, which manipulate the GTP-binding proteins by covalent modifications, including ADP ribosylation, glycosylation, adenylylation, proteolytic cleavage and deamidation. Bacterial toxins are important virulence factors but are also potent and efficient pharmacological tools to study the physiological functions of their eukaryotic targets. Recent studies indicate that amazing variations exist in the molecular mechanisms by which toxins attack Rho proteins, which are discussed here.

  1. [Use of botulinum toxin in strabismus].

    Science.gov (United States)

    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

  2. Application of Botulinum Toxin in Pain Management

    OpenAIRE

    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,...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  6. Application of botulinum toxin in pain management.

    Science.gov (United States)

    Sim, Woo Seog

    2011-03-01

    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, and neuropathic pain. Further studies are needed to understand the exact analgesic mechanisms, efficacy and complications of botulinum toxin in chronic pain disorders.

  7. Comparative effect of methioninyl adenylate on the growth of Salmonella typhimurium and Pseudomonas aeruginosa.

    Science.gov (United States)

    Enouf, J; Laurence, F; Farrugia, G; Blanchard, P; Robert-Gero, M

    1976-10-11

    The bacteriostatic effect of methioninyl adenylate(MAMP)--a specific inhibitor of the enzyme methionyl-tRNA synthetase--was investigated on Salmonella typhimurium and Pseudomonas aeruginosa. 0.1 mM of this molecule added to the culture, inhibits the growth of S. typhimurium. The inhibition is specifically reversible by 0.1 mM L-methionine. In the same conditions even 1-2 mM MAMP has a very slight effect on the growth rate of P. aeruginosa and only during the first two generations. The same observation was made with the two other members of the fluorescens group P.fluorescens and P.putida. The growth rate of P. testosteroni with 1 mM MAMP in the medium is similar to the growth rate of P. aeruginosa but the other member of the acidovorans group P. acidovorans is much more affected by the smae concentration of the inhibitor. --P. multivorans is inhibited by MAMP like P. acidovorans but with a somewhat higher yield at the end of the culture. --MAMP has no effect on P. alcaligenes. The possible reasons for the weak bacteriostatic effect of MAMP on P. aeruginosa were investigated. It was established that the inhibitor enters the cells and is not used as a carbon and energy source. The intracellular methionine concentration in S. typhimurium and in P. aeruginosa is about the same and does not increase when bacteria are cultivated with MAMP. The MTS of the two microorganisms is inhibited by MAMP in vitro to about the same extent. Furthermore the tRNAmet from P. aeruginosa are fully acylated after 3 to 4 generations with this compound. Nevertheless MAMP elicits higher MTS activity in P. aeruginosa and in P. acidovorans after 1 h of incubation. The most striking difference between S. typhimurium and P. aeruginosa is that the intra and extracellular level of 5'phosphodiesterase which degrades MAMP is 10-20 fold higher in the second than in the first species.

  8. Designing Inhibitors of Anthrax Toxin

    Science.gov (United States)

    Nestorovich, Ekaterina M.; Bezrukov, Sergey M.

    2014-01-01

    Introduction Present-day rational drug design approaches are based on exploiting unique features of the target biomolecules, small- or macromolecule drug candidates, and physical forces that govern their interactions. The 2013 Nobel Prize in chemistry awarded “for the development of multiscale models for complex chemical systems” once again demonstrated the importance of the tailored drug discovery that reduces the role of the trial and error approach to a minimum. The “rational drug design” term is rather comprehensive as it includes all contemporary methods of drug discovery where serendipity and screening are substituted by the information-guided search for new and existing compounds. Successful implementation of these innovative drug discovery approaches is inevitably preceded by learning the physics, chemistry, and physiology of functioning of biological structures under normal and pathological conditions. Areas covered This article provides an overview of the recent rational drug design approaches to discover inhibitors of anthrax toxin. Some of the examples include small-molecule and peptide-based post-exposure therapeutic agents as well as several polyvalent compounds. The review also directs the reader to the vast literature on the recognized advances and future possibilities in the field. Expert opinion Existing options to combat anthrax toxin lethality are limited. With the only anthrax toxin inhibiting therapy (PA-targeting with a monoclonal antibody, raxibacumab) approved to treat inhalational anthrax, in our view, the situation is still insecure. The FDA’s animal rule for drug approval, which clears compounds without validated efficacy studies on humans, creates a high level of uncertainty, especially when a well-characterized animal model does not exist. Besides, unlike PA, which is known to be unstable, LF remains active in cells and in animal tissues for days. Therefore, the effectiveness of the post-exposure treatment of the individuals

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

    OpenAIRE

    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...

  10. A kinase-anchoring proteins and adenylyl cyclase in cardiovascular physiology and pathology.

    Science.gov (United States)

    Efendiev, Riad; Dessauer, Carmen W

    2011-10-01

    3'-5'-Cyclic adenosine monophosphate (cAMP), generated by adenylyl cyclase (AC), serves as a second messenger in signaling pathways regulating many aspects of cardiac physiology, including contraction rate and action potential duration, and in the pathophysiology of hypertrophy and heart failure. A kinase-anchoring proteins localize the effect of cAMP in space and time by organizing receptors, AC, protein kinase A, and other components of the cAMP cascade into multiprotein complexes. In this review, we discuss how the interaction of A kinase-anchoring proteins with distinct AC isoforms affects cardiovascular physiology.

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

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  13. MARTX toxins as effector delivery platforms.

    Science.gov (United States)

    Gavin, Hannah E; Satchell, Karla J F

    2015-12-01

    Bacteria frequently manipulate their host environment via delivery of microbial 'effector' proteins to the cytosol of eukaryotic cells. In the case of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin, this phenomenon is accomplished by a single, >3500 amino acid polypeptide that carries information for secretion, translocation, autoprocessing and effector activity. MARTX toxins are secreted from bacteria by dedicated Type I secretion systems. The released MARTX toxins form pores in target eukaryotic cell membranes for the delivery of up to five cytopathic effectors, each of which disrupts a key cellular process. Targeted cellular processes include modulation or modification of small GTPases, manipulation of host cell signaling and disruption of cytoskeletal integrity. More recently, MARTX toxins have been shown to be capable of heterologous protein translocation. Found across multiple bacterial species and genera--frequently in pathogens lacking Type 3 or Type 4 secretion systems--MARTX toxins in multiple cases function as virulence factors. Innovative research at the intersection of toxin biology and bacterial genetics continues to elucidate the intricacies of the toxin as well as the cytotoxic mechanisms of its diverse effector collection.

  14. Target-Driven Evolution of Scorpion Toxins.

    Science.gov (United States)

    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

  15. Plant insecticidal toxins in ecological networks.

    Science.gov (United States)

    Ibanez, Sébastien; Gallet, Christiane; Després, Laurence

    2012-04-01

    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-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.

  16. [Axillary hyperhidrosis, botulinium A toxin treatment: Review].

    Science.gov (United States)

    Clerico, C; Fernandez, J; Camuzard, O; Chignon-Sicard, B; Ihrai, T

    2016-02-01

    Injection of type A botulinum toxin in the armpits is a temporary treatment for axillary hyperhidrosis. This technique described in 1996 by Bushara et al., is known to be efficient and safe. The purpose of this article was to review the data concerning the treatment of axillary hyperhidrosis with botulinum toxin type A, and discuss the other treatment modalities for this socially disabling entity.

  17. The Ins and Outs of Anthrax Toxin.

    Science.gov (United States)

    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

  18. Plant Insecticidal Toxins in Ecological Networks

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    DEFF Research Database (Denmark)

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

    1994-01-01

    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...

  2. Interplay between toxin transport and flotillin localization

    DEFF Research Database (Denmark)

    Pust, Sascha; Dyve, Anne Berit; Torgersen, Maria L;

    2010-01-01

    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...... investigated whether toxin binding and uptake were associated with flotillin relocalization. We observed a toxin-induced redistribution of the flotillins, which seemed to be regulated in a p38-dependent manner. Our experiments provide no evidence for a changed endocytic uptake of Stx or ricin in cells silenced...... 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...

  3. Stealth and mimicry by deadly bacterial toxins

    DEFF Research Database (Denmark)

    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 Pseudomonas toxin with an NAD+ analogue and eukaryotic elongation factor 2 have provided new insights into the mechanism of inactivation of protein synthesis caused by these protein factors.  Concomitantly, rigorous steady-state and stopped flow kinetic analyses of the toxin-catalyzed reaction, in combination...... with inhibitor studies, has resulted in a quantum leap in our understanding of the mechanistic details of this deadly enzyme mechanism.  Furthermore, it is now apparent that these toxins use stealth and molecular mimicry in unleashing their toxic strategy within the infected host eukaryotic cell....

  4. Toxins and Secretion Systems of Photorhabdus luminescens

    Directory of Open Access Journals (Sweden)

    Athina Rodou

    2010-06-01

    Full Text Available Photorhabdus luminescens is a nematode-symbiotic, gram negative, bioluminescent bacterium, belonging to the family of Enterobacteriaceae.Recent studies show the importance of this bacterium as an alternative source of insecticides, as well as an emerging human pathogen. Various toxins have been identified and characterized in this bacterium. These toxins are classified into four major groups: the toxin complexes (Tcs, the Photorhabdus insect related (Pir proteins, the “makes caterpillars floppy” (Mcf toxins and the Photorhabdus virulence cassettes (PVC; the mechanisms however of toxin secretion are not fully elucidated. Using bioinformatics analysis and comparison against the components of known secretion systems, multiple copies of components of all known secretion systems, except the ones composing a type IV secretion system, were identified throughout the entire genome of the bacterium. This indicates that Photorhabdus luminescens has all the necessary means for the secretion of virulence factors, thus it is capable of establishing a microbial infection.

  5. Immunotoxins: The Role of the Toxin

    Directory of Open Access Journals (Sweden)

    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.

  6. Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii*

    Science.gov (United States)

    Sugai, Yoshinori; Ueno, Yohei; Hayashi, Ken-ichiro; Oogami, Shingo; Toyomasu, Tomonobu; Matsumoto, Sadamu; Natsume, Masahiro; Nozaki, Hiroshi; Kawaide, Hiroshi

    2011-01-01

    Diterpenes show diverse chemical structures and various physiological roles. The diversity of diterpene is primarily established by diterpene cyclases that catalyze a cyclization reaction to form the carbon skeleton of cyclic diterpene. Diterpene cyclases are divided into two types, monofunctional and bifunctional cyclases. Bifunctional diterpene cyclases (BDTCs) are involved in hormone and defense compound biosyntheses in bryophytes and gymnosperms, respectively. The BDTCs catalyze the successive two-step type-B (protonation-initiated cyclization) and type-A (ionization-initiated cyclization) reactions of geranylgeranyl diphosphate (GGDP). We found that the genome of a lycophyte, Selaginella moellendorffii, contains six BDTC genes with the majority being uncharacterized. The cDNA from S. moellendorffii encoding a BDTC-like enzyme, miltiradiene synthase (SmMDS), was cloned. The recombinant SmMDS converted GGDP to a diterpene hydrocarbon product with a molecular mass of 272 Da. Mutation in the type-B active motif of SmMDS abolished the cyclase activity, whereas (+)-copalyl diphosphate, the reaction intermediate from the conversion of GGDP to the hydrocarbon product, rescued the cyclase activity of the mutant to form a diterpene hydrocarbon. Another mutant lacking type-A activity accumulated copalyl diphosphate as the reaction intermediate. When the diterpene hydrocarbon was enzymatically synthesized from [U-13C6]mevalonate, all carbons were labeled with 13C stable isotope (>99%). The fully 13C-labeled product was subjected to 13C-13C COSY NMR spectroscopic analyses. The direct carbon-carbon connectivities observed in the multidimensional NMR spectra demonstrated that the hydrocarbon product by SmMDS is miltiradiene, a putative biosynthetic precursor of tanshinone identified from the Chinese medicinal herb Salvia miltiorrhiza. Hence, SmMDS functions as a bifunctional miltiradiene synthase in S. moellendorffii. In this study, we demonstrate that one-dimensional and

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

    International Nuclear Information System (INIS)

    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)

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

    OpenAIRE

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

  10. Bicarbonate-sensitive soluble and transmembrane adenylyl cyclases in peripheral chemoreceptors.

    Science.gov (United States)

    Nunes, Ana R; Holmes, Andrew P S; Sample, Vedangi; Kumar, Prem; Cann, Martin J; Monteiro, Emília C; Zhang, Jin; Gauda, Estelle B

    2013-08-15

    Stimulation of the carotid body (CB) chemoreceptors by hypercapnia triggers a reflex ventilatory response via a cascade of cellular events, which includes generation of cAMP. However, it is not known if molecular CO2/HCO3(-) and/or H(+) mediate this effect and how these molecules contribute to cAMP production. We previously reported that the CB highly expresses HCO3(-)-sensitive soluble adenylyl cyclase (sAC). In the present study we systematically characterize the role of sAC in the CB, comparing the effect of isohydric hypercapnia (IH) in cAMP generation through activation of sAC or transmembrane-adenylyl cyclase (tmAC). Pharmacological deactivation of sAC and tmAC decreased the CB cAMP content in normocapnia and IH with no differences between these two conditions. Changes from normocapnia to IH did not effect the degree of PKA activation and the carotid sinus nerve discharge frequency. sAC and tmAC are functional in CB but intracellular elevations in CO2/HCO3(-) in IH conditions on their own are insufficient to further activate these enzymes, suggesting that the hypercapnic response is dependent on secondary acidosis.

  11. Adenylyl cyclase 3 haploinsufficiency confers susceptibility to diet-induced obesity and insulin resistance in mice

    Science.gov (United States)

    Tong, Tao; Shen, Ying; Lee, Han-Woong; Yu, Rina; Park, Taesun

    2016-01-01

    Adenylyl cyclase 3 (Adcy3), a member of the mammalian adenylyl cyclase family responsible for generating the second messenger cAMP, has long been known to play an essential role in olfactory signal transduction. Here, we demonstrated that Adcy3 heterozygous null mice displayed increased visceral adiposity in the absence of hyperphagia and developed abnormal metabolic features characterized by impaired insulin sensitivity, dyslipidemia, and increased plasma levels of proinflammatory cytokines on both chow and high-fat diet (HFD). Of note, HFD decreased the Adcy3 expression in white adipose tissue, liver, and muscle. We also report for the first time that Adcy3 haploinsufficiency resulted in reduced expression of genes involved in thermogenesis, fatty acid oxidation, and insulin signaling, with enhanced expression of genes related to adipogenesis in peripheral tissues of mice. In conclusion, these findings suggest that cAMP signals generated by Adcy3 in peripheral tissues may play a pivotal role in modulating obesity and insulin sensitivity. PMID:27678003

  12. Phosphorylation-independent regulation of the diguanylate cyclase WspR.

    Directory of Open Access Journals (Sweden)

    Nabanita De

    2008-03-01

    Full Text Available Environmental signals that trigger bacterial pathogenesis and biofilm formation are mediated by changes in the level of cyclic dimeric guanosine monophosphate (c-di-GMP, a unique eubacterial second messenger. Tight regulation of cellular c-di-GMP concentration is governed by diguanylate cyclases and phosphodiesterases, which are responsible for its production and degradation, respectively. Here, we present the crystal structure of the diguanylate cyclase WspR, a conserved GGDEF domain-containing response regulator in Gram-negative bacteria, bound to c-di-GMP at an inhibitory site. Biochemical analyses revealed that feedback regulation involves the formation of at least three distinct oligomeric states. By switching from an active to a product-inhibited dimer via a tetrameric assembly, WspR utilizes a novel mechanism for modulation of its activity through oligomerization. Moreover, our data suggest that these enzymes can be activated by phosphodiesterases. Thus, in addition to the canonical pathways via phosphorylation of the regulatory domains, both product and enzyme concentration contribute to the coordination of c-di-GMP signaling. A structural comparison reveals resemblance of the oligomeric states to assemblies of GAF domains, widely used regulatory domains in signaling molecules conserved from archaea to mammals, suggesting a similar mechanism of regulation.

  13. Characterization of Plasmodium falciparum adenylyl cyclase-β and its role in erythrocytic stage parasites.

    Directory of Open Access Journals (Sweden)

    Eric Salazar

    Full Text Available The most severe form of human malaria is caused by the parasite Plasmodium falciparum. The second messenger cAMP has been shown to be important for the parasite's ability to infect the host's liver, but its role during parasite growth inside erythrocytes, the stage responsible for symptomatic malaria, is less clear. The P. falciparum genome encodes two adenylyl cyclases, the enzymes that synthesize cAMP, PfACα and PfACβ. We now show that one of these, PfACβ, plays an important role during the erythrocytic stage of the P. falciparum life cycle. Biochemical characterization of PfACβ revealed a marked pH dependence, and sensitivity to a number of small molecule inhibitors. These inhibitors kill parasites growing inside red blood cells. One particular inhibitor is selective for PfACβ relative to its human ortholog, soluble adenylyl cyclase (sAC; thus, PfACβ represents a potential target for development of safe and effective antimalarial therapeutics.

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

    Science.gov (United States)

    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

  15. Botulinum toxin: yesterday, today, tomorrow

    Directory of Open Access Journals (Sweden)

    A. R. Artemenko

    2013-01-01

    Full Text Available Botulinum toxin (BoNT is a bacterial neurotoxin presented with seven serotypes that inhibit neurotransmitter release from nerve endings. The serotypes of BoNT are antigenically dissimilar, act via different, but interconnected mechanisms, and are not interchangeable. The activity of BoNT is associated with impaired neuroexocytosis occurring in several steps: from the binding of BoNT to its specific receptor on the axon terminal membrane to the proteolytic enzymatic cleavage of SNARE substrate. The effect of BoNT is considered to be restricted to the peripheral nervous system, but when given in particularly high doses, it has been recently shown to affect individual brain structures. In addition, by modulating peripheral afferentation, BoNT may influence the excitability of central neuronal structures at both spinal and cortical levels. Only BoNT serotypes A and B are used in clinical practice and aesthetic medicine. The type A has gained the widest acceptance as a therapeutic agent for more than 100 abnormalities manifesting themselves as muscular hyperactivity, hyperfunction of endocrine gland, and chronic pain. The effect of BoNT preparations shows itself 2-5 days after injection, lasts 3 months or more, and gradually decreases with as a result of pharmacokinetic and intracellular reparative processes. Biotechnology advances and potentialities allow purposefully modification of the protein molecular structure of BoNT, which expands the use and efficiency of performed therapy with neurotoxins. Recombinant technologies provide a combination of major therapeutic properties of each used BoNT serotype and expand indications for recombinant chimeric toxins.

  16. The Enterotoxicity of Clostridium difficile Toxins

    OpenAIRE

    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...

  17. Retrograde transport of protein toxins through the Golgi apparatus

    DEFF Research Database (Denmark)

    Sandvig, Kirsten; Skotland, Tore; van Deurs, Bo;

    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 into and through...

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

    Institute of Scientific and Technical Information of China (English)

    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

  19. The magnesium-protoporphyrin IX (oxidative) cyclase system. Studies on the mechanism and specificity of the reaction sequence.

    Science.gov (United States)

    Walker, C J; Mansfield, K E; Rezzano, I N; Hanamoto, C M; Smith, K M; Castelfranco, P A

    1988-10-15

    Mg-protoporphyrin IX monomethyl ester cyclase activity was assayed in isolated developing cucumber (Cucumis sativus L. var. Beit Alpha) chloroplasts [Chereskin, Wong & Castelfranco (1982) Plant Physiol. 70, 987-993]. The presence of both 6- and 7-methyl esterase activities was detected, which permitted the use of diester porphyrins in a substrate-specificity study. It was found that: (1) the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester was inactive as a substrate for cyclization; (2) only one of the two enantiomers of 6-beta-hydroxy-Mg-protoporphyrin dimethyl ester had detectable activity as a substrate for the cyclase; (3) the 2-vinyl-4-ethyl-6-beta-oxopropionate derivatives of Mg-protoporphyrin mono- or di-methyl ester were approx. 4 times more active as substrates for cyclization than the corresponding divinyl forms; (4) at the level of Mg-protoporphyrin there was no difference in cyclase activity between the 4-vinyl and 4-ethyl substrates; (5) reduction of the side chain of Mg-protoporphyrin in the 2-position from a vinyl to an ethyl resulted in a partial loss of cyclase activity. This work suggests that the original scheme for cyclization proposed by Granick [(1950) Harvey Lect. 44, 220-245] should now be modified by the omission of the 6-methyl acrylate derivative of Mg-protoporphyrin monomethyl ester and the introduction of stereo-specificity at the level of the hydroxylated intermediate.

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

    OpenAIRE

    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...

  1. Towards Rational Design of a Toxoid Vaccine against the Heat-Stable Toxin of Escherichia coli

    Science.gov (United States)

    Taxt, Arne M.; Diaz, Yuleima; Aasland, Rein; Clements, John D.; Nataro, James P.; Sommerfelt, Halvor

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and death in children <5 years old. ETEC strains that express the heat-stable toxin (ST), with or without the heat-labile toxin, are among the four most important diarrhea-causing pathogens. This makes ST an attractive target for an ETEC vaccine. An ST vaccine should be nontoxic and elicit an immune response that neutralizes native ST without cross-reacting with the human endogenous guanylate cyclase C receptor ligands. To identify variants of ST with no or low toxicity, we screened a library of all 361 possible single-amino-acid mutant forms of ST by using the T84 cell assay. Moreover, we identified mutant variants with intact epitopes by screening for the ability to bind neutralizing anti-ST antibodies. ST mutant forms with no or low toxicity and intact epitopes are termed toxoid candidates, and the top 30 candidates all had mutations of residues A14, N12, and L9. The identification of nontoxic variants of L9 strongly suggests that it is a novel receptor-interacting residue, in addition to the previously identified N12, P13, and A14 residues. The screens also allowed us to map the epitopes of three neutralizing monoclonal antibodies, one of which cross-reacts with the human ligand uroguanylin. The common dominant epitope residue for all non-cross-reacting antibodies was Y19. Our results suggest that it should be possible to rationally design ST toxoids that elicit neutralizing immune responses against ST with minimal risk of immunological cross-reactivity. PMID:26883587

  2. NNDSS - Table II. Shiga toxin to Shigellosis

    Data.gov (United States)

    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...

  3. NNDSS - Table II. Shiga toxin to Shigellosis

    Data.gov (United States)

    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...

  4. Bacterial Toxins as Pathogen Weapons Against Phagocytes.

    Science.gov (United States)

    do Vale, Ana; Cabanes, Didier; Sousa, Sandra

    2016-01-01

    Bacterial toxins are virulence factors that manipulate host cell functions and take over the control of vital processes of living organisms to favor microbial infection. Some toxins directly target innate immune cells, thereby annihilating a major branch of the host immune response. In this review we will focus on bacterial toxins that act from the extracellular milieu and hinder the function of macrophages and neutrophils. In particular, we will concentrate on toxins from Gram-positive and Gram-negative bacteria that manipulate cell signaling or induce cell death by either imposing direct damage to the host cells cytoplasmic membrane or enzymatically modifying key eukaryotic targets. Outcomes regarding pathogen dissemination, host damage and disease progression will be discussed.

  5. Toxin Detection by Surface Plasmon Resonance

    Directory of Open Access Journals (Sweden)

    2009-02-01

    Full Text Available Significant efforts have been invested in the past years for the development of analytical methods for fast toxin detection in food and water. Immunochemical methods like ELISA, spectroscopy and chromatography are the most used in toxin detection. Different methods have been linked, e.g. liquid chromatography and mass spectrometry (LC-MS, in order to detect as low concentrations as possible. Surface plasmon resonance (SPR is one of the new biophysical methods which enables rapid toxin detection. Moreover, this method was already included in portable sensors for on-site determinations. In this paper we describe some of the most common methods for toxin detection, with an emphasis on SPR.

  6. Bacterial toxins as pathogen weapons against phagocytes

    Directory of Open Access Journals (Sweden)

    Ana edo Vale

    2016-02-01

    Full Text Available Bacterial toxins are virulence factors that manipulate host cell functions and take over the control of vital processes of living organisms to favour microbial infection. Some toxins directly target innate immune cells, thereby annihilating a major branch of the host immune response. In this review we will focus on bacterial toxins that act from the extracellular milieu and hinder the function of macrophages and neutrophils. In particular, we will concentrate on toxins from Gram-positive and Gram-negative bacteria that manipulate cell signalling or induce cell death by either imposing direct damage to the host cells cytoplasmic membrane or enzymatically modifying key eukaryotic targets. Outcomes regarding pathogen dissemination, host damage and disease progression will be discussed.

  7. [Botulinum toxin in disabling dermatological diseases].

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  9. Updates on tetanus toxin: a fundamental approach

    Directory of Open Access Journals (Sweden)

    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.

  10. Nanoanalysis of the arthropod neuro-toxins

    OpenAIRE

    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...

  11. Target-Driven Evolution of Scorpion Toxins

    OpenAIRE

    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...

  12. Botulinum toxin treatment of hemifacial spasm.

    OpenAIRE

    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.

  13. Toxicological Perspective on Climate Change: Aquatic Toxins.

    Science.gov (United States)

    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

  14. Toxin-Antitoxin Systems of Staphylococcus aureus.

    Science.gov (United States)

    Schuster, Christopher F; Bertram, Ralph

    2016-05-05

    Toxin-antitoxin (TA) systems are small genetic elements found in the majority of prokaryotes. They encode toxin proteins that interfere with vital cellular functions and are counteracted by antitoxins. Dependent on the chemical nature of the antitoxins (protein or RNA) and how they control the activity of the toxin, TA systems are currently divided into six different types. Genes comprising the TA types I, II and III have been identified in Staphylococcus aureus. MazF, the toxin of the mazEF locus is a sequence-specific RNase that cleaves a number of transcripts, including those encoding pathogenicity factors. Two yefM-yoeB paralogs represent two independent, but auto-regulated TA systems that give rise to ribosome-dependent RNases. In addition, omega/epsilon/zeta constitutes a tripartite TA system that supposedly plays a role in the stabilization of resistance factors. The SprA1/SprA1AS and SprF1/SprG1 systems are post-transcriptionally regulated by RNA antitoxins and encode small membrane damaging proteins. TA systems controlled by interaction between toxin protein and antitoxin RNA have been identified in S. aureus in silico, but not yet experimentally proven. A closer inspection of possible links between TA systems and S. aureus pathophysiology will reveal, if these genetic loci may represent druggable targets. The modification of a staphylococcal TA toxin to a cyclopeptide antibiotic highlights the potential of TA systems as rather untapped sources of drug discovery.

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

    Science.gov (United States)

    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

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

    KAUST Repository

    Irving, Helen R.

    2012-02-01

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

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

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

    2010-10-01

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

  19. Characterization of a Fungal Thioesterase Having Claisen Cyclase and Deacetylase Activities in Melanin Biosynthesis

    Science.gov (United States)

    Vagstad, Anna L; Hill, Eric A; Labonte, Jason W; Townsend, Craig A

    2012-01-01

    Summary Melanins are a broad class of darkly-pigmented macromolecules formed by oxidative polymerization of phenolic monomers. In fungi, melanins are known virulence factors that contribute to pathogenicity. Their biosynthesis generally involves polymerization of 1,8-dihydroxynaphthalene via a 1,3,6,8- tetrahydroxynaphthalene (THN) precursor assembled by multidomain, nonreducing polyketide synthases. Multiple, convergent routes to THN have evolved in fungi. Parallel heptaketide and hexaketide pathways exist that utilize conventional C-terminal thioesterase/Claisen cyclase domains and separate side-chain deacylases. Here, in vitro characterization of Pks1 from Colletotrichum lagenarium establishes a true THN synthase with a bifunctional thioesterase (TE) catalyzing both cyclization and deacetylation of an enzyme-bound hexaketide substrate. Chimeric TE domains were generated by swapping lid regions of active sites between classes of melanin TEs to gain insight into this unprecedented catalysis of carbon–carbon bond making and breaking by an α/β-hydrolase fold enzyme. PMID:23261597

  20. 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

    OpenAIRE

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

    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...

  1. New structural forms of a mycobacterial adenylyl cyclase Rv1625c

    Directory of Open Access Journals (Sweden)

    Deivanayaga Barathy

    2014-09-01

    Full Text Available Rv1625c is one of 16 adenylyl cyclases encoded in the genome of Mycobacterium tuberculosis. In solution Rv1625c exists predominantly as a monomer, with a small amount of dimer. It has been shown previously that the monomer is active and the dimeric fraction is inactive. Both fractions of wild-type Rv1625c crystallized as head-to-head inactive domain-swapped dimers as opposed to the head-to-tail dimer seen in other functional adenylyl cyclases. About half of the molecule is involved in extensive domain swapping. The strain created by a serine residue located on a hinge loop and the crystallization condition might have led to this unusual domain swapping. The inactivity of the dimeric form of Rv1625c could be explained by the absence of the required catalytic site in the swapped dimer. A single mutant of the enzyme was also generated by changing a phenylalanine predicted to occur at the functional dimer interface to an arginine. This single mutant exists as a dimer in solution but crystallized as a monomer. Analysis of the structure showed that a salt bridge formed between a glutamate residue in the N-terminal segment and the mutated arginine residue hinders dimer formation by pulling the N-terminal region towards the dimer interface. Both structures reported here show a change in the dimerization-arm region which is involved in formation of the functional dimer. It is concluded that the dimerization arm along with other structural elements such as the N-terminal region and certain loops are vital for determining the oligomeric nature of the enzyme, which in turn dictates its activity.

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

    Directory of Open Access Journals (Sweden)

    Xiao-Hong eWen

    2014-06-01

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

  3. Interaction of retinal guanylate cyclase with the alpha subunit of transducin: potential role in transducin localization.

    Science.gov (United States)

    Rosenzweig, Derek H; Nair, K Saidas; Levay, Konstantin; Peshenko, Igor V; Crabb, John W; Dizhoor, Alexander M; Slepak, Vladlen Z

    2009-02-01

    Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Janet M Young

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

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

    Directory of Open Access Journals (Sweden)

    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.

  7. Clostridium perfringens Delta-Toxin Induces Rapid Cell Necrosis

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

  9. 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*

    OpenAIRE

    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, ...

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

    Science.gov (United States)

    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

  11. A minor conformation of a lanthanide tag on adenylate kinase characterized by paramagnetic relaxation dispersion NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    2010-01-01

    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 to -437 bp 5' to ATG led to

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

    Directory of Open Access Journals (Sweden)

    Chinnusamy Viswanathan

    2010-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Teresa eDuda

    2014-03-01

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

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

    OpenAIRE

    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 ...

  17. Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

    OpenAIRE

    SATO, Tsutomu; Kouda, Masanori; HOSHINO, Tsutomu; 佐藤, 努

    2004-01-01

    To provide insight into the catalytic mechanism for the final deprotonation reaction of squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius, mutagenesis experiments were conducted for the following ten residues: Thr41, Glu45, Glu93, Arg127, Trp133, Gln262, Pro263, Tyr267, Phe434 and Phe437. An X-ray analysis of SHC has revealed that two types of water molecules ("front water" and "back waters") were involved around the deprotonation site. The results of these mutagenesis experi...

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

    Directory of Open Access Journals (Sweden)

    Kabir Hassan Biswas

    2015-04-01

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

  19. Clostridium perfringens delta toxin is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences.

    Directory of Open Access Journals (Sweden)

    Maria Manich

    Full Text Available Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside G(M2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da is a basic protein (pI 9.1 which shows a significant homology with C. perfringens Beta toxin (43% identity, with C. perfringens NetB (40% identity and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to G(M2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes G(M2 as receptor and forms anion-selective channels.

  20. Marine toxins and their toxicological significance: An overview

    Digital Repository Service at National Institute of Oceanography (India)

    Sarkar, A.

    This article presents an overview of various types of marine toxins and their toxicological significance in the context of biotechnological research and development. The characteristics and toxic potentials of different marine toxins highlighted...

  1. Marine Toxins Targeting Ion Channels

    Directory of Open Access Journals (Sweden)

    Hugo R. Arias

    2006-04-01

    Full Text Available Abstract: This introductory minireview points out the importance of ion channels for cell communication. The basic concepts on the structure and function of ion channels triggered by membrane voltage changes, the so-called voltage-gated ion channels (VGICs, as well as those activated by neurotransmitters, the so-called ligand-gated ion channel (LGICs, are introduced. Among the most important VGIC superfamiles, we can name the voltage-gated Na+ (NaV, Ca2+ (CaV, and K+ (KV channels. Among the most important LGIC super families, we can include the Cys-loop or nicotinicoid, the glutamate-activated (GluR, and the ATP-activated (P2XnR receptor superfamilies. Ion channels are transmembrane proteins that allow the passage of different ions in a specific or unspecific manner. For instance, the activation of NaV, CaV, or KV channels opens a pore that is specific for Na+, Ca2+, or K+, respectively. On the other hand, the activation of certain LGICs such as nicotinic acetylcholine receptors, GluRs, and P2XnRs allows the passage of cations (e.g., Na+, K+, and/or Ca2+, whereas the activation of other LGICs such as type A γ-butyric acid and glycine receptors allows the passage of anions (e.g., Cl− and/or HCO3−. In this regard, the activation of NaV and CaV as well as ligand-gated cation channels produce membrane depolarization, which finally leads to stimulatory effects in the cell, whereas the activation of KV as well as ligand-gated anion channels induce membrane hyperpolarization that finally leads to inhibitory effects in the cell. The importance of these ion channel superfamilies is emphasized by considering their physiological functions throughout the body as well as their pathophysiological implicance in several neuronal diseases. In this regard, natural molecules, and especially marine toxins, can be potentially used as modulators (e.g., inhibitors or prolongers of ion channel functions to treat or to alleviate a specific

  2. Inhibiting bacterial toxins by channel blockage.

    Science.gov (United States)

    Bezrukov, Sergey M; Nestorovich, Ekaterina M

    2016-03-01

    Emergent rational drug design techniques explore individual properties of target biomolecules, small and macromolecule drug candidates, and the physical forces governing their interactions. In this minireview, we focus on the single-molecule biophysical studies of channel-forming bacterial toxins that suggest new approaches for their inhibition. We discuss several examples of blockage of bacterial pore-forming and AB-type toxins by the tailor-made compounds. In the concluding remarks, the most effective rationally designed pore-blocking antitoxins are compared with the small-molecule inhibitors of ion-selective channels of neurophysiology.

  3. Streptococcal toxins: role in pathogenesis and disease.

    Science.gov (United States)

    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.

  4. Streptococcal toxins: role in pathogenesis and disease.

    Science.gov (United States)

    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

  5. Natural Toxins for Use in Pest Management

    Directory of Open Access Journals (Sweden)

    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.

  6. Positive regulation of Clostridium difficile toxins.

    OpenAIRE

    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...

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

    OpenAIRE

    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...

  8. 77 FR 9888 - Shiga Toxin-Producing Escherichia coli

    Science.gov (United States)

    2012-02-21

    ... Food Safety and Inspection Service Shiga Toxin-Producing Escherichia coli in Certain Raw Beef Products... manufacturing trimmings for six non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45..., non-intact product, that are contaminated with Shiga toxin-producing Escherichia coli (STEC) O26,...

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

    NARCIS (Netherlands)

    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

  10. Anthrax toxin-induced rupture of artificial lipid bilayer membranes

    Science.gov (United States)

    Nablo, Brian J.; Panchal, Rekha G.; Bavari, Sina; Nguyen, Tam L.; Gussio, Rick; Ribot, Wil; Friedlander, Art; Chabot, Donald; Reiner, Joseph E.; Robertson, Joseph W. F.; Balijepalli, Arvind; Halverson, Kelly M.; Kasianowicz, John J.

    2013-08-01

    We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

  11. Effect of treatment with botulinum toxin on spasticity.

    OpenAIRE

    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.

  12. Bacterial protein toxins : tools to study mammalian molecular cell biology

    NARCIS (Netherlands)

    Wüthrich, I.W.

    2014-01-01

    Bacterial protein toxins are genetically encoded proteinaceous macromolecules that upon exposure causes perturbation of cellular metabolism in a susceptible host. A bacterial toxin can work at a distance from the site of infection, and has direct and quantifiable actions. Bacterial protein toxins ca

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

    OpenAIRE

    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.

  14. Expression of nitric oxide synthase and guanylate cyclase in the human ciliary body and trabecular meshwork

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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

  17. Soluble Guanylate Cyclase Stimulators: a Novel Treatment Option for Heart Failure Associated with Cardiorenal Syndromes?

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2014-08-18

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

  19. Disruption of Epac1 protects the heart from adenylyl cyclase type 5-mediated cardiac dysfunction.

    Science.gov (United States)

    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

  20. The Functional State of Hormone-Sensitive Adenylyl Cyclase Signaling System in Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    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.

  1. In silico prediction of tyrosinase and adenylyl cyclase inhibitors from natural compounds.

    Science.gov (United States)

    Fong, Pedro; Tong, Henry H Y; Chao, Chi M

    2014-02-01

    Although many herbal medicines are effective in the treatment of hyperpigmentation, the potency of different constituents remains unknown. In this work, more than 20,000 herbal ingredients from 453 herbs were docked into the crystal structures of adenylyl cyclase and a human homology tyrosinase model using Surflex-Dock. These two enzymes are responsible for melanin production and inhibition of them may attain a skin-whitening effect superior to currently available agents. The essential drug properties for topical formulation of the herbal ingredients, including skin permeability, sensitization, irritation, corrosive and carcinogenic properties were predicted by Dermwin, Skin Sensitization Alerts (SSA), Skin Irritation Corrosion Rules Estimation Tool (SICRET) and Benigni/Bossa rulebase module of Toxtree. Moreover, similarity ensemble and pharmacophore mapping approaches were used to forecast other potential targets for these herbal compounds by the software, SEArch and PharmMapper. Overall, this study predicted seven compounds to have advanced drug-like properties over the well-known effective tyrosinase inhibitors, arbutin and kojic acid. These seven compounds have the highest potential for further in vitro and in vivo investigation with the aim of developing safe and high-efficacy skin-whitening agents.

  2. Identification of small molecules inhibiting diguanylate cyclases to control bacterial biofilm development.

    Science.gov (United States)

    Sambanthamoorthy, Karthik; Luo, Chunyuan; Pattabiraman, Nagarajan; Feng, Xiarong; Koestler, Benjamin; Waters, Christopher M; Palys, Thomas J

    2014-01-01

    Biofilm formation by pathogenic bacteria is an important virulence factor in the development of numerous chronic infections, thereby causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. An urgent need for new strategies to treat biofilm-based infections is critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. The absence of this signalling system in higher eukaryotes makes it an attractive target for the development of new anti-biofilm agents. In this study, the results of an in silico pharmacophore-based screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP are described. Four small molecules, LP 3134, LP 3145, LP 4010 and LP 1062 that antagonize these enzymes and inhibit biofilm formation by Pseudomonas aeruginosa and Acinetobacter baumannii in a continuous-flow system are reported. All four molecules dispersed P. aeruginosa biofilms and inhibited biofilm development on urinary catheters. One molecule dispersed A. baumannii biofilms. Two molecules displayed no toxic effects on eukaryotic cells. These molecules represent the first compounds identified from an in silico screen that are able to inhibit DGC activity to prevent biofilm formation. PMID:24117391

  3. Structural features of Escherichia coli heat-stable enterotoxin that activates membrane-associated guanylyl cyclase.

    Science.gov (United States)

    Sato, T; Shimonishi, Y

    2004-03-01

    Heat-stable enterotoxin (ST), a small peptide of 18 or 19 amino acid residues produced by enterotoxigenic Escherichia coli, is the cause of acute diarrhea in infants and travelers in developing countries. ST triggers a biological response by binding to a membrane-associated guanylyl cyclase C (GC-C) which is located on intestinal epithelial cell membranes. This binding causes an increase in the concentration of cGMP as a second messenger in cells and activates protein kinase A and cystic fibrosis transmembrane conductance regulator. Here we describe the crystal structure of an ST at 0.89 A resolution. The molecule has a ring-shaped molecular architecture consisting of six peptide molecules with external and internal diameters of approximately 35 and 7 A, respectively and a thickness of approximately 11 A. The conserved residues at the central portion of ST are distributed on the outer surface of the ring-shaped peptide hexamer, suggesting that the hexamer may be implicated in the association with GC-C through these invariant residues. PMID:15049831

  4. Molecular Cloning, and Characterization of an Adenylyl Cyclase-Associated Protein from Gossypium arboreum L.

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

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

    2014-08-18

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

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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...

  9. Immunogenicity of toxins during Staphylococcus aureus infection

    NARCIS (Netherlands)

    N.J. Verkaik (Nelianne); O. Dauwalder (Olivier); K. Antri (Kenza); I. Boubekri (Ilhem); C.P. de Vogel (Corné); C. Badiou (Cédric); M. Bes (Michèle); F. Vandenesch (François); M. Tazir (Mohammed); H. Hooijkaas (Herbert); H.A. Verbrugh (Henri); A.F. van Belkum (Alex); J. Etienne (Jerome); G. Lina (Gérard); N. Ramdani-Bouguessa (Nadjia); W.J.B. van Wamel (Willem)

    2010-01-01

    textabstractAB - BACKGROUND: Toxins are important Staphylococcus aureus virulence factors, but little is known about their immunogenicity during infection. Here, additional insight is generated. METHODS: Serum samples from 206 S. aureus-infected patients and 201 hospital-admitted control subjects we

  10. Natural toxins for use in pest management

    Science.gov (United States)

    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 ...

  11. Treatment of Frontal Hyperhidrosis With Botulinum Toxin

    Directory of Open Access Journals (Sweden)

    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.

  12. Noncosmetic periocular therapeutic applications of botulinum toxin

    Directory of Open Access Journals (Sweden)

    Kaynak-Hekimhan Pelin

    2010-01-01

    Full Text Available Botulinum toxin blocks acetylcholine release at the neuromuscular junction. The drug which was initially found to be useful in the treatment of strabismus has been extremely effective in the treatment of variety of conditions, both cosmetic and noncosmetic. Some of the noncosmetic uses of botulinum toxin applications include treatment of spastic facial dystonias, temporary treatment of idiopathic or thyroid dysfunction-induced upper eyelid retraction, suppression of undesired hyperlacrimation, induction of temporary ptosis by chemodenervation in facial paralysis, and correction of lower eyelid spastic entropion. Additional periocular uses include control of synchronic eyelid and extraocular muscle movements after aberrant regeneration of cranial nerve palsies. Cosmetic effects of botulinum toxin were discovered accidentally during treatments of facial dystonias. Some of the emerging nonperiocular application for the drug includes treatment of hyperhidrosis, migraine, tension-type headaches, and paralytic spasticity. Some of the undesired side effects of periocular applications of botulinum toxin inlcude ecchymosis, rash, hematoma, headache, flu-like symptoms, nausea, dizziness, loss of facial expression, lower eyelid laxity, dermatochalasis, ectropion, epiphora, eyebrow and eyelid ptosis, lagophthalmos, keratitis sicca, and diplopia.

  13. Okadaic Acid: More than a Diarrheic Toxin

    Directory of Open Access Journals (Sweden)

    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.

  14. Toxins and antimicrobial peptides: interactions with membranes

    Science.gov (United States)

    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 peptide potency, was monitored with a sensitive fluorescence leakage assay. Detailed molecular information on peptidemembrane interactions and peptide structure was further gained through vibrational spectroscopy combined with circular dichroism. Finally, steady-state fluorescence experiments yielded insight into the local environment of native or engineered tryptophan residues in 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.

  15. Mutant with diphtheria toxin receptor and acidification function but defective in entry of toxin

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-10-06

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

  18. Mode of action of mosquitocidal Bacillus thuringiensis toxins.

    Science.gov (United States)

    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

  19. General synthesis of β-alanine-containing spider polyamine toxins and discovery of nephila polyamine toxins 1 and 8 as highly potent inhibitors of ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Lucas, Simon; Poulsen, Mette H; Nørager, Niels G;

    2012-01-01

    Certain spiders contain large pools of polyamine toxins, which are putative pharmacological tools awaiting further discovery. Here we present a general synthesis strategy for this class of toxins and prepare five structurally varied polyamine toxins. Electrophysiological testing at three ionotropic...

  20. Covalent aspartylation of aspartyl-tRNA synthetase from Bakers' yeast by its cognat aspartyl adenylate: identification of the labeled residues

    Energy Technology Data Exchange (ETDEWEB)

    Mejdoub, H.; Kern, D.; Giege, R.; Ebel, J.P.; Boulanger, Y.; Reinbolt, J.

    1987-04-07

    Aspartyl-tRNA synthetase from bakers' yeast gives an unstable complex with the cognate adenylate, which reacts after dissociation with amino acid side chains of the protein. This leads to a covalent incorporation of (/sup 14/C)-aspartic acid into aspartyl-tRNA synthetase via amide or ester bonds formed between the ..cap alpha..-carboxyl group of activated aspartic acid and accessible lysines, serines, and threonines. This property is used to label the peptides at the surface of the enzyme. The main labeled residues have been identified, and their location in the primary structure is discussed in relation to structural properties of aspartyl-tRNA synthetase.

  1. Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene.

    Science.gov (United States)

    Rodriguez-Centeno, Javier; Sastre, Leandro

    2016-01-01

    Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation.

  2. A human skeletal overgrowth mutation increases maximal velocity and blocks desensitization of guanylyl cyclase-B☆

    Science.gov (United States)

    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

  3. Analgesic effects of adenylyl cyclase inhibitor NB001 on bone cancer pain in a mouse model

    Science.gov (United States)

    Kang, Wen-bo; Yang, Qi; Guo, Yan-yan; Wang, Lu; Wang, Dong-sheng; Cheng, Qiang; Li, Xiao-ming; Tang, Jun; Zhao, Jian-ning; Liu, Gang; Zhuo, Min

    2016-01-01

    Background Cancer pain, especially the one caused by metastasis in bones, is a severe type of pain. Pain becomes chronic unless its causes and consequences are resolved. With improvements in cancer detection and survival among patients, pain has been considered as a great challenge because traditional therapies are partially effective in terms of providing relief. Cancer pain mechanisms are more poorly understood than neuropathic and inflammatory pain states. Chronic inflammatory pain and neuropathic pain are influenced by NB001, an adenylyl cyclase 1 (AC1)-specific inhibitor with analgesic effects. In this study, the analgesic effects of NB001 on cancer pain were evaluated. Results Pain was induced by injecting osteolytic murine sarcoma cell NCTC 2472 into the intramedullary cavity of the femur of mice. The mice injected with sarcoma cells for four weeks exhibited significant spontaneous pain behavior and mechanical allodynia. The continuous systemic application of NB001 (30 mg/kg, intraperitoneally, twice daily for three days) markedly decreased the number of spontaneous lifting but increased the mechanical paw withdrawal threshold. NB001 decreased the concentrations of cAMP and the levels of GluN2A, GluN2B, p-GluA1 (831), and p-GluA1 (845) in the anterior cingulate cortex, and inhibited the frequency of presynaptic neurotransmitter release in the anterior cingulate cortex of the mouse models. Conclusions NB001 may serve as a novel analgesic to treat bone cancer pain. Its analgesic effect is at least partially due to the inhibition of AC1 in anterior cingulate cortex. PMID:27612915

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

    Science.gov (United States)

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

    2013-11-15

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

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

    KAUST Repository

    Turek, Ilona

    2016-03-05

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

  6. Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis

    Science.gov (United States)

    Mann, Francis M.; Prisic, Sladjana; Hu, Huayou; Xu, Meimei; Coates, Robert M.; Peters, Reuben J.

    2009-01-01

    Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans. PMID:19574210

  7. Characterization and phylogenetic epitope mapping of CD38 ADPR cyclase in the cynomolgus macaque

    Directory of Open Access Journals (Sweden)

    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.

  8. A peptide against soluble guanylyl cyclase α1: a new approach to treating prostate cancer.

    Directory of Open Access Journals (Sweden)

    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.

  9. Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) dunal.

    Science.gov (United States)

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

    2014-06-13

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

  10. Toxin(s), Other Than Cholera Toxin, Produced by Environmental Non O1 Non O139 Vibrio cholerae

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Catalina Sanz

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

  13. Sea Anemone (Cnidaria, Anthozoa, Actiniaria Toxins: An Overview

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    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.

  14. Toxins for Transgenic Resistance to Hemipteran Pests

    Directory of Open Access Journals (Sweden)

    Bryony C. Bonning

    2012-06-01

    Full Text Available 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 has provided effective plant protection against some insect pests, Bt toxins exhibit little toxicity against sap sucking insects. Indeed, the pest status of some Hemiptera on Bt-transgenic plants has increased in the absence of pesticide application. The increased pest status of numerous hemipteran species, combined with increased prevalence of resistance to chemical insecticides, provides impetus for the development of biologically based, alternative management strategies. Here, we provide an overview of approaches toward transgenic resistance to hemipteran pests.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  16. Tyrosine Phosphatase TpbA Controls Rugose Colony Formation in Pseudomonas aeruginosa by Dephosphorylating Diguanylate Cyclase TpbB

    OpenAIRE

    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...

  17. Shiga Toxin Interaction with Human Intestinal Epithelium

    OpenAIRE

    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 ...

  18. Abstract Action Potential Models for Toxin Recognition

    OpenAIRE

    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...

  19. Why do we study animal toxins?

    OpenAIRE

    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,...

  20. Toxins for Transgenic Resistance to Hemipteran Pests

    OpenAIRE

    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...

  1. Natural Toxins for Use in Pest Management

    OpenAIRE

    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...

  2. The Biology of the Cytolethal Distending Toxins

    Directory of Open Access Journals (Sweden)

    Teresa Frisan

    2011-03-01

    Full Text Available The cytolethal distending toxins (CDTs, produced by a variety of Gram-negative pathogenic bacteria, are the first bacterial genotoxins described, since they cause DNA damage in the target cells. CDT is an A-B2 toxin, where the CdtA and CdtC subunits are required to mediate the binding on the surface of the target cells, allowing internalization of the active CdtB subunit, which is functionally homologous to the mammalian deoxyribonuclease I. The nature of the surface receptor is still poorly characterized, however binding of CDT requires intact lipid rafts, and its internalization occurs via dynamin-dependent endocytosis. The toxin is retrograde transported through the Golgi complex and the endoplasmic reticulum, and subsequently translocated into the nuclear compartment, where it exerts the toxic activity. Cellular intoxication induces DNA damage and activation of the DNA damage responses, which results in arrest of the target cells in the G1 and/or G2 phases of the cell cycle and activation of DNA repair mechanisms. Cells that fail to repair the damage will senesce or undergo apoptosis. This review will focus on the well-characterized aspects of the CDT biology and discuss the questions that still remain unanswered.

  3. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    Directory of Open Access Journals (Sweden)

    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.

  4. Fast collapse but slow formation of secondary structure elements in the refolding transition of E. coli adenylate kinase.

    Science.gov (United States)

    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

  5. Cobalt-, zinc- and iron-bound forms of adenylate kinase (AK) from the sulfate-reducing bacterium Desulfovibrio gigas: purification, crystallization and preliminary X-ray diffraction analysis

    International Nuclear Information System (INIS)

    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

  6. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    Science.gov (United States)

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyo Joon; Nishikawa, Satoshi; Tokutomi, Yuiko; Uesugi, Seiichi (Osaka Univ. (Japan)); Takenaka, Hitoshi; Hamada, Minoru (Miyazaki Medical College (Japan)); Kuby, S.A. (Univ. of Utah, Salt Lake City (USA))

    1990-02-06

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

  8. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

    Science.gov (United States)

    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

  9. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

    Science.gov (United States)

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-04-01

    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

  10. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

    Directory of Open Access Journals (Sweden)

    Leonie Schnell

    2016-04-01

    Full Text Available 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-dimethylphenylsemicarbazone (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.

  11. Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements.

    Science.gov (United States)

    Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

    2014-04-11

    Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈ 14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4'-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr(112) (hydrogen bonding of ATP adenine to K in the closed active center), His(221) (covalent anchoring of dihydroxyacetone to K), Asp(401) and Asp(403) (metal coordination to L), and Asp(556) (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His(221) point mutant acted specifically as a cyclase without kinase activity.

  12. Fold modulating function: Bacterial toxins to functional amyloids

    Directory of Open Access Journals (Sweden)

    Adnan Khawaja Syed

    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.

  13. Cytoskeleton as an Emerging Target of Anthrax Toxins

    Directory of Open Access Journals (Sweden)

    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.

  14. Recent Insights into Clostridium perfringens Beta-Toxin

    Directory of Open Access Journals (Sweden)

    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.

  15. Fusarial toxins: secondary metabolites of Fusarium fungi.

    Science.gov (United States)

    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

  16. Fusarial toxins: secondary metabolites of Fusarium fungi.

    Science.gov (United States)

    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. Transcriptional Stimulation of Anthrax Toxin Receptors by Anthrax Edema Toxin and Bacillus anthracis Sterne Spore

    OpenAIRE

    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 ...

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

    DEFF Research Database (Denmark)

    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 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...

  19. Staphylococcus aureus α-Toxin: Nearly a Century of Intrigue

    Directory of Open Access Journals (Sweden)

    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.

  20. Military Importance of Natural Toxins and Their Analogs.

    Science.gov (United States)

    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

  1. ArachnoServer: a database of protein toxins from spiders

    OpenAIRE

    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...

  2. ATDB: a uni-database platform for animal toxins

    OpenAIRE

    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...

  3. ClanTox: a classifier of short animal toxins

    OpenAIRE

    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...

  4. Military Importance of Natural Toxins and Their Analogs

    Directory of Open Access Journals (Sweden)

    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.

  5. Molecular Cloning and Characterization of an Allene Oxide Cyclase Gene Associated with Fiber Strength in Cotton

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Directory of Open Access Journals (Sweden)

    Rameshwar K Sharma

    2014-07-01

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

  7. Pharmacokinetic interaction profile of riociguat, a new soluble guanylate cyclase stimulator, in vitro.

    Science.gov (United States)

    Rickert, Verena; Haefeli, Walter Emil; Weiss, Johanna

    2014-08-01

    Riociguat is a new soluble guanylate cyclase stimulator under development for pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension. So far, the interaction potential of riociguat with other drugs is nearly unknown. Therefore, we assessed in vitro the potency of riociguat to inhibit important drug metabolising enzymes (cytochrome P450 (CYP) 3A4, CYP2C19, and CYP2D6) and drug transporters (P-glycoprotein (P-gp/ABCB1), breast cancer resistance protein (BCRP/ABCG2), and organic anion transporting polypeptides (OATP) 1B1 and 1B3). In addition we evaluated its substrate characteristics for P-gp, BCRP, and the multidrug resistance-associated protein 1 (MRP1/ABCC1). We also assessed riociguat's inducing properties on important drug metabolising enzymes and transporters and investigated its ability to activate the pregnane-X-receptor (PXR). Riociguat was identified as a weak to moderate inhibitor of P-gp (f2-value: 11.7 ± 4.8 μM), BCRP (IC50 = 46.2 ± 20.3 μM), OATP1B1 (IC50 = 34.1 ± 3.15 μM), OATP1B3 (IC50 = 50.3 ± 7.5 μM), CYP2D6 (IC50 = 12.4 ± 0.74 μM), and CYP2C19 (IC50 = 46.1 ± 7.14 μM). Furthermore, it induced mRNA expression of BCRP/ABCG2 (3-fold at 20 μM) and to a lesser extent of CYP3A4 (2.3-fold at 20 μM), UGT1A4, and ABCB11. The only weak inducing properties were confirmed by weak activation of PXR. Considering its systemic concentrations its interaction potential as a perpetrator drug seems to be low. In contrast, our data suggest that riociguat is a P-gp substrate and might therefore act as a victim drug when co-administered with strong P-gp inductors or inhibitors. PMID:24657506

  8. Guanylate cyclase C deficiency causes severe inflammation in a murine model of spontaneous colitis.

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    Cruz Diana P

    2012-07-01

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

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

    KAUST Repository

    Wong, Aloysius Tze

    2013-07-08

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

  11. A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity.

    Science.gov (United States)

    Hess, Kenneth C; Liu, Jingjing; Manfredi, Giovanni; Mühlschlegel, Fritz A; Buck, Jochen; Levin, Lonny R; Barrientos, Antoni

    2014-10-01

    Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.

  12. Cloning and Functional Analysis of Lycopeneε-Cyclase (IbLCYe) Gene from Sweetpotato, Ipomoea batatas (L.) Lam

    Institute of Scientific and Technical Information of China (English)

    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.

  13. Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

    Science.gov (United States)

    Devitt, Luke C; Fanning, Kent; Dietzgen, Ralf G; Holton, Timothy A

    2010-01-01

    The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.

  14. A Single Residue Switch for Mg2+-dependent Inhibition Characterizes Plant Class II Diterpene Cyclases from Primary and Secondary Metabolism*

    Science.gov (United States)

    Mann, Francis M.; Prisic, Sladjana; Davenport, Emily K.; Determan, Mara K.; Coates, Robert M.; Peters, Reuben J.

    2010-01-01

    Class II diterpene cyclases mediate the acid-initiated cycloisomerization reaction that serves as the committed step in biosynthesis of the large class of labdane-related diterpenoid natural products, which includes the important gibberellin plant hormones. Intriguingly, these enzymes are differentially susceptible to inhibition by their Mg2+ cofactor, with those involved in gibberellin biosynthesis being more sensitive to such inhibition than those devoted to secondary metabolism, which presumably limits flux toward the potent gibberellin phytohormones. Such inhibition has been suggested to arise from intrasteric Mg2+ binding to the DXDD motif that cooperatively acts as the catalytic acid, whose affinity must then be modulated in some fashion. While further investigating class II diterpene cyclase catalysis, we discovered a conserved basic residue that seems to act as a counter ion to the DXDD motif, enhancing the ability of aspartic acid to carry out the requisite energetically difficult protonation of a carbon-carbon double bond and also affecting inhibitory Mg2+ binding. Notably, this residue is conserved as a histidine in enzymes involved in gibberellin biosynthesis and as an arginine in those dedicated to secondary metabolism. Interchanging the identity of these residues is sufficient to switch the sensitivity of the parent enzyme to inhibition by Mg2+. These striking findings indicate that this is a single residue switch for Mg2+ inhibition, which not only supports the importance of this biochemical regulatory mechanism in limiting gibberellin biosynthesis, but the importance of its release, presumably to enable higher flux, into secondary metabolism. PMID:20430888

  15. Somatic 'soluble' adenylyl cyclase isoforms are unaffected in Sacy tm1Lex/Sacy tm1Lex 'knockout' mice.

    Directory of Open Access Journals (Sweden)

    Jeanne Farrell

    Full Text Available BACKGROUND: Mammalian Soluble adenylyl cyclase (sAC, Adcy10, or Sacy represents a source of the second messenger cAMP distinct from the widely studied, G protein-regulated transmembrane adenylyl cyclases. Genetic deletion of the second through fourth coding exons in Sacy(tm1Lex/Sacy(tm1Lex knockout mice results in a male sterile phenotype. The absence of any major somatic phenotype is inconsistent with the variety of somatic functions identified for sAC using pharmacological inhibitors and RNA interference. PRINCIPAL FINDINGS: We now use immunological and molecular biological methods to demonstrate that somatic tissues express a previously unknown isoform of sAC, which utilizes a unique start site, and which 'escapes' the design of the Sacy(tm1Lex knockout allele. CONCLUSIONS/SIGNIFICANCE: These studies reveal increased complexity at the sAC locus, and they suggest that the known isoforms of sAC play a unique function in male germ cells.

  16. Adenylyl cyclase types I and VI but not II and V are selectively inhibited by nitric oxide

    Directory of Open Access Journals (Sweden)

    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.

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

    OpenAIRE

    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...

  18. Tumor Targeting and Drug Delivery by Anthrax Toxin.

    Science.gov (United States)

    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

  19. ArachnoServer: a database of protein toxins from spiders

    Directory of Open Access Journals (Sweden)

    Kaas Quentin

    2009-08-01

    Full Text Available 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 the largest pool of toxic peptides. However, at present, there are no databases dedicated to spider toxins and hence it is difficult to realise their full potential as drugs, insecticides, and pharmacological probes. Description We have developed ArachnoServer, a manually curated database that provides detailed information about proteinaceous toxins from spiders. Key features of ArachnoServer include a new molecular target ontology designed especially for venom toxins, the most up-to-date taxonomic information available, and a powerful advanced search interface. Toxin information can be browsed through dynamic trees, and each toxin has a dedicated page summarising all available information about its sequence, structure, and biological activity. ArachnoServer currently manages 567 protein sequences, 334 nucleic acid sequences, and 51 protein structures. Conclusion ArachnoServer provides a single source of high-quality information about proteinaceous spider toxins that will be an invaluable resource for pharmacologists, neuroscientists, toxinologists, medicinal chemists, ion channel scientists, clinicians, and structural biologists. ArachnoServer is available online at http://www.arachnoserver.org.

  20. Role of receptors in Bacillus thuringiensis crystal toxin activity.

    Science.gov (United States)

    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

  1. Botulinum toxin for treatment of glandular hypersecretory disorders.

    LENUS (Irish Health Repository)

    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.

  2. Nanoparticle-detained toxins for safe and effective vaccination

    Science.gov (United States)

    Hu, Che-Ming J.; Fang, Ronnie H.; Luk, Brian T.; Zhang, Liangfang

    2013-12-01

    Toxoid vaccines--vaccines based on inactivated bacterial toxins--are routinely used to promote antitoxin immunity for the treatment and prevention of bacterial infections. Following chemical or heat denaturation, inactivated toxins can be administered to mount toxin-specific immune responses. However, retaining faithful antigenic presentation while removing toxin virulence remains a major challenge and presents a trade-off between efficacy and safety in toxoid development. Here, we show a nanoparticle-based toxin-detainment strategy that safely delivers non-disrupted pore-forming toxins for immune processing. Using erythrocyte membrane-coated nanoparticles and staphylococcal α-haemolysin, we demonstrate effective virulence neutralization via spontaneous particle entrapment. Compared with vaccination with heat-denatured toxin, mice vaccinated with the nanoparticle-detained toxin showed superior protective immunity against toxin-mediated adverse effects. We find that the non-disruptive detoxification approach benefited the immunogenicity and efficacy of toxoid vaccines. We anticipate that this study will open new possibilities in the preparation of antitoxin vaccines against the many virulence factors that threaten public health.

  3. Gene therapy for carcinoma of the breast: Genetic toxins

    International Nuclear Information System (INIS)

    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

  4. 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

    OpenAIRE

    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 ...

  5. SVM-Based Prediction of Propeptide Cleavage Sites in Spider Toxins Identifies Toxin Innovation in an Australian Tarantula

    OpenAIRE

    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...

  6. First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

    Science.gov (United States)

    M'Barek, Sarrah; Fajloun, Ziad; Cestèle, Sandrine; Devaux, Christiane; Mansuelle, Pascal; Mosbah, Amor; Jouirou, Besma; Mantegazza, Massimo; Van Rietschoten, Jurphaas; El Ayeb, Mohamed; Rochat, Hervé; Sabatier, Jean-Marc; Sampieri, François

    2004-11-01

    Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.

  7. A common origin for the bacterial toxin-antitoxin systems parD and ccd, suggested by analyses of toxin/target and toxin/antitoxin interactions.

    Directory of Open Access Journals (Sweden)

    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.

  8. Tetanus toxin : primary structure, expression in E. coli, and homology with botulinum toxins

    NARCIS (Netherlands)

    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

  9. Binding of Diphtheria Toxin to Phospholipids in Liposomes

    Science.gov (United States)

    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

  10. Botulinum toxin in the treatment of sialorrhea

    Directory of Open Access Journals (Sweden)

    Svetel Marina

    2009-01-01

    Full Text Available Background/Aim. Botulinum toxin-A (BTX-A is known to block the release of acetylcholine from motor and autonomic nerve terminals and may significantly decrease saliva production when injected intraglandulary. The aim of this study was to evaluate effects of BTX-A injections in the treatment of disabling sialorrhea in various neurological disorders. Methods. This study included 19 consecutive patients with significant sialorrhea associated with various neurological disorders. Out of them 13 patients were with Parkinson's disease, two with pantothenate kinase-associated neurodegeneration, two with multiple system atrophy, one with Wilson's disease, and one patient with postoperative sialorrhea. Botulinum toxin-A (Dysport®, Ipsen Pharma was injected into the parotid glands with (n = 7 patients or without (n = 12 patients ultrasound guidance. All the patients were scored before and after the treatment and in weekly intervals thereafter using the salivation item of the part II (Activities of Daily Living of the Unified Parkinson's Disease Rating Scale (UPDRS. Results. Thirteen patients (68% reported beneficial effect of BTX-A injection, while 6 of them (32% had no response at all. The sialorrhea scores before and after the treatment were 3.1 ± 0.1 (range 2-4 and 1.8 ± 0.1 (range 0- 3, respectively (t = 5.636; p < 0.001. There was no difference in the magnitude of response between the groups with (t = 4.500; p = 0.004 and without (t = 3.674; p = 0.005 ultrasound control of injection sites. Adverse effects were registered in 5 patients (26%. Conclusions. Botulinum toxin-A injections to easily accessible parotid glands, without necessity for ultrasound guidance, are safe and efficaceous treatment for sialorrhea in different neurological disorders.

  11. Ultrasound-guided botulinum toxin injections

    Directory of Open Access Journals (Sweden)

    S. E. Khatkova

    2016-01-01

    Full Text Available One of the key conditions for achieving the desirable result during botulinum toxin therapy for muscular dystonia, spasticity, and other diseases accompanied by spasm, pain, and autonomic dysfunction (dystonias, spasticity, etc. is the proper administration of the agent into the muscles directly involved in the pathological process. The exact entry of botulinum toxin into the target muscles is essential for successful and safe treatment because its injection into a normal muscle may cause side effects. The most common errors are the incorrect depth and incorrect direction of a needle on insertion. Therefore, the exact injection of the agent particularly into the shallow and deep muscles is a difficult task even for an experienced specialist and requires the use of controlling methods.The European Consensus on Botulinum Toxin Therapy points out that various injection techniques are needed for the better identification of necessary muscles. However, there are currently no reports on the clear advantage of any technique. In our country, injections using palpation and anatomical landmarks have been widely used in routine practice so far; electromyographic monitoring and electrostimulation have been less frequently applied. In recent years, the new method ultrasound-guided injection has continued to grow more popular. This effective, accessible, and easy-to-use method makes it possible to manage a real-time injection process and to ensure the exact entry of the agent into the muscle. This paper is dedicated to a comparative analysis of different injection methods and to a description of the ultrasound-guided technique and its advantages over others. 

  12. Glycolipid binding preferences of Shiga toxin variants.

    Directory of Open Access Journals (Sweden)

    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.

  13. Prevention, control and detection of Fusarial toxins

    Directory of Open Access Journals (Sweden)

    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

  14. Shared Binding Sites in Lepidoptera for Bacillus thuringiensis Cry1Ja and Cry1A Toxins

    OpenAIRE

    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...

  15. Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027.

    Science.gov (United States)

    Van Lanen, Steven G; Lin, Shuangjun; Dorrestein, Pieter C; Kelleher, Neil L; Shen, Ben

    2006-10-01

    C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety that is derived from l-alpha-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-beta-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any alpha-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[(32)P]PP(i) exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated beta-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-beta-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic beta-amino acids such as beta-phenylalanine, 3-chloro-beta-tyrosine, and 3-hydroxy-beta-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all alpha-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-beta-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-beta-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-beta-tyrosyl-S-SgcC2 thioester substrate, and represents the first beta-amino acid-specific adenylation enzyme characterized biochemically. PMID:16887797

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

    Science.gov (United States)

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

    1988-05-17

    The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and

  17. Overview of Botulinum Toxins for Aesthetic Uses.

    Science.gov (United States)

    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

  18. Metal Ion Activation of Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B

    Science.gov (United States)

    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

  19. Distribution of the Toxin following Medial Rectus Muscular Injection of Botulinum Toxin Gel in Rabbits

    Institute of Scientific and Technical Information of China (English)

    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.

  20. Diarrhetic Shellfish Toxins and Other Lipophilic Toxins of Human Health Concern in Washington State

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  2. NCBI nr-aa BLAST: CBRC-OLAT-04-0016 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OLAT-04-0016 ref|NP_001098686.1| pituitary adenylate cyclase-activating polype...ptide 1B [Takifugu rubripes] emb|CAD38842.1| pituitary adenylate cyclase-activating polypeptide 1B [Takifugu rubripes] NP_001098686.1 0.0 88% ...

  3. NCBI nr-aa BLAST: CBRC-TNIG-22-0273 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TNIG-22-0273 ref|NP_001098686.1| pituitary adenylate cyclase-activating polype...ptide 1B [Takifugu rubripes] emb|CAD38842.1| pituitary adenylate cyclase-activating polypeptide 1B [Takifugu rubripes] NP_001098686.1 0.0 89% ...

  4. NCBI nr-aa BLAST: CBRC-GACU-08-0006 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-GACU-08-0006 ref|NP_001098686.1| pituitary adenylate cyclase-activating polype...ptide 1B [Takifugu rubripes] emb|CAD38842.1| pituitary adenylate cyclase-activating polypeptide 1B [Takifugu rubripes] NP_001098686.1 0.0 89% ...

  5. NCBI nr-aa BLAST: CBRC-DRER-06-0099 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DRER-06-0099 ref|NP_001098686.1| pituitary adenylate cyclase-activating polype...ptide 1B [Takifugu rubripes] emb|CAD38842.1| pituitary adenylate cyclase-activating polypeptide 1B [Takifugu rubripes] NP_001098686.1 0.0 82% ...

  6. NCBI nr-aa BLAST: CBRC-TSYR-01-0228 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-TSYR-01-0228 ref|NP_001098686.1| pituitary adenylate cyclase-activating polype...ptide 1B [Takifugu rubripes] emb|CAD38842.1| pituitary adenylate cyclase-activating polypeptide 1B [Takifugu rubripes] NP_001098686.1 8e-04 40% ...

  7. NCBI nr-aa BLAST: CBRC-XTRO-01-0270 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0270 ref|NP_001098685.1| pituitary adenylate-cyclase activating polype...ptide receptor 1A [Takifugu rubripes] emb|CAD35690.1| pituitary adenylate-cyclase activating polypeptide receptor 1A [Takifugu rubripes] NP_001098685.1 2e-07 30% ...

  8. NCBI nr-aa BLAST: CBRC-OLAT-17-0031 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OLAT-17-0031 ref|NP_001098686.1| pituitary adenylate cyclase-activating polype...ptide 1B [Takifugu rubripes] emb|CAD38842.1| pituitary adenylate cyclase-activating polypeptide 1B [Takifugu rubripes] NP_001098686.1 0.0 79% ...

  9. Bordetella pertussis Strain Lacking Pertactin and Pertussis Toxin.

    Science.gov (United States)

    Williams, Margaret M; Sen, Kathryn; Weigand, Michael R; Skoff, Tami H; Cunningham, Victoria A; Halse, Tanya A; Tondella, M Lucia

    2016-02-01

    A Bordetella pertussis strain lacking 2 acellular vaccine immunogens, pertussis toxin and pertactin, was isolated from an unvaccinated infant in New York State in 2013. Comparison with a French strain that was pertussis toxin-deficient, pertactin wild-type showed that the strains carry the same 28-kb deletion in similar genomes.

  10. Botulinum Toxin in the Treatment of Facial Paralysis.

    Science.gov (United States)

    Mehdizadeh, Omid B; Diels, Jacqueline; White, William Matthew

    2016-02-01

    This article reviews the current literature supporting the use of botulinum toxin in producing symmetric facial features and reducing unwanted, involuntary movements. Methods, protocols, and adverse events are discussed. Additionally, the authors suggest that using botulinum toxin A therapy in postparalytic facial synkinesis can provide long-term results when used in conjunction with other treatment modalities.

  11. Detection of shiga toxins by lateral flow assay

    Science.gov (United States)

    Shiga toxin-producing Escherichia coli (STEC) produce Shiga toxins (Stxs) that can cause human disease and death. The contamination of food products with STEC represents a food safety problem that necessitates rapid and effective detection strategies to mitigate risk. In this manuscript we report ...

  12. Anthrax Toxin Delivers a One-Two Punch

    OpenAIRE

    Bradley, Kenneth A; LeVine, Steven M

    2010-01-01

    Although identified as a major Bacillus anthracis virulence factor over 50 years ago, defining the physiologically relevant targets of anthrax toxin has been challenging. Liu et al. demonstrate that intoxication of myeloid-derived cells contributes to establishing infection, but is not required for mortality resulting from high toxin concentrations associated with end-stage disease.

  13. Serum Antibody Response to Clostridium botulinum Toxin in Infant Botulism

    OpenAIRE

    Rubin, Lorry G.; Dezfulian, Manuchehr; Yolken, Robert H.

    1982-01-01

    A serum antibody response has not been previously demonstrated after infection with Clostridium botulinum. We developed an enzyme immunoassay for measuring serum antibody to C. botulinum toxins A, B, and E. This assay system detected a specific immunoglobulin G and immunoglobulin M antibody response to C. botulinum toxin in two patients with infant botulism.

  14. Recent advances in the medicinal chemistry of polyamine toxins

    DEFF Research Database (Denmark)

    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 ...

  15. Materials used in toxin detection, from A to Z

    Science.gov (United States)

    Whether it is during extraction, isolation, or detection the analysis of toxins invariably entails the interactions with materials that can bind to or otherwise interact with the toxin or the matrix in which it is lodged. As a result an enormous variety of materials have been developed to facilitate...

  16. Oxidative Stress in Shiga Toxin Production by Enterohemorrhagic Escherichia coli

    Directory of Open Access Journals (Sweden)

    Katarzyna Licznerska

    2016-01-01

    Full Text Available Virulence of enterohemorrhagic Escherichia coli (EHEC strains depends on production of Shiga toxins. These toxins are encoded in genomes of lambdoid bacteriophages (Shiga toxin-converting phages, present in EHEC cells as prophages. The genes coding for Shiga toxins are silent in lysogenic bacteria, and prophage induction is necessary for their efficient expression and toxin production. Under laboratory conditions, treatment with UV light or antibiotics interfering with DNA replication are commonly used to induce lambdoid prophages. Since such conditions are unlikely to occur in human intestine, various research groups searched for other factors or agents that might induce Shiga toxin-converting prophages. Among other conditions, it was reported that treatment with H2O2 caused induction of these prophages, though with efficiency significantly lower relative to UV-irradiation or mitomycin C treatment. A molecular mechanism of this phenomenon has been proposed. It appears that the oxidative stress represents natural conditions provoking induction of Shiga toxin-converting prophages as a consequence of H2O2 excretion by either neutrophils in infected humans or protist predators outside human body. Finally, the recently proposed biological role of Shiga toxin production is described in this paper, and the “bacterial altruism” and “Trojan Horse” hypotheses, which are connected to the oxidative stress, are discussed.

  17. Oxidative Stress in Shiga Toxin Production by Enterohemorrhagic Escherichia coli.

    Science.gov (United States)

    Licznerska, Katarzyna; Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Dydecka, Aleksandra; Topka, Gracja; Gąsior, Tomasz; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2016-01-01

    Virulence of enterohemorrhagic Escherichia coli (EHEC) strains depends on production of Shiga toxins. These toxins are encoded in genomes of lambdoid bacteriophages (Shiga toxin-converting phages), present in EHEC cells as prophages. The genes coding for Shiga toxins are silent in lysogenic bacteria, and prophage induction is necessary for their efficient expression and toxin production. Under laboratory conditions, treatment with UV light or antibiotics interfering with DNA replication are commonly used to induce lambdoid prophages. Since such conditions are unlikely to occur in human intestine, various research groups searched for other factors or agents that might induce Shiga toxin-converting prophages. Among other conditions, it was reported that treatment with H2O2 caused induction of these prophages, though with efficiency significantly lower relative to UV-irradiation or mitomycin C treatment. A molecular mechanism of this phenomenon has been proposed. It appears that the oxidative stress represents natural conditions provoking induction of Shiga toxin-converting prophages as a consequence of H2O2 excretion by either neutrophils in infected humans or protist predators outside human body. Finally, the recently proposed biological role of Shiga toxin production is described in this paper, and the "bacterial altruism" and "Trojan Horse" hypotheses, which are connected to the oxidative stress, are discussed. PMID:26798420

  18. 9 CFR 121.3 - VS select agents and toxins.

    Science.gov (United States)

    2010-01-01

    ... genetically modified. (d) VS select agents or toxins that meet any of the following criteria are excluded from... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS POSSESSION, USE, AND... recombinant organisms: (1) Nucleic acids that can produce infectious forms of any of the select agent...

  19. Comparative genomics of Shiga toxin encoding bacteriophages

    Directory of Open Access Journals (Sweden)

    Smith Darren L

    2012-07-01

    Full Text Available Abstract Background Stx bacteriophages are responsible for driving the dissemination of Stx toxin genes (stx across their bacterial host range. Lysogens carrying Stx phages can cause severe, life-threatening disease and Stx toxin is an integral virulence factor. The Stx-bacteriophage vB_EcoP-24B, commonly referred to as Ф24B, is capable of multiply infecting a single bacterial host cell at a high frequency, with secondary infection increasing the rate at which subsequent bacteriophage infections can occur. This is biologically unusual, therefore determining the genomic content and context of Ф24B compared to other lambdoid Stx phages is important to understanding the factors controlling this phenomenon and determining whether they occur in other Stx phages. Results The genome of the Stx2 encoding phage, Ф24B was sequenced and annotated. The genomic organisation and general features are similar to other sequenced Stx bacteriophages induced from Enterohaemorrhagic Escherichia coli (EHEC, however Ф24B possesses significant regions of heterogeneity, with implications for phage biology and behaviour. The Ф24B genome was compared to other sequenced Stx phages and the archetypal lambdoid phage, lambda, using the Circos genome comparison tool and a PCR-based multi-loci comparison system. Conclusions The data support the hypothesis that Stx phages are mosaic, and recombination events between the host, phages and their remnants within the same infected bacterial cell will continue to drive the evolution of Stx phage variants and the subsequent dissemination of shigatoxigenic potential.

  20. Cardiovascular-Active Venom Toxins: An Overview.

    Science.gov (United States)

    Rebello Horta, Carolina Campolina; Chatzaki, Maria; Rezende, Bruno Almeida; Magalhães, Bárbara de Freitas; Duarte, Clara Guerra; Felicori, Liza Figueiredo; Ribeiro Oliveira-Mendes, Bárbara Bruna; do Carmo, Anderson Oliveira; Chávez-Olórtegui, Carlos; Kalapothakis, Evanguedes

    2016-01-01

    Animal venoms are a mixture of bioactive compounds produced as weapons and used primarily to immobilize and kill preys. As a result of the high potency and specificity for various physiological targets, many toxins from animal venoms have emerged as possible drugs for the medication of diverse disorders, including cardiovascular diseases. Captopril, which inhibits the angiotensin-converting enzyme (ACE), was the first successful venom-based drug and a notable example of rational drug design. Since captopril was developed, many studies have discovered novel bradykinin-potentiating peptides (BPPs) with actions on the cardiovascular system. Natriuretic peptides (NPs) have also been found in animal venoms and used as template to design new drugs with applications in cardiovascular diseases. Among the anti-arrhythmic peptides, GsMTx-4 was discovered to be a toxin that selectively inhibits the stretch-activated cation channels (SACs), which are involved in atrial fibrillation. The present review describes the main components isolated from animal venoms that act on the cardiovascular system and presents a brief summary of venomous animals and their venom apparatuses. PMID:26812904